Membrane Biophysics

Fundamental Principles of Membrane Biophysics
David Njus Department of Biological Sciences Wayne State University
D. Njus, 2000
Table of Contents Chapter 1. Biological Membranes Chapter 2. Thermodynamics of Micelle Formation Chapter 3. The Fluid Mosaic Membrane Chapter 4. Membrane Electrostatics Chapter 5. Specific and Non-Specific Binding Chapter 6. Permeability and Conductance Chapter 7. Permeability and Conductance of Electrolytes Chapter 8. Channels and Excitable Membranes Chapter 9. Active Transport Chapter 10. Facilitated Diffusion Chapter 11. Coupled Transport Chapter 12. Energy Coupling Chapter 13. Epithelial Transport Appendices I. Glossary of Symbols II. Abbreviations III. Fundamental Constants IV. Conversion Factors V. Mathematical Formulae
Glossary of Symbols A C c cmc D d E E e F F f f G g H h I J Ki Kp k ki m N n P Q q R r S T t u V v W X area molar concentration, capacitance velocity of light critical micelle concentration diffusion coefficient derivative energy, reduction potential electric field electronic charge force Faraday constant frictional coefficient fugacity Gibbs free energy conductance, acceleration of gravity enthalpy Plancks constant current flow equilibrium constant for reaction i partition coefficient Boltzmann constant rate constant for reaction i mass, aggregation number Avogadro's number amount in moles permeability coefficient, pressure, power heat charge gas constant, resistance radius entropy absolute temperature time mobility volume velocity work mole fraction x Z z o distance collision factor valence
activity coefficient difference dipole moment dielectric constant permittivity of vacuum viscosity, efficiency wavelength of light electrochemical potential density reflection coefficient electrical potential
Abbreviations atm C cal coul D Da eq esu F g j K l M m min mol S sec V Angstroms atmospheres degrees centigrade calories coulombs Debyes Daltons equivalents electrostatic units farads (coul/volt) grams joules degrees Kelvin liters moles/liter meters minutes moles Siemens seconds volts 103 10-2 10-3 10-6 10-9 10-12 10-15 10-18
k c m n p f a
kilo centi milli micro nano pico femto atto
Fundamental Constants Gas constant R = 8.3144 j.K-1.mol-1 1.9872 cal.K-1.mol-1 8.3144 x 107 ergs.K-1.mol-1 0.082054 l.atm.K-1.mol-1 k = 1.38044 x 10-16 erg.K-1 N = 6.0230 x 1023 molecules.mole-1 To = 273.15 K F = 96,490 coul.eq-1 o = 8.854 x 10-12 coul.m-1.volt-1 Vo = 22.4138 l.mol-1 e = 4.80286 x 10-10 esu 1.602 x 10-19 coul me = 9.1083 x 10-28 g c = 2.997930 x 1010 cm.sec-1 g = 980.665 cm.sec-2 h = 6.6252 x 10-27 erg.sec = 103 l.m-3 Z = 1011 M-1.sec-1
Boltzmann constant Avogadro's Number Ice point Faraday constant Permittivity of vacuum Molar volume, ideal gas, 0C, 1 atm Electronic charge Electron mass Velocity of light Standard acceleration of gravity Planck's constant Volume conversion factor Collision factor
Conversion Factors 1 atm = 760 mm = 1.01325 x 106 dyne.cm-2 1 cal = 4.184000 j 1 j = 107 ergs = 1 volt.coul 1 erg = 1 dyne.cm 1 ev = 1.60206 x 10-12 erg 1 l.atm = 24.22 cal 1 = 10-10 m 1 Debye = 10-18 esu.cm.molecule-1 = 2 x 10-6 coul.m.mol-1 1 kcal/eq = 0.043362 volts
Mathematical Formulae sinh x = 1/2 (ex - e-x) sinh x = x + x3/3! + x5/5! + x7/7! + ... ex = 1 + x + x2/2! + x3/3! + ... Surface Area Cylinder (minus ends) Sphere Volume Cylinder Sphere
A = 2rh A = 4r2 V = r2h V = (4/3) r3

CHAPTER 1: BIOLOGICAL MEMBRANES
D. Njus, 2000
CHAPTER 1: BIOLOGICAL MEMBRANES
Section 1.1. Biological Membranes Biological membranes maintain the spatial organization of life. Membranes defined the boundaries of the first living cells and still work to shield cellular metabolism from changes in the environment. Membranes prevent undesirable agents from entering cells and keep needed molecules on the inside. They also organize the interior of eukaryotic cells by separating compartments for specialized purposes. Membranes are not static barriers, but active structures. To function effectively, they must selectively pass molecules, ions, and signals from one side to the other. The strategy underlying biological membrane function is that the best barrier between aqueous compartments is a hydrophobic layer. The water-soluble compounds present within cells and in their environments are not soluble in the lipid milieu of the membrane and pass slowly or not at all through even a very thin lipid layer. This mechanism has a number of advantages which life has exploited. First, the lipid bilayer is a natural structure and assembles spontaneously. Second, the structure is flexible and allows for growth and movement as well as for the insertion and operation of protein machinery. Finally, the structure has a low dielectric constant giving the membrane electrical properties which are used in signalling, transport and energy transduction. To understand how biological membranes function, we will begin by analyzing their structure. The structure determines the fundamental properties of fluidity, permeability, and membrane potential. The origin and characterization of these properties will be analyzed next. Finally, we will discuss how these properties contribute to the various functions of biological membranes: signal transduction, energy transduction, and transport. Life, like all other processes in our universe, obeys the laws of physics and chemistry. Consequently, the theoretical framework of physical chemistry provides powerful tools for understanding living systems. Especially important is the requirement imposed by the second law of thermodynamics: processes must result in a net decrease in free energy in order to occur spontaneously. Free energy changes govern all metabolic processes, but they are particularly apparent in common phenomena of biological membranes. For example, membrane structure is governed by the distribution of compounds between the hydrophobic interior of the membrane and the aqueous spaces on either side. Free energy also determines the movement of molecules and ions across membranes in response to concentration gradients and membrane potentials. Because membranes have a well defined planar geometry, the mathematics is simpler than it might otherwise be. Thus, to understand in depth the structure and function of biological membranes, it is essential to understand and apply principles of physical chemistry. The purpose of this course is to construct a coherent framework to do that.
Section 1.2. The Fluid Mosaic Model of Membrane Structure Early on, it was recognized that hydrophobic compounds passed more readily than water-soluble compounds through biological membranes. This, coupled with the identification of lipids as a major component, led to the notion that biological membranes have a hydrophobic character. The calculation (erroneous, as it turns out) that the lipid Page 1.1
content was twice that needed for a single layer of lipid led to the concept (correct, as luck would have it) of the lipid bilayer (Gorter and Grendel, 1925; Danielli and Davson, 1935). Lipids are amphiphilic compounds with a small hydrophilic headgroup attached to long hydrocarbon chains. In the lipid bilayer, lipids are aligned with the headgroups facing the water on either surface of the membrane and the hydrophobic hydrocarbons sandwiched in between. The lipid bilayer concept did not establish the location of the protein components of the membrane. Originally, for lack of a better site, the proteins were stuck on to the membrane surface. This was not tenable, of course, because proteins are responsible for moving molecules and messages across membranes and they could not perform those functions without being a integral part of the membrane itself. This realization gave rise to the concept of integral and peripheral proteins. Peripheral proteins are loosely associated with the membrane and located on the surface of the lipid bilayer. Integral proteins are inserted into the membrane and pass all the way (or much of the way) across the membrane. Originally, the integral proteins were thought to form a well defined matrix with the lipid bilayer filling in the spaces in between. In the late 1960's, however, it became clear that many proteins are not rigidly fixed in the membrane, but can diffuse across the surfaces of cells relatively easily and independently. Membranes came to be viewed as fluid structures with proteins and lipids arranged in their thermodynamically most favorable structure. Lipids exist in a bilayer and provide the milieu in which the integral membrane proteins float. The proteins are oriented so that their hydrophobic surfaces are immersed in the hydrophobic interior of the lipid bilayer. Hydrophilic amino acids are exposed only in the aqueous regions on either side of the membrane. The organization of the membrane is a direct consequence of the partitioning of its components, both lipid and protein, so that hydrophobic regions are kept within the membrane and hydrophilic parts are exposed to the water on either side. Because the components are not held together by bonds, they are free to diffuse and move independently within the plane of the membrane. Singer and Nicolson (1972) captured this view in the fluid mosaic model. The fluid mosaic model persists as the accepted view of membrane structure. The recognition of linkages between membrane proteins and components of the cytoskeletal system has modified the concept somewhat. The cytoskeleton does impose some constraints on the distribution of membrane proteins. As we shall see, phase separation also can create separate domains with different characteristics within membranes. Consequently, the fluid mosaic model does not imply that all the components of a particular membrane are randomly and homogeneously distributed.
Section 1.3. Classes of Biological Membranes Biological membranes perform many different functions in all kinds of cells. They can be divided, however, into four classes based on differences in their fundamental energetics. These differences arose during the course of evolution as different organisms adopted different strategies to cope with their environments. The first class includes prokaryotic cell membranes, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts. These membranes, which share a common evolutionary origin, do not contain cholesterol. A proton gradient drives the functions of these membranes. The proton gradient is generated by a variety of
Page 1.2
mechanisms, but a redox chain is common. These membranes can use the proton gradient to generate ATP using an ATP synthase of the F1F0 type. The second class consists of plasma membranes of animal cells. These have a Na+/K+ ATPase which pumps Na+ out of and K+ into the cells. The Na+ and K+ concentration gradients created thereby participate in many functions of the membrane including transport, excitability, and signalling. The third class of membranes includes the plasma membranes of plant and fungal cells. These differ from animal cell membranes in that they lack a Na+/K+ ATPase and instead have a proton-translocating ATPase of the P-type. The proton gradient created by this ATPase drives transport and other functions of the plant plasma membrane. This fundamental difference between plant and animal cell membranes reflects a basic difference between plant and animal lifestyles. Unlike animal cells, plant cells cannot rely on seawater or a circulatory fluid to provide external Na+, so the substitution of a proton pump for a sodium pump is necessary. Moreover, because plants are immobile, plant cells can have a rigid cell wall to support the plasma membrane in times of osmotic imbalance. Animal cells, by contrast, must maintain osmotic balance by regulating the concentrations of internal osmolytes such as Na+ and K+ to balance the external salt concentration. The fourth class of membranes includes membranes of the vacuolar system. This comprises the membranes of Golgi-derived organelles including lysosomes, endosomes, secretory vesicles in animal cells and peroxisomes, vacuoles and tonoplasts in plants and fungi. These membranes have a proton-translocating ATPase of the V-type. The proton gradient created by this ATPase drives processes in the membrane and also makes the interior of the organelle acidic, a property frequently important in the function of the organelle.
Section 1.4. Membrane Biosynthesis and Asymmetry Although the lipid bilayer is basically a symmetrical structure, natural membranes are not. The two sides of the membrane differ, so the membrane has a functional polarity. Molecules, ions and signals will be moved one way but not the other. The two sides of the membrane differ because of the way the membrane is synthesized. In animal and plant cells, the plasma membrane and vacuolar membranes share a common synthetic pathway. The proteins are inserted into the endoplasmic reticulum. The membranes are transferred to the Golgi apparatus where carbohydrates are attached and processed. The membranes are also sorted and leave the Golgi targeted to their final destinations. This process has a number of consequences. First, the proteins are inserted into the membranes with a defined orientation; they are inserted from the cytoplasmic side of the membrane. Second, the carbohydrates are attached only to the other side, the interior of the Golgi. Thus, the carbohydrates face only the interior of organelles and the exterior of the cell; they do not appear on the cytosolic side of a membrane. This, along with the processes of exocytosis and endocytosis, emphasize that the interior of vacuolar organelles is equivalent to the exterior of the cell in terms of membrane polarity. In bacterial cells, the proteins and lipids are synthesized inside the cell and inserted into the membrane. The biosynthesis of the mitochondrial inner membrane and the thylakoid membrane are more complex because some of the proteins are synthesized in the cytoplasm and then inserted into the organelle. The end result, however, is that the
Page 1.3
proteins are placed in the membrane with a defined orientation and this gives the membrane polarity. The structure of a biological membrane is a consequence of both spontaneous assembly and programmed development. The fluid mosaic model emphasizes the spontaneous assembly of the lipid bilayer with the proteins orienting to accommodate their own hydrophobic and hydrophilic surfaces. At the same time, the membrane is asymmetrical largely because of its history. The structure of each protein is determined by the amino acid sequence and the direction in which that sequence was inserted into the membrane. The further processing of that protein, particularly the attachment of carbohydrates, is also asymmetrical since the relevant enzymes are confined to one side of the membrane or the other. The asymmetry is obviously crucial because it gives membranes a polarity essential for function. Transport occurs in defined directions. This in turn creates the membrane potential, a polarity that plays a fundamental role in many membrane processes.
Section 1.5. Summary Lipids in biological membranes are arranged predominantly in a bilayer structure. Proteins are oriented so that hydrophobic amino acids are buried inside the membrane and hydrophilic residues are exposed on the aqueous surfaces. Proteins are inserted with a defined orientation and give polarity to the membrane. Four classes of biological membranes may be distinguished on the basis of their different primary ion pumps. All of these considerations should be kept in mind as we proceed to analyze the structure and function of biological membranes.
References S.J. Singer and G.L. Nicolson (1972) The fluid mosaic model of the structure of cell membranes, Science 175, 720-731.
Page 1.4

CHAPTER 2: THERMODYNAMICS OF MICELLE FORMATION
D. Njus, 2000
CHAPTER 2: THERMODYNAMICS OF MICELLE FORMATION
Section 2.1. Properties of Water Water is a remarkable substance in many ways, and its unusual properties are crucial in the functioning of biological membranes. First, water molecules dissociate into H+ and OH-. This property allows H+ to equilibrate among protonatable groups in all of the molecules in a solution. As we shall see, it also makes H+ a convenient ion to use for creating electrical potential differences across biological membranes. A second important property of water is its polarity. The dipole moment of the OH bond is 1.51 Debyes and the water molecule itself has a dipole moment of 1.84 Debyes. As a consequence, water has a high dielectric constant (80.37 at 20C) and polarizes to neutralize electric fields. For this reason, electric fields and associated differences in electrical potential exist primarily across biological membranes rather than across aqueous regions of cells. Finally, water molecules participate in hydrogen bonding. The hydrogen bond is primarily electrostatic (a dipole-dipole interaction) with an energy of 4.5-6 kcal/mol. The bond is linear with the hydrogen atom situated directly between two electronegative atoms (two oxygen atoms in the case of the H2O-H2O bond). Hydrogen bonding accounts for the relatively low freezing and boiling points of water relative to other compounds with comparable molecular weights. Most importantly, hydrogen bonding is responsible for the hydrophobic effect. Because hydrogen bonds are fairly strong, water molecules will orient so as to hydrogen bond even if this orientation restricts their mobility. For example, water molecules on the surface (an air-water interface) will have fewer other water molecules with which to interact than will molecules in the interior of the solution. Molecules on the surface will nevertheless hydrogen bond to other water molecules, but the smaller number of possible bonding partners means that they will have a lower entropy than molecules in the interior. To increase the entropy of the system, water will minimize its surface area. This effect is responsible for the high surface tension of water. Because nonpolar molecules will not hydrogen bond, they reduce the bonding possibilities of adjacent water molecules. Thus, just as increasing the surface area decreases the entropy of an aqueous solution, so does introducing nonpolar molecules. Exclusion of these nonpolar substances from the aqueous solution increases the entropy and decreases the free energy. It is this entropy-driven effect that causes nonpolar compounds to be excluded from aqueous solutions. It is important to recognize that nonpolar molecules do not attract each other; they are pushed together because they are mutually excluded from water. This phenomenon is known as the hydrophobic effect.
Page 2.1
Figure 2.1
Page 2.2
Section 2.2. Structures Formed by Amphiphilic Molecules Amphiphiles are those molecules that are polar on one end and nonpolar on the other. These promiscuous molecules have affinities for both aqueous and nonpolar phases. At low concentrations, they dissolve in water. At some critical concentration, however, they reach their solubility limit and begin to aggregate into micellar structures. The micelle structure allows the molecule to keep its polar region in the aqueous phase on the surface of the micelle and the nonpolar portion in the nonpolar interior of the micelle. The limiting monomer solubility is called the critical micelle concentration (cmc). At concentrations below the cmc, the amphiphile will exist as monomers. At concentrations above this level, the excess amphiphile will aggregate to form micelles. Two factors are involved in the spontaneous formation of micelles. First, the hydrophobic effect causes the nonpolar portion of the molecule to be separated from water and sequestered in the interior of the structure. Second, interactions between the head groups determine how closely the molecules may be packed. Amphiphiles with a single hydrocarbon chain, such as dodecyl sulfate, must pack a number of head groups around a relatively small volume of hydrocarbon. This large surface area to volume ratio is achieved by forming a spherical micelle structure. By contrast, amphiphiles with two hydrocarbon chains, such as phospholipids, must pack the same number of headgroups around twice as large a volume of hydrocarbon. This smaller surface area to volume ratio is achieved by forming the bilayer structure (Figure 2.2).
Figure 2.2 It should be recognized that the spherical micelle and the planar bilayer are really two extremes of a continuum. Micelles in the shape of oblate spheroids will exhibit intermediate ratios of surface area to volume. Under a particular set of conditions, an amphiphile will form micelles with a particular surface area to volume ratio and thus will form micelles of a particular size. This characteristic of the micelle is described by the aggregation number m, the average number of amphiphile molecules in a single micelle. The critical micelle concentration and the aggregation number together characterize the micelle that a particular amphiphile will form under a given set of conditions. We can make some intuitive generalizations about how these parameters should respond to a variety of changes. Increasing the chain length of the amphiphile should lower the aqueous solubility and decrease the critical micelle concentration. For similar reasons, amphiphiles with two hydrocarbon chains (phospholipids) should have a lower cmc than those with a single chain (detergents). Ionic detergents should have a greater water solubility than nonionic detergents and therefore should have a higher critical micelle concentration. Repulsive forces between polar groups should be less for nonionic detergents than for ionic detergents. Therefore, nonionic detergents should form micelles with smaller surface areas per amphiphile (larger aggregation numbers). Increasing the ionic strength should diminish
Page 2.3
repulsive forces between polar groups of ionic detergents thereby increasing the aggregation number. These characteristics of the cmc and the aggregation number are illustrated by the data in Table I. Deoxycholate, cholate and sodium dodecyl sulfate are ionic detergents; Lubrol WX and Triton X-100 are nonionic. TABLE 2.1 Detergent cmc (mM) m ________________________________________________________________ Sodium dodecylsulfate 50 mM NaCl 2.3 72 500 mM NaCl 0.51 126 Deoxycholate 10 mM NaCl, pH 7.5 4 4 300 mM NaCl, pH 7.5 6 29 Cholate 45 2 Lubrol WX 0.125 96 Triton X-100 0.24 140 ________________________________________________________________
Section 2.3. The Hydrophobic Effect The thermodynamics of micelle formation has been analyzed in an elegant fashion by Tanford (1980). The two factors determining micelle structure, the hydrophobic effect and head group interaction, are each assumed to contribute separately to the free energy of the micelle. A summary of this analysis will be presented here. To understand the contribution of the hydrophobic effect to micelle structure, let us first consider the solubility of hydrocarbons in water. The chemical potential of the hydrocarbon in the aqueous phase is (2.1) w = w + RT ln Xw + RT ln fw We assume that ln fw = 0 because the hydrocarbon concentration in water is extremely low. Now consider the chemical potential of the hydrocarbon in a pure hydrocarbon phase: (2.2) HC = HC + RT ln XHC + RT ln fHC
In pure hydrocarbon, XHC = 1 so ln XHC = ln fHC = 0. When the hydrocarbon partitions between water and the hydrocarbon phase, equilibrium is reached when the chemical potentials are equal (w = HC). Therefore, (2.3) HC = w + RT ln Xw
Since Xw is the saturating concentration (in mole fraction) of the hydrocarbon in water, the free energy change for transferring a hydrocarbon molecule from water into the hydrocarbon phase can be determined from the compound's water solubility:
Page 2.4
(2.4)
HC - w = RT ln Xw
For a series of n-alkanes, the following empirical relationship is found: (2.5) HC - w = -2436 - 884 nc cal/mol
where nc is the number of carbon atoms in the molecule. Of course, as nc increases, the water solubility of the hydrocarbon decreases. Double bonds increase the water solubility (decrease the hydrophobicity) of hydrocarbons. We can use a similar analysis to examine the partitioning of hydrocarbon molecules between water and the interior of a micelle. The chemical potential of the hydrocarbon molecule in a micelle is (2.6) mic = mic + RT ln Xmic
Setting mic equal to the chemical potential of the hydrocarbon in water (equation 2.1) allows us to solve for the free energy change for transfer of the hydrocarbon from water to the interior of the micelle: (2.7) mic - w = RT ln Xw - RT ln Xmic = RT ln (Xw/Xmic)
If Xw is the solubility of the hydrocarbon in water, then Xmic can be calculated from the increase in solubility observed in the presence of micelles. For a series of n-alkanes and micelles formed from sodium dodecyl sulfate, the following empirical relationship is observed: (2.8) mic - w = -1934 - 771 nc cal/mol As in the case of the transfer of hydrocarbon from water to the pure hydrocarbon phase, the free energy change is proportional to the number of carbon atoms in the compound and the energy contribution of each carbon atom is about 800 cal/mol.
Section 2.4. Thermodynamics of Single-Component Micelles There is a dynamic tension at work in the micelle structure. The polar groups tend to repel each other because they have similar charges and dipole moments. Nevertheless, they must remain close enough together to prevent water from gaining access to the hydrophobic interior of the micelle. Let us consider the chemical potential of an amphiphile in water (w) and in a micelle of size m (mic,m). (2.9) and (2.10) mic,m = mic,m + (RT/m) ln (Xm/m) RT ln (Xm/m) is the contribution of the whole micelle to the free energy, so this term is divided by m to determine the free energy contribution of each molecule of amphiphile. w = w + RT ln Xw + RT ln fw
Page 2.5
Since amphiphiles will equilibrate between the aqueous phase and micelles, these chemical potentials must be equal. Therefore, (2.11) mic,m - w = RT ln Xw + RT ln fw - (RT/m) ln (Xm/m)
If the aggregation number m is large or the mole fraction of amphiphile in micelles Xm is small, then the final term can be ignored. This allows us to calculate the cmc knowing mic,m - w. (2.12) mic,m - w = RT ln Xw = RT ln cmc
Alternatively, equation 2.11 can be solved for ln Xm (2.13) ln Xm = -(m/RT)(mic,m - w) + m ln Xw + m ln fw + ln m
This allows us to calculate the concentration of amphiphile in micelles of aggregation number m at any given aqueous amphiphile concentration Xw. Again, we must know mic,m - w. To analyze mic,m - w, we will separate this energy into two parts: that contributed by the hydrophobic effect (Um - w) and that caused by head group repulsion (Wm). (2.14) mic,m - w = Um - w + Wm
Tanford determines each of these components semiempirically, although the expressions can be rationalized to some extent. The contribution of the hydrophobic effect is specified as (2.15) Um - w = -2100 - 700 (nc -2) + 25 (A-21) + Constant
This expression attributes 700 cal/mol to each carbon atom in the hydrocarbon chain in rough agreement with the values given in Section 2.3. The hydrophobic effect is also assumed to depend linearly on the surface area of the micelle per molecule of amphiphile (A). A is measured in square angstroms, so each square angstrom of hydrophobic surface area per molecule adds about 25 cal/mol to the free energy. This dependence of the free energy on micelle surface area per molecule is crucial since it helps determine the size of the micelle. While the volume of the micelle increases in proportion to the aggregation number m, the surface area increases only in proportion to m2/3. Therefore, larger micelles will have a smaller surface area per molecule and smaller micelles will have a larger surface area per molecule. Under a given set of conditions, an amphiphile molecule will have an optimum surface area, and this will determine the size of the micelle that it will form. Having estimated the contribution of the hydrophobic effect to the free energy of micelle formation, we must estimate the contribution of the head group interaction (Wm). This may be calculated from pressure vs. area curves measured for monolayers of amphiphile. If an amphiphile is added to water, it will form a monolayer on the surface of the water with the polar end in the water and the nonpolar region extending into the air. Page 2.6
The pressure required to compress this monolayer will depend on the repulsive forces between the head groups (Phg) and on the intrinsic pressure exerted by ideal molecules by virtue of their kinetic energy (Pke). The latter pressure is (2.16) Pke = kT/A
where A is the surface area per molecule and k is the Boltzmann constant. This expression is the two-dimensional correlate of the ideal gas law. The pressure attributable to head group interaction is therefore (2.17) Phg = P - (kT/A)
The work done against this head group repulsion is (2.18) Wm = - (P-kT/A) dA
This work function can be evaluated by integrating pressure vs. area curves determined from monolayer compression experiments and corrected for kT/A (Figure 2.2). For the trimethylammonium and sulfate head groups, the work functions as evaluated by Tanford are (2.19) (2.20) Wm = 1.51 x 105/A - 8.3 x 104/A2 - 2.4 x 107/A3 Wm = 1.07 x 105/A + 5.4 x 105/A2 - 3.6 x 107/A3
50
P (erg/cm 2 )
40 30 20 10 0 40
Ptotal Phg
60
80
100
A ( 2 )
Figure 2.3
We now have semiempirical expressions that can be used to calculate the free energies involved in the formation of dodecyl sulfate and cetyltrimethylammonium
Page 2.7
micelles. Table II compares experimental results to results of Tanford's semiempirical calculations based on equations 2.12 and 2.13. If head group interactions are calculated theoretically (Debye-Huckel theory), agreement with experimental results is not as good.
TABLE 2.2 m cmc (M) _________________________________________________________ Experimental 59 0.0066 N+(CH3)3 OSO395 0.0015 Semiempirical (Monolayer data) N+(CH3)3 60 0.0062 OSO3 93 0.0018 Theoretical (Debye-Huckel) 39 0.0042 OSO3_________________________________________________________ The hydrophobic effect and the head group interaction have both been cast as functions of the micelle surface area per molecule (equations 2.15, 2.19 and 2.20). It is instructive to plot these energies as functions of the surface area (Figure 2.4). This shows that there is a molecular surface area which minimizes the total free energy. The surface area giving this minimum free energy determines the aggregation number of the micelle. A larger surface area per molecule implies smaller micelles. A smaller surface area per molecule implies larger micelles with the bilayer structure being the limiting case (infinitely large micelle). The minimum free energy itself is RT ln cmc (equation 2.12). This plot illustrates effects of changes in the energies. For example, decreasing the work function by increasing the ionic strength will lower the cmc and increase the aggregation number (Figure 2.5).
Page 2.8
4 2 Wm
(kcal/mol)
0 -2 -4 RT ln cmc -6 -8 50 larger micelles Um - w 75 A min 2 smaller micelles 100 U mic,m - w
A ( )
Figure 2.4
(kcal/mol)
W m (low ionic strength) 0 Wm (high ionic strength) U mic,m - w
U m- w -10 50 100
A ( 2 )
Figure 2.5
Page 2.9
Lauryl Sulfate (Dodecyl sulfate)

-
O 3S O CH2
(CH2 )10
CH 3
Cetyltrimethylammonium (CTAB) CH 3 H3C N+ CH 3 CH 2 (CH2 )14 CH 3
Cholic Acid: X = OH Deoxycholic Acid: X = H CH 3 OH CH 3

-
OOC H CH 3
H
7
H OH H
X Fig 2.6. Some ionic detergents
Page 2.10
Triton X-100 (Octylphenoxypolyoxyethanol)
HO
(CH2 -CH2 -O)10
(CH2 )7
CH3
Lubrol W HO (CH2 -CH2 -O)7 (CH2 )15 CH3
Fig. 2.7. Some nonionic detergents
References Stillinger, F.H. (1980) Water revisited, Science 209, 451-457. C. Tanford (1974) Theory of micelle formation in aqueous solutions, J. Phys. Chem. 78, 2469-2479. C. Tanford (1974) Thermodynamics of micelle formation: Prediction of micelle size and size distribution, Proc. Natl. Acad. Sci. USA 71, 1811-1815. C. Tanford (1977) The hydrophobic effect and the organization of living matter, Science 200, 1012-1018. C. Tanford (1979) Interfacial free energy and the hydrophobic effect, Proc. Natl. Acad. Sci. USA 76, 4175-4176. C. Tanford (1980) The Hydrophobic Effect, Second Edition, John Wiley & Sons, New York.
Page 2.11

CHAPTER 3: THE FLUID MOSAIC MEMBRANE
D. Njus, 2000
CHAPTER 3: THE FLUID MOSAIC MEMBRANE
Section 3.1. Characteristics of Lipid Bilayers Naturally occurring phospholipids have a very low critical micelle concentration. For example, the cmc for dipalmitoyl phosphatidylcholine is 4.7 x 10-10 M. Therefore, phospholipids will virtually always form into a bilayer structure. The lipid bilayer has a thickness of approximately 40 , determined principally by the chain length of the fatty acids in the phospholipids. Each phospholipid molecule occupies a surface area of about 70 2. As in a micelle, the surface area occupied by each phospholipid molecule in the lipid bilayer is determined by the balance between head-group interactions and the hydrophobic effect. If the head groups favor greater separation than the hydrocarbon chains will permit, then the hydrocarbon chains may tilt so that they are not aligned perpendicular to the surface of the membrane. This decreases the thickness of the bilayer and increases the cross-sectional area occupied by each fatty-acid chain. If the head-groups tend to pack more tightly than the hydrocarbon chains, the fatty acid chains will be aligned perpendicular to the plane of the membrane and there may also be a force on the bilayer favoring formation a concave bend. The hydrocarbon chains in diacyl phospholipids undergo a phase transition from an ordered (crystalline) to a disordered (fluid) state. Some phase transition temperatures are given in Table 3.1. Increasing unsaturation and decreasing fatty acid chain length lower the phase transition temperature. Cholesterol generally causes the phase transition to
Table 3.1. Phase transition temperatures for the transition from ordered to disordered hydrocarbon chains in diacyl phospholipids in hydrated multilayers Phospholipid Transition temperature (C) ______________________________________________________________________ 75 diC22 phosphatidyl choline 54.9 diC18 phosphatidyl choline diC16 phosphatidyl choline 41.4 23.9 diC14 phosphatidyl choline -22 diC18:1 phosphatidyl choline diC16 phosphatidyl serine (low pH) 72 55 diC16 phosphatidyl serine (high pH) diC16 phosphatidyl ethanolamine 60 49.5 diC14 phosphatidyl ethanolamine ______________________________________________________________________ occur over a broader range of temperatures. As described by the fluid mosaic model, the lipids in a biological membrane are typically in a fluid state at physiological temperatures. In the ordered state, the fatty acid chains occupy less volume than in the fluid state. In the case of phosphatidylcholine, this means that the hydrocarbon chains must tilt to achieve adequate separation of the head groups.
Page 3.1
Bilayers composed of mixed lipids may exhibit phase separation. If the chemical potentials of the individual lipids are higher in the mixed phase than in homogeneous phases, then the homogeneous phases will separate out. This can be detected as a spatial separation of different lipids or as domains having different characteristics (e.g., fluidity). A striking example of this is the ripple phase exhibited by phosphatidylcholine bilayers. At low temperatures, the bilayer forms a homogeneous ordered phase and, at high temperatures, it forms a single fluid phase. At intermediate temperatures, however, the bilayer exhibits a periodic pattern showing an undulating pattern or ripples on its surface. The molecular basis for this is not yet clear, but some inferences can be made. In the intermediate temperature range, phospholipids with tilted chains coexist with phospholipids with extended chains. The differences in chain tilting and bilayer thickness discourage intermixing of phospholipids in different phases and causes the two groups to segregate (Marden et al., 1984). Lipids in natural membranes are characteristically distributed asymmetrically in the two halves of the bilayer. For example, human red cells are mostly PC on the outside and mostly PE on the inside (Table 3.2). This asymmetry can be maintained because phospholipids are very slow to redistribute (flip-flop) between the two sides of a bilayer. The original cause of the asymmetry may lie in the biosynthetic history of the membrane, but there also appear to be ATPases that invest cellular energy in the transport of phospholipid head groups across a variety of natural membranes (Devaux, 1992).
Table 3.2. Distribution of lipids in natural membranes Outer Monolayer Inner Monoayer Membrane SM PC PE PS SM PC PE PS ___________________________________________________________________ Human RBC 40 42 10 0 8 13 45 25 Rat RBC 42 35 15 0 6 20 40 25 Bovine ROS 10 40 40 - >80 10 10 ___________________________________________________________________ Values are percentages of total lipid in that layer of the membrane. RBC = red blood cell; ROS = rod outer segment; SM = sphingomyelin.
Section 3.2. Model Lipid Membranes Because phospholipids naturally form bilayer structures (at least at low lipid:water ratios), artificial membranes can be produced in a number of ways. Phospholipid vesicles are easily made by sonicating lipids (Huang, 1969), by reverse phase vaporization (Deamer and Bangham, 1976), or by dialyzing away detergent (Milsmann et al., 1978). Sonication is convenient but produces rather small unilamellar vesicles so the membranes have a high radius of curvature. Reverse phase vaporization produces larger vesicles but the solvent in which the lipids are initially dissolved contaminates the preparation and may alter permeability and other properties of the bilayer. Sonication and reverse phase vaporization are both rather harsh treatments for proteins, so reconstitution of membrane proteins is generally accomplished by some variation of the dialysis method.
Page 3.2
Measuring the electrical properties of membranes requires a planar membrane separating two aqueous spaces large enough to accommodate electrodes. Planar bilayers can be made by spreading lipid in solvent over an aperture (Mueller et al., 1963) or by raising two monolayers past the aperture (Montal and Mueller, 1972). This produces artificial membranes with much less total surface area than a liposome suspension, but it does permit electrical recording of artificial membrane properties. In the solvent/aperture method, the solvent in which the lipid is dissolved moves to the rim of the aperture and the bilayer across the opening thins out to form a "black lipid membrane." Nevertheless, the solvent can form lenses in the artificial membrane and there is always some question about the influence of remaining solvent on the properties of the membrane. The monolayer method corrects this. Even using great care, planar membranes formed by either method are relatively unstable and their short lifetime is an experimental handicap. To study membrane proteins, liposomes formed by dialysis have proven most convenient. The proteins can be solubilized in the detergent and then conveniently reconstituted. Monitoring effects of these proteins on electrical properties of the membrane is not possible, however, because liposomes are too small to insert electrodes. To overcome this problem a number of new approaches have been tried. First, liposomes containing the reconstituted protein may be fused into a black lipid membrane (Miller and Racker, 1976). Patch clamping technology has introduced a new approach. A patch pipette can be raised through a lipid monolayer on the surface of a solution and then lowered back down. A planar bilayer forms across the opening of the patch pipette and this bilayer will contain proteins dispersed in the lipid monolayer (Tank et al., 1982; SuarezIsla et al., 1983).
Page 3.3
Section 3.3. Lipid Components Fatty acids: Fatty acids have two characteristics that affect the physical properties of the membrane: chain length and degree of unsaturation. Fatty acids may be identified by a common name, by the standard nomenclature, or by the w nomenclature. According to the standard nomenclature, fatty acids are represented as x:y, z1,z2 ... zn where x represents the number of carbon atoms, y represents the number of double bonds, and z1,z2 ... zn represent the carbon atoms preceding double bonds counting from the carboxy end. According to the w nomenclature, fatty acids are represented as x:yz' where x represents the number of carbons, y represents the number of double bonds, and z' represents the position of the first double bond counting from the carbon (methyl terminus). For example, the structure of palmitoleic acid (16:1, 9-cis or 17) is: CH3-(CH2)4-CH2CH=CH(CH2)7COOH TABLE 3.3. Some Common Fatty acids Saturated Unsaturated No. of Common Common Nomenclature Carbons Name Name Standard ___________________________________________________________________ 10 Capric Palmitoleic 16:1, 9-cis 17 12 Lauric Oleic 18:1, 9-trans 19 14 Myristic Vaccenic 18:1, 11-cis 17 16 Palmitic Linoleic 18:2, 9-cis,12-cis 26 18 Stearic -Linolenic 18:3, 9-cis,12-cis,15-cis33 20 Arachidic -Linolenic 18:3, 6-cis,9-cis,12-cis36 22 Behenic Arachidonic 20:4, 5,8,11,14 (all cis) 46 24 Lignoceric ___________________________________________________________________
Phospholipids: Phospholipids have the general structure shown below: OHeadgroup-O-P-O- CH2 O O CH-O-C-CH2...CH3 CH2-O-C-CH2...CH3 O
Page 3.4
The head groups - which differ in charge, polarity and reactivity - give the phospholipids different characteristics: Phosphat idylcholine (PC or lecit hin) CH3 CH3 N+ CH3 CH2 CH2 O OP O O
Phosphat idylet hanolamine (PE) NH3 + CH2 CH2
OP O O
Phosphat idylser ine (PS) NH3 + CH COOPhosphat idylinosit ol (PI) HOHC CHOH HOCH HOHC CH CHOH O
OCH2 O P O O
OP O O
Phosphat idic A cid (PA ) OHO P O O
Page 3.5
Cholesterol: Cholesterol is present in plasma membranes, lysosomes and storage granules. The cholesterol content of the Golgi complex increases on moving from the cis to the trans cisternae. Cholesterol is not present in bacterial, inner mitochondrial or chloroplast thylakoid membranes. CH3
21 12 18 11 1 2 19 13 20 22 24 23 16 26 9 14 8 7 5 4 6 10 15 25
CH3
17
CH3
CH3
27
CH3
A HO
3
Cholesterol Sphingomyelin: The sphingomyelin content is high in lysosomes and storage granules: rat liver lysosomes, 24%; bovine chromaffin granules,15%; serotonin granules from pig platelets, 24.9%; bovine pituitary neurosecretory vesicles, 21.7%.
Section 3.4. Structure of Membrane Proteins Because they are typically hydrophobic, membrane proteins have been notoriously difficult to study using traditional protein chemistry techniques. The advent of molecular biology has made it far easier to clone and sequence a membrane proteins gene than to study the protein itself. The usefulness of this approach depends to a large extent on how much we can deduce about the structure of the protein from its amino acid sequence. Three-dimensional structures of membrane proteins are difficult to determine, but those that have been established seem to follow one of two patterns: the helix bundle which includes most integral membrane proteins and the beta barrel which includes some proteins in the outer membrane of gram negative bacteria and the outer mitochondrial membrane (von Heijne, 1994). The helix bundle type follow the pattern set by bacteriorhodopsin; the membrane-spanning segments are both hydrophobic and alpha-helical. These membranespanning segments are separated by hydrophilic domains that are exposed to the aqueous environments at either membrane surface. The -helical nature of the membrane spanning regions can be rationalized. Hydrophobic side chains will tend not to interact with each other or with lipid components of the membrane. That means that the structure of a hydrophobic domain will be determined primarily by the backbone hydrogen bonding pattern. Accordingly, the a-helix is the expected pattern. The -helix consists of 3.61 amino acids per turn spanning a distance of 1.5 per residue. Given a membrane thickness of 30-40 , a transmembrane a-helix should contain 20-27 amino acid residues.
Page 3.6
To predict which segments in a protein are membrane-spanning regions, it has become common to use a hydropathy index. The most widely used is that described by Kyte and Doolittle (1982). A variety of thermodynamic parameters could be used to assign a hydropathy value to each amino acid. The validity of any particular choice may be debated, but the only important consideration is that some consensus index of hydropathy is established for each amino acid side chain. Kyte and Doolittle based their hydrophathy index on the water-vapor transfer free energies of the side chains and on the interiorexterior distribution of amino acid side chains. The hydropathy value of a span of amino acids is then determined by summing the hydropathy indices for each amino acid in that span. It is convenient to choose an odd number for the span length so the hydropathy index can be associated with the amino acid in the middle of the span. For example, if a span of 7 is chosen, the hydropathy value at amino acid 40 is the sum of the hydropathy indices of amino acids 37-43.
Table 3.4. Kyte-Doolittle Hydropathy Scale Amino Acid Single Kyte-Doolittle Residue Letter Code Hydropathy Index ________________________________________________________________ Isoleucine I 4.5 Valine V 4.2 Leucine L 3.8 Phenylalanine F 2.8 Cysteine/cystine C 2.5 Methionine M 1.9 Alanine A 1.8 Glycine G -0.4 Threonine T -0.7 Tryptophan W -0.9 Serine S -0.8 Tyrosine Y -1.3 Proline P -1.6 Histidine H -3.2 Glutamic acid E -3.5 Glutamine Q -3.5 Aspartic acid D -3.5 Asparagine N -3.5 Lysine K -3.9 Arginine R -4.5 ________________________________________________________________
Section 3.5. Solubilization and Reconstitution of Membrane Proteins Integral membrane proteins, by nature, have hydrophobic surfaces that allow them to penetrate into the hydrophobic center of lipid bilayers. To get these proteins out of a membrane, therefore, these hydrophobic surfaces must be protected by detergent molecules Page 3.7
or the protein will denature. The strategy used to solubilize membrane proteins is to add detergent to the membrane. The detergent intercalates into the lipid bilayer forming a mixed micelle with the phospholipids. When enough detergent has entered the membrane, the structure changes from the bilayer favored by phospholipids to the oblate or spherical micelles favored by the detergent. As this happens, the bilayer structure breaks down and the proteins escape into soluble particles consisting of the protein along with detergent and residual phospholipid. In principle, any detergent can be used to break down the lipid bilayer and solubilize membrane proteins. In practice, however, strong detergents may also enter into the protein itself and denature it. Dodecyl sulfate is a good example. It is useful in gel electrophoresis because it disrupts the secondary and tertiary structure of a protein causing it to migrate on the gel according to size alone. Dodecyl sulfate definitely solubilizes membrane proteins, but it also denatures them destroying their structure and activity. Many membrane proteins (e.g., channels, transporters) have no assayable function after they are solubilized and removed from the membrane. In order to study their functions, they must be reconstituted into some sort of a membrane structure. Reconstitution is typically a matter of simply reversing the solubilization process. Phospholipids are reintroduced, detergent is removed, and the particle in which the protein is situated changes from a micelle back into a bilayer. The objective here is to remove the detergent but not the phospholipid. This can be accomplished by dialysis or gel filtration. Because phospholipids have an extremely low critical micelle concentration, the rate at which free phospholipids are removed is extremely slow. Obviously, the same will be true for a detergent with a low cmc, making such a detergent (Triton X-100 is an example) difficult to use in reconstitution. A detergent with a higher cmc can be removed relatively rapidly, however, permitting reconstitution to occur successfully. The bile acids, cholic acid and deoxycholic acid, have been particularly useful in solubilization and reconstitution of membrane proteins. They have a high cmc, making it easy to remove them by dialysis. Because they are ionic, their effectiveness as detergents depends on the ionic strength, and the salt concentration can be manipulated to shift the balance between solubilization and reconstitution. The bile acids also have a structure that is particularly suited to solubilizing membrane proteins without denaturing them. These molecules have a generally planar structure with a polar side and a nonpolar side. The nonpolar side protects the hydrophobic protein surface while the other side faces the water. At the same time, this asymmetrical polarity does not favor penetration by the detergent into the hydrophobic core of the protein.
References D. Deamer and A.D. Bangham (1976) Large volume liposomes by an ether vaporization method, Biochim. Biophys. Acta 443, 629-634. P.F. Devaux (1992) Protein involvement in transmembrane lipid asymmetry, Ann. Rev. Biophys. Biomol. Struct. 21, 417-439. C.H. Huang (1969) Studies on phosphatidylcholine vesicles. Formation and physical characteristics, Biochemistry 8, 344-352. J. Kyte and R.F. Doolittle (1982) A simple method for displaying the hydropathic character of a protein, J. Mol. Biol. 157, 105-132. Page 3.8
M. Marder, H.L. Frisch, J.S. Langer, and H.M. McConnell (1984) Theory of the intermediate rippled phase of phospholipid bilayers, Proc. Natl. Acad. Sci. USA 81, 6559-6561. C. Miller and E. Racker (1976) Ca2+-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers, J. Memb. Biol. 30, 283-300. M.H.W. Milsmann, R.A. Schwendener and H.G. Weder (1978) The preparation of large single bilayer liposomes by a fast and controlled dialysis, Biochim. Biophys. Acta 512, 147-155. M. Montal and P. Mueller (1972) Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties, Proc. Natl. Acad. Sci. USA 69, 3561-3566. P. Mueller, D.O. Rudin, H.T. Tien, and W.C. Wescott (1963) Methods for the formation of single bimolecular lipid membranes in aqueous solution, J. Phys. Chem 67, 534535. J.R. Silvius (1992) Solubilization and functional reconstitution of biomembrane components, Ann. Rev. Biophys. Biomol. Struct. 21, 323-348. B.A. Suarez-Isla, K. Wan, J. Lindstrom, and M. Montal (1983) Single-channel recordings from purified acetylcholine receptors reconstituted in bilayers formed at the tip of patch pipets, Biochemistry 22, 2319-2323. D.W. Tank, C. Miller, and W. Webb (1982) Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax, Proc. Natl. Acad. Sci. USA 79, 7749-7753. G. von Heijne (1994) Membrane proteins: From sequence to structure, Ann. Rev. Biophys. Biomol. Struct. 23, 167-192.
Page 3.9

CHAPTER 4: MEMBRANE ELECTROSTATICS
D. Njus, 2000
CHAPTER 4: MEMBRANE ELECTROSTATICS
Section 4.1. Electrostatics in One Dimension To understand the electrostatics of biological membranes, one must understand the concepts of charge, electric field and electrical potential. This section, therefore, reviews some basic concepts of electrostatic theory. The units to be used in this discussion require some attention because the choice of units determines the nature of the constants appearing in the equations. Physics textbooks commonly employ cgs units (statcoulombs, ergs, etc.). Most of the practical measurements made in biological research, however, are in rationalized MKS units (coulombs, volts, etc.). For that reason, rationalized MKS units will be used throughout this discussion. Electrostatic forces are so strong that positive and negative charges are generally paired and overall electroneutrality is strictly observed in nature. If a positive and negative charge are separated, there will be an attractive force between them. According to Coulomb's Law, this force will be proportional to the absolute magnitudes of the two charges (q1 and q2) and to the inverse square of the distance between them (x): (4.1) F = (q1q2)/(4ox2)
o, the permittivity of a vacuum, is a constant equal to 8.854 x 10-12 coul/m.volt. Increasing the distance between the two charges will require us to do work, which (following the usual physical definition) is (4.2) W = - F dx
If a charge is located in the vicinity of many other charges, the force on it will be the sum of the forces exerted by the other charges. In such a case, it is often more convenient to define the electric field at a point x as the force exerted by all of the charges in the neighborhood on a unit charge at that point. Thus, the electric field created by a point charge q at a distance x is (4.3) E(x) = q/4ox2
The electrical potential (x) is the work that must be done to move a unit charge from a reference point to a point x. (4.4) (x) = -
x ref
E dx
Because we are interested in the electrical properties of biological membranes, we can take advantage of their planar symmetry. All points the same distance from the surface of the membrane will have the same electric field and the same electrical potential, so the dimension perpendicular to the membrane surface is the only significant one. We will consider charge not to be distributed as points in space but to be spread smoothly on planes parallel to the membrane. If positive charge is smoothly distributed on a plane of charge,
Page 4.1
as in Figure 4.1A, it will create an electric field perpendicular to the plane. The electric field will be proportional to the charge density on the plane (qs): (4.5) E = qs/2o
If we have two parallel planes of charge, one with a positive surface charge density (+qs) and one with an equal density of negative charge (-qs), their electric fields will be additive in the space between them and will cancel in the spaces on either side (Figure 4.1B). Between the planes, therefore, the total electric field will be qs/o; outside of that space, the total electric field will be zero. This arrangement is the common textbook example, the parallel plate capacitor. Because there is an electric field between the two plates of the parallel plate capacitor, there will be a difference in electrical potential (4.6) = - 0 E dx = - 0 (qs/o) dx = - qsx/o
x x
This is the potential energy that must be expended to move a unit charge from plate 1 (at 0) to plate 2 (at x).
A +q s B +q s -q s
E =
-q s 2
E =
+q s 2
E =0
E =
qs
E =0
0 Single plane of charge Parallel plate capacitor
Figure 4.1. Electric fields associated with planes of charge
The preceding equations apply to charges arranged in a vacuum. In the real world, charges will be separated by a medium composed of polarizable molecules. These molecules will tend to align with the electric field and cancel it. The electric field will be diminished in proportion to the polarizability of the medium, expressed as the dielectric constant . Therefore, if the plates of a parallel plate capacitor are separated by a medium of dielectric constant , the electric field between the plates will be (4.7) E = qs/o
Page 4.2
Of course, the electrical potential difference between the plates will be correspondingly smaller as well: (4.8) = - qsx/o
Water, which has a dielectric constant of 80, is very polarizable. For that reason, charge separation across water will tend to create a relatively small electric field and small electrical potential differences. By contrast, hydrocarbons such as hexane ( = 1.89) have a very low dielectric constant. For that reason, charge separation across organic phases (such as the interior of biological membranes) will create large electric fields. It is this low dielectric constant that makes it possible to create significant electrical potential differences across biological membranes.
Section 4.2. Membrane Potential Separation of electrical charges by biological membranes creates an electrical potential difference across the membrane. This total difference in electrical potential, commonly called the membrane potential, plays a crucial role in many membrane functions. It is the force that drives ions across the membrane. Because it defines the electrical energy lost when an ion crosses the membrane, the membrane potential partly determines the energy stored in ion concentration gradients. Finally, the electric field associated with the membrane potential acts on dipolar groups in membrane proteins and may regulate the activities of these proteins. Voltage-dependent channels, for example, open and close in response to changes in the membrane potential. It is conceptually helpful to divide the charge separation created by biological membranes into three components. First, charges may be separated by moving ions all the way across the membrane from the aqueous medium on one side to the aqueous medium on the other. This separation of capacitative charge is probably the most important component of the membrane potential. Second, fixed charge bound to the membrane surface will be neutralized by counterions present in the adjacent aqueous solution. Because these counterions will tend to diffuse away from the membrane surface, there will be a consequent charge separation. This leads to the surface potential. Finally, because the ester linkages between fatty acids and the glycerol backbones of the membrane lipids are dipolar in character, alignment of these dipoles creates a charge separation which gives rise to the dipole potential.
Page 4.3
Outside
Membrane
Inside
dipole potential (outside) surface potential (outside) dipole potential (inside)
potential created by capacitative charge
membrane potential surface potential (inside)
Figure 4.2. Components of the membrane potential
When charge is moved from one side of a biological membrane to the other, we assume that the net charge on one side is equal and opposite to the net charge on the other. This is strictly required to maintain overall electroneutrality. These equal and opposite charges separated by the low dielectric medium of the lipid membrane form an electrical arrangement like the parallel plate capacitor. We will call the charge transferred across the membrane the capacitative charge qc. The separation of this capacitative charge will create an electric field in the membrane (4.9) E = qc/Ao
and an electrical potential difference across the membrane (4.10) = qcx/Ao The capacitance C is the ratio of the capacitative charge to the potential difference: (4.11) C = qc/ = Ao/x
The capacitance, therefore, is proportional to the area of the membrane and inversely proportional to the thickness. For a biological membrane, the capacitance is typically
Page 4.4
about 1 F/cm2. This corresponds to a membrane 35 thick with a dielectric constant of 4. The total membrane potential will include contributions from the capacitative charge, from the surface charge on each side of the membrane, and from the surface dipole on each side of the membrane (Figure 4.2). Because the contributions of surface charge and surface dipole on one side of the membrane will tend to cancel the contributions of those components on the other side, the capacitative charge is generally the primary determinant of the total membrane potential. Section 4.3. Surface Potential When fixed charges are bound to a surface, and the counter ions are dissolved in the adjacent solution, the counter ions will tend to move away from the surface because of diffusion. This creates a charge separation and consequently an electrical potential difference called the surface potential. Whereas the relationship between the capacitative charge and the membrane potential is a simple proportionality (the capacitance), the relationship between the surface charge and the surface potential is quite complex. The magnitude of the surface potential depends on the amount of fixed surface charge and also on the separation between the fixed charge and the diffusable charge. The charge separation depends on a dynamic tension with diffusion pushing the counterions away from the surface and electrical attraction pulling them toward the surface. The theoretical analysis of surface potentials and surface charge was originally developed by Gouy and Chapman. Just as the DeBye-Hckel theory describes ion distributions as a function of distance from a fixed point charge, the Gouy-Chapman theory describes ion distributions as a function of distance from the membrane surface. The analysis rests on three basic principles: the Boltzmann distribution, the Poisson equation, and electroneutrality. qs q v (x) ( ) = 0 (x) o
0 Figure 4.3. The electrical potential as a function of distance from the surface.
Page 4.5
First, we imagine a plane of fixed charge with a surface charge density qs. Adjacent to it is a medium with a space charge density qv(x) where x is the distance from the surface (Figure 4.3). Overall electroneutrality requires that the total space charge be equal and opposite to the sum of the fixed charge qs and the capacitative charge qc where the capacitative charge is the net charge on the other side of the membrane. As we shall see, the capacitative charge is usually small compared to the surface charge. For simplicity, we shall simply include it as part of qs: (4.12) - 0 qv(x) dx = qs
The space charge at any point x is obtained by summing over all of the ionic species present in the solution: (4.13) qv(x) = F i ziCi(x)
For each ion species i, zi is the valence and Ci is the concentration in moles/liter. F is the Faraday constant and is the constant 103 l/m3. Since the ion concentrations should depend on the electrical potential according to the Boltzmann distribution: (4.14) Ci(x) = Cio exp(-ziF (x)/RT)
where Cio is the concentration of ion species i far from the membrane (x=). The Boltzmann distribution (eq. 4.14) together with equation 4.13 relates qv(x) to (x). (4.15) qv(x) = F i ziCio exp(-ziF (x)/RT)
Another relationship between qv(x) and (x) is provided by Poisson's equation: (4.16) qv(x)/o = -d2/dx2
Poisson's equation can be rationalized by considering the single plane of charge (Figure 4.1A). If the plane is assumed to have a thickness dx, the change in the electric field dE/dx upon crossing the plane is qs/o. If equation 4.4 is differentiated twice, it is apparent that (4.17) d2/dx2 = -dE/dx = -qs/o
Poisson's equation (eq. 4.16) can be integrated in two ways. First, using eq. 4.12, (4.18) qs = - 0 qv(x) dx = o 0 (d2/dx2) dx

We define () = 0, so (4.19) qs = o (do/dx)
Page 4.6
Second, using eq. 4.15 and 4.16, (4.20) d2/dx2 = - (F/o) i ziCio exp(-ziF/RT)
To integrate equation 4.20, we multiply both sides by 2d/dx. We can then integrate this equation to obtain (4.21) (d/dx)2 = (2RT/o) i Cio exp(-ziF/RT) + Constant
At x = , = 0 and d/dx = 0, so Constant = -(2RT/o) i Cio and equation 4.21 becomes (4.22) (d/dx)2 = (2RT/o) i Cio [exp(-ziF/RT) - 1]
Combining this with equation 4.19 gives (4.23) qs2 = 2RTo (i Cio [exp(-ziFo/RT) - 1])
Equation 4.23 is the general form of the Gouy-Chapman equation relating the surface charge to the surface potential. A more convenient form is obtained by assuming that all of the ions in the aqueous phase are univalent. We will let Cio = Co for both anions and cations. Then equation 4.23 simplifies to (4.24) qs = (8RTCoo)1/2 [sinh (Fo/2RT)]
It is apparent that the surface potential depends on the magnitude of the surface charge (Figure 4.4) and also on the ionic strength of the aqueous medium (Figure 4.5). Clearly, the counterions necessary to neutralize the fixed charge will have a much greater effect on the ion concentration near the membrane surface if the ion concentration is low. At low ionic strength, therefore, the concentration gradient will be greater and diffusion forces will be stronger. This will drive the counterions farther from the surface leading to a greater charge separation and a larger potential difference. Another way of visualizing this is to simplify equation 4.24 by expanding the sinh as a power series and dropping higher order terms (assume o is small). Equation 4.24 then reduces to (4.25) qs = (8RTCoo)1/2(Fo/2RT) This may be rewritten as (4.26) qs/oo = (2CoF2/RTo)1/2
The left-hand side of this equation is equal to the inverse of the distance x between plates of a parallel plate capacitor (equation 4.8). A constant with units of m-1 is customarily defined as
Page 4.7
(4.27)
2 = (F2/RTo) i Cio zi2
If all of the ions in the aqueous phase are univalent, then is equal to the right-hand side of equation 4.26. Therefore, (4.28) 1/x =
One may think of 1/ as a measure of the thickness of the diffuse double layer. That is, the same surface potential o would result if all of the diffusable charge were placed at a distance 1/ from the surface of the membrane. In the foregoing analysis, we have incorporated the capacitative charge into the surface charge. In actual practice, the capacitative charge is usually negligible compared to the fixed surface charge and contributes little to the surface potential. To illustrate this, we may note that the capacitative charge will rarely be greater than 10-7 coul/cm2 since that charge will create a 100 mV membrane potential given the usual membrane capacitance of 1 F/cm2. Typical surface charge densities are much larger than this (chromaffin granules have a surface charge density of -1.38 x 10-6 coul/cm2).
Figure 4.4. The surface potential as a function of the surface charge density, according to the Gouy-Chapman equation. The curve is for a univalent electrolyte at 10 mM concentration. The dielectric constant has been taken as 80 and the temperature as 20C.
Page 4.8
Figure 4.5. The decay of potential from a surface. The surface charge density has been assumed to be 0.0158 coul/m2. The electrolyte is univalent and the dielectric constant and temperature have been taken as 80 and 20C respectively. Surface charge has a number of interesting effects. Because a negative surface charge attracts cations to the membrane surface, it increases the conductance of the membrane to cations (McLaughlin et al., 1970) and enhances the binding of cations to the membrane surface (McLaughlin and Harary, l976). It should be noted that the surface potential has a greater affect on the distributions of divalent ions than on distributions of monovalent ions. For this reason, high Ca2+ concentrations at the membrane surface, sometimes attributed to binding, may actually be caused by the surface potential. The surface potential also changes the pH near the surface of the membrane; this may shift the apparent pK values of protonatable groups and the apparent pH optima of membranebound enzymes. Finally, the surface potential may act to repel or attract other surfaces thereby functioning in phenomena such as exocytosis and intercellular communication.
Section 4.4. Dipole Potential A third kind of charge separation that can be created by biological membranes is that of the surface dipole. Phospholipids are dipolar in character. These dipoles, all oriented in the same direction, lead to a charge separation, which creates the dipole potential. The dipole potential may not be a significant component of the membrane potential because the dipoles on opposite surfaces of the membrane are oriented in opposite Page 4.9
directions and tend to cancel each other. However, as we shall see, the dipole potential may be a major factor in determining the ionic permeability of the lipid bilayer. The dipolar character of phospholipids seems to arise from the ester linkage between the fatty acid groups and the glycerol backbone. The head groups of the phospholipids are certainly polar. However, they seem not to contribute appreciably to the dipole potential. This is partly attributable to the fact that the head groups lie on the aqueous surface of the membrane where the dielectric constant of the medium tends to neutralize the dipoles. Moreover, the head groups seem to lie flat on the surface of the membrane so the dipole moment in the direction perpendicular to the membrane surface is small. The dipole potential is (4.29) = D/o
where D is the surface dipole density in coul/m. To estimate the potential magnitude of surface dipole effects, let us assume that a typical phospholipid has a dipole moment of 1.5 Debyes (1.5 x 10-18 esu.cm or 5 x 10-30 coul.m) and occupies a membrane area of 60 2. This yields a surface dipole density of 8.34 x 10-12 coul/m and implies a dipole potential of (1000/) mV. If we assume that the dielectric constant of membrane in the region of the dipole layer membrane is between 4 and 10, the surface dipole should create a potential difference of 100-250 mV. The electric field set up by the capacitative charge may cause the dipoles to orient in a direction perpendicular to the membrane. Thus, the surface dipole may change in response to a membrane potential. In this way, the surface dipole will affect the electric field near the membrane surface and may be important in modulating the response of membranebound enzymes to the membrane potential.
References R.G. Ashcroft, H.G.L. Coster, and J.R. Smith (1981) The molecular organisation of bimolecular lipid membranes. The dielectric structure of the hydrophilic/hydrophobic interface, Biochim. Biophys. Acta 643, 191-204. R. Aveyard and D.A. Haydon (1973) An Introduction to the Principles of Surface Chemistry, Cambridge University Press, Cambridge. R.F. Flewelling and W.L. Hubbell (1986) Hydrophobic ion interactions within membranes, Biophys. J. 49, 531-540. R.F. Flewelling and W.L. Hubbell (1986) The membrane dipole potential in a total membrane potential model, Biophys. J. 49, 541-552. D.A. Haydon and S.B. Hladky (1972) Ion transport across thin lipid membranes: A critical discussion of mechanisms in selected systems, Quart. Rev. Biophys. 5, 187-282. S. McLaughlin (1977) Electrostatic potentials at membrane-solution interfaces, Current Topics in Membrane Transport 9, 71-144. S. McLaughlin and H. Harary (1976) The hydrophobic adsorption of charged molecules to bilayer membranes: A test of the applicability of the Stern equation, Biochemistry 15, 1941-1948.
Page 4.10
P.L. Yeagle (1979) Effect of transmembrane electrical potential and micelle geometry on phospholipid head group conformation, Arch. Biochem. Biophys. 198, 501-505.
Page 4.11

CHAPTER 5: SPECIFIC AND NON-SPECIFIC BINDING
D. Njus, 2000
CHAPTER 5: SPECIFIC AND NON-SPECIFIC BINDING
Section 5.1. The Langmuir Adsorption Isotherm Small molecules may bind to membranes either non-specifically or specifically. Non-specific binding occurs when small amphiphilic molecules adsorb to the surface of the lipid bilayer or to the equivalent hydrophobic/hydrophilic interfaces of membrane proteins. Specific binding occurs when ligands bind to selective binding sites of specific receptors. In either case, similar analyses can be used to describe the binding process. The simplest description of the relationship between bound and free ligand assumes an equilibrium defined by the law of mass action: (5.1) L + M = LM
where L is the free ligand, M signifies empty binding sites on the membrane, and LM represents ligand bound to the membrane. The equilibrium constant K is (5.2) K = [L][M]/[LM]
Let us define Lm as the concentration of bound ligand ([LM]) and Lmax as the total number of binding sites on the membrane ([M]+[LM]). Then the equilibrium constant becomes (5.3) K = [L](Lmax-Lm)/Lm
Equation 5.3 may be solved to yield an expression for the amount of bound ligand Lm (5.4) Lm = Lmax [L] / ([L] + K)
The amount of bound ligand Lm depends upon the binding constant K, the number of binding sites Lmax, and the concentration of ligand [L] at the surface of the membrane (Figure 5.1). As described in Section 5.2, if the ligand is charged, its concentration at the surface of the membrane [L] will not necessarily equal its concentration in the bulk of the aqueous phase but will be related to it by the surface potential. As described in Section 5.3, the binding constant K reflects the change in free energy occurring upon binding. Equation 5.3 may also be rearranged to yield the Scatchard equation: (5.5) Lm/[L] = Lmax/K - Lm/K
This form is particularly useful because a plot of Lm/[L] vs. Lm is linear and may be used to define the parameters Lmax and K (Figure 5.2).
Page 5.1
Lmax Lmax Lm Lm Lmax 2 [L] K
K [L] Lm
Lmax
Figure 5.1
Figure 5.2
Section 5.2. Effect of Surface Potential [L] is the concentration of ligand at the surface of the membrane. As described earlier, this is related to [L], the concentration far from the membrane, by the Boltzmann equation: (5.6) [L] = [L] exp (-ziFo/RT)
Obviously, the known ligand concentration in the bulk of the aqueous phase [L] may be used in place of the ligand concentration at the membrane surface [L] if the ligand is uncharged or if the surface potential o is negligible. Since the surface potential depends upon both the ionic strength of the solution and upon the surface charge density, it can be considered negligible if the ionic strength is high or if the surface charge density is low. If the surface potential is greater than a few millivolts, however, [L] will be significantly different from [L]. For example, if the surface potential is -18 mV and the ligand is a univalent anion, [L] will be only 50% of [L], and using [L] in place of [L] will significantly change the appearance of the binding curve (Figure 5.3) and the Scatchard plot (Figure 5.4). A special problem occurs when a charged ligand binds to the membrane to such an extent that the bound ligand itself affects the membrane surface charge. This is generally not a problem for ligands that bind to receptors since the receptor density (Lmax) is typically insignificant compared to the surface charge density of the membrane. Ligands that adsorb to the membrane, however, may have a significant effect on the surface charge density. This situation, first considered by Stern, has been analyzed more recently by McLaughlin and Harary (1976). If we assume that the surface charge density of the membrane is initially zero, then [L] = [L] for low values of Lm. As Lm increases, however, the surface potential will increase and binding will deviate in the direction expected in the presence of a surface potential. Thus, the Scatchard plot will appear to curve (Figure 5.4).
Page 5.2
Section 5.3. The Binding Constant The binding constant K reflects the standard free energy change upon binding. This can be seen by recognizing that the free energy change for the reaction in equation 1 is (5.7) G = G + RT ln ([LM]/[L][M])
At equilibrium, G = 0 and [LM]/([L][M]) = 1/K. Therefore, (5.8) G = RT ln K
For adsorption, the free energy change represents the free energy change for the transfer of the ligand from water into the membrane. For receptors, the free energy change carries additional significance. In this case, ligand binding not only changes the location of the ligand, but it changes the conformation of the receptor to which the ligand binds. Thus, the free energy of binding includes not just an affinity between the binding site and the protein but a change in the conformation of the protein. The conformational change is crucial, of course, because it activates the receptor thereby transmitting the ligand-binding signal.
Section 5.4. Specific vs. Non-Specific Binding Often, it is desirable to study the binding of a ligand to a receptor or other membrane-bound protein, but the ligand (because it is hydrophilic or amphiphilic) exhibits considerable non-specific binding to the membrane. This may be analyzed as follows: Total binding to the membrane (from eq. 5.4) will be the sum of the specific (sp) and nonspecific (ns) contributions: (5.9) Lm = Lmsp + Lmns = Lmaxsp [L]/([L] + Ksp) + Lmaxns [L]/([L] + Kns)
Let us assume that the affinity of the non-specific binding sites is very weak compared to the affinity of the specific sites (Kns >> Ksp). To measure specific binding, one uses ligand
Page 5.3
concentrations that range about Ksp, so [L] will be very small relative to the non-specific binding constant [L] << Kns). Equation 5.9 then reduces to (5.10) Lm = Lmaxsp [L]/([L] + Ksp) + Lmaxns [L]/Kns so non-specific binding will be directly proportional to the ligand concentration over this range. This non-specific binding can be measured by adding a high concentration of ligand (Kns >> [L] >> Ksp). Under these conditions, equation 5.9 reduces to (5.11) Lm = Lmaxsp + Lmaxns [L] / Kns Because Lmaxsp << Lmaxns, the measured binding Lm will all be non-specific. This gives the slope of the non-specific binding line (Lmaxns / Kns). Then specific binding can be calculated by subtracting non-specific binding from total binding as in Figure 5.5.
References S. McLaughlin and H. Harary (1976) The hydrophobic adsorption of charged molecules to bilayer membranes: A test of the applicability of the Stern equation, Biochemistry 15, 1941-1948. T.M. DeLorey and R.W. Olsen (1992) -Aminobutyric acidA receptor structure and function, J. Biol. Chem. 267, 16747-16750. C.D. Strader, T.M. Fong, M.R. Tota, D. Underwood, and R.A. F. Dixon (1994) Structure and function of G-protein-coupled receptors, Ann. Rev. Biochem. 63, 101-132. M. Hollmann and S. Heinemann (1994) Cloned glutamate receptors, Ann. Rev. Neurosci. 17,
Page 5.4

CHAPTER 6: PERMEABILITY AND CONDUCTANCE
D. Njus, 2000
CHAPTER 6: PERMEABILITY AND CONDUCTANCE
Section 6.1. Formal Analysis of Permeability and Conductance The passage of ions and molecules across membranes is a phenomenon often divided into two parts: permeability and conductance. Conductance describes the movement of electrolytes (ions and charged molecules) across the membrane in response to a membrane potential. Permeability describes the movement of uncharged molecules (nonelectrolytes) as well as the movement of electrolytes in the absence of a membrane potential. Three different approaches may be used to analyze permeability and conductance: classical thermodynamics, nonequilibrium thermodynamics and statistical mechanics. Strictly speaking, classical thermodynamics and statistical mechanics apply only to equilibrium situations. Nonequilibrium thermodynamics, by contrast, was developed to describe non-equilibrium phenomena such as permeability and conductance. Many purists, therefore, prefer to discuss permeability and conductance in terms of nonequilibrium thermodynamics. Unfortunately, this approach generally provides little insight into the physical events occurring as molecules or ions cross biological membranes. For that reason, we will focus here on the other two approaches: We will see that they both yield the same results and provide different insights into the processes involved. The analysis of permeability and conductance using classical thermodynamics is based on the concept of the electrochemical potential: (6.1) = + RT ln C + zF
At equilibrium, a given ion or molecule must have the same electrochemical potential on both sides of the membrane. If we use the subscript i to denote one side of the membrane (inside) and the subscript o to denote the other side (outside), then (6.2) i + RT ln Ci + zF i = o + RT ln Co + zF o
Since i = o, equation 6.2 can be rearranged to yield (6.3) Ci/Co = exp [-zF(i - o)/RT]
This is the famous Nernst equation, which gives the equilibrium concentration gradient of an electrolyte in the presence of a membrane potential. If the molecule is a non-electrolyte (z=0) or if the membrane potential is zero, then equation 6.3 simplifies to (6.4) Ci = C o
The analysis of permeability and conductance using statistical mechanics is based on two concepts: the concept of unidirectional fluxes and transition state theory. The notion that net flux across the membrane is simply the sum of two individual unidirectional fluxes was first introduced by Ussing. Transition state theory, championed by Eyring, argues that the rate at which a given transition will occur depends on the probability that the ion or molecule has enough energy to cross the transition barrier. The probability of Page 6.1
having this activation energy is given by the Boltzmann distribution. Let us imagine that the activation energy for a particular ion or molecule crossing the membrane is Ea (Figure 6.1). Then the rate of influx (Joi) is (6.5) Joi = Co exp [-(Ea-zF o)/RT] x constant
The rate of efflux (Jio) is (6.6) Jio = Ci exp [-(Ea-zF i)/RT] x constant
These two rates must be equal at equilibrium, so (6.7) Ci exp [-Ea/RT + zF i/RT] = Co exp [-Ea/RT + zF o/RT]
Equation 6.7 reduces to the Nernst equation (equation 6.3), so the statistical mechanics approach predicts the same equilibrium as does the thermodynamics approach. Energy Ea zF o Outside Membrane Inside
zF
i
Figure 6.1. Transition-state energy for membrane permeation.
Section 6.2. Equilibria Permeation and conduction are passive processes and will proceed in the direction of equilibrium. For electrolytes in the presence of a membrane potential, we have shown that the equilibrium concentration gradient is given by the Nernst equation (equation 6.3). For non-electrolytes and for electrolytes when the membrane potential is zero, the equilibrium reduces to Co = Ci (equation 6.4). An interesting exception to these rules is found in the case of weak acids and weak bases. At neutral pH, weak acids and weak bases are predominantly in their charged forms (A- and BH+). These charged species do not permeate across the hydrophobic barrier presented by biological membranes. The charged species, however, are in equilibrium with uncharged species that will permeate the membrane. Permeation of the uncharged species causes the charged species to reach the following equilibria: (6.8) [BH+]i/[BH+]o = [H+]i/[H+]o Page 6.2
(6.9)
[A-]i/[A-]o = [H+]o/[H+]i
To understand the logic behind this, let us consider the case of a weak base (Figure 6.2). The uncharged species (B) will reach the equilibrium expected of nonelectrolytes (Bo = Bi). On either side of the membrane, this unprotonated species will be in equilibrium with the protonated form: (6.10) K = [B]o[H+] o /[BH+] o = [B]i[H+] i /[BH+] i
Since [B]o = [B]i, equation 6.10 reduces to equation 6.8. A similar analysis may be applied to weak acids to establish equation 6.9. Outside BH + Inside BH +
Membrane
H++ B
B + H +
Figure 6.2. Permeation of a weak base.
Section 6.3. Permeation of Non-Electrolytes The velocity (v) at which a molecule or ion will diffuse is proportional to the gradient of its electrochemical potential (d/dx): (6.11) v = -(1/Nf) (d/dx)
N is Avogadro's number and f is the frictional coefficient. The total rate of flow J is the velocity multiplied by the concentration C: (6.12) J = vC = -(C/Nf)(d/dx)
For a non-electrolyte, d/dx = d(RT ln C)/dx, so (6.13) J = (-C/Nf)(RT/C)(dC/dx) = -(RT/Nf)(dC/dx)
If we define RT/Nf as the diffusion coefficient D, equation 6.13 reduces to Fick's Law of diffusion.
Page 6.3
Membrane permeation involves diffusion across the membrane. To analyze this, we must integrate Fick's Law (equation 6.13) from one side of the membrane to the other (Figure 3). (6.14) 0 J dx = - 0 (RT/Nf)(dC/dx)dx Membrane Inside Ci Cmi Co C mo 0 d
d d
Outside
Figure 6.3. Concentration profile for steady-state flow across a membrane This integration, which allows us to determine how the flow depends on the overall concentration gradient (Ci - Co) across the membrane, is relatively simple. As we shall see (Section 7.1), the corresponding integration for the case of non-electrolytes is much more difficult. To integrate equation 6.14, we assume that the flow of non-electrolyte across the membrane reaches a steady state. This means that the flow J must be the same at all points within the membrane so that the concentration profile across the membrane does not change with time. Steady-state is a natural assumption because it is a stable condition. Suppose that J varied within the membrane so that the flow into a particular region was greater than the flow out of that region. The concentration of non-electrolyte in that region would then rise. This, in turn, would cause the flow out of the region to increase and the flow into the region to decrease. Thus, the concentration would spontaneously change to maintain equality between the flows into and out of the region. This assures steady state. We may then integrate equation 6.14 assuming that J is independent of x (constant). (6.15) Jd = -(RT/Nf)(Cmi-Cmo)
Cmi is the concentration in the membrane at the inner surface and Cmo is the concentration in the membrane at the outer surface. We imagine that, at each surface of the membrane, molecules in the aqueous phase are in equilibrium with molecules in the membrane phase. Therefore, the chemical potential in the water phase (w) must equal the chemical potential in the membrane (m): (6.16) w = w + RT ln Cw = m = m + RT ln Cm
Page 6.4
The concentration at the surface of the membrane (Cm) is then (6.17) Cm = Cw exp [(w- m)/RT]
An expression for J is obtained by substituting equation 6.17 into equation 6.15 and rearranging: (6.18) J = -(RT/Nfd){exp [(w - m)/RT]}(Ci - Co)
The flux is proportional to the concentration difference across the membrane (Ci - Co). It is also proportional to the permeability coefficient P defined as (6.19) P = (RT/Nfd){exp [(w - m)/RT]}
The permeability coefficient includes the term RT/Nf, which is the diffusion coefficient of the molecule in the lipid phase of the membrane. This term will depend on the size of the molecule. The term exp[(w-m)/RT] describes the partitioning of the molecule between water and the membrane and will depend on the lipid solubility of the molecule. Since Cm/Cw is the membrane:water partition coefficient (Kp), equation 6.17 implies that (6.20) Kp = exp [(w - m)/RT]
A comparison of equations 6.19 and 6.20 shows that P and Kp should be linearly related. Walter and Gutknecht (1984) tested this prediction in a study of the permeability of lipid bilayers to a series of carboxylic acids. After correcting for unstirred layer effects and assuming that only the protonated form of the carboxylic acid permeates, they found that the permeability coefficient is related to the hexadecane:water partition coefficient (Kp') as follows: (6.21) log P = 0.90 log Kp' + 0.87
The observed slope of 0.90 is close to the predicted slope of 1.0. Moreover, the free energy change for the transfer of the carboxylic acid from water into the membrane (m-w) can be determined from either the permeability coefficient P or the partition coefficient Kp. The incremental change in the free energy per methylene group for the series of carboxylic acids (acetic, propionic, butyric, hexanoic) is -898 159 cal/mole determined from the partition coefficient and -764 54 cal/mole determined from the permeability coefficient. These numbers agree very well with the energies described in the discussion on micelle formation (Section 2.4).
Section 6.4. Unstirred Layers When a compound diffuses from one side of a membrane to the other, the membrane may be the principal barrier to flow but not the only barrier. Passage of the molecule across the membrane may also be slowed by diffusion across the aqueous layers adjacent to either surface of the membrane. These so-called unstirred layers may range in thickness from 1 m to 500 m (Remember that the membrane itself is only 4 x 10-3 m Page 6.5
thick). The unstirred layer effect will generally be most prominent for relatively nonpolar compounds. For these compounds, the permeability coefficient will be large and diffusion across the membrane itself will be relatively fast. Diffusion across the aqueous layers, therefore, may be partially rate-limiting. For compounds that are quite water soluble, permeation across the membrane will be slow, and diffusion across the unstirred layers will have relatively less effect. To consider the effect of unstirred layers, imagine that a compound has a membrane permeability coefficient P and an aqueous diffusion constant D. Let the unstirred layers have thicknesses, di and do (Figure 6.4), let the bulk concentrations of the compound be Ci (inside) and Co (outside) and let the concentrations at the surface of the membrane be Cmi and Cmo. The flow through the membrane then is (6.22) Jm = P (Cmi - Cmo)
The flow through the unstirred layers will be (6.23) (6.24) Ji = (D/di) (Ci - Cmi) Jo = (D/do) (Cmo - Co)
At steady-state, the flows must all be the same (Jm = Ji = Jo = J). Therefore, equations 6.22, 6.23 and 6.24 can be transformed to (6.25) (6.26) (6.27) J/P = Cmi - Cmo Jdi/D = Ci - Cmi Jdo/D = Cmo - Co
By summing these three equations, we obtain (6.28) J (1/P + di/D + do/D) = Ci - Co Unstirred Layer do Unstirred Layer d
i
Membrane
Ci C mi
C mo Co
Page 6.6
Figure 6.4. Permeation and diffusion through unstirred layers Therefore, the effect of unstirred layers is to decrease the permeability so the apparent permeability coefficient (Papp) is smaller than P: (6.29) 1/Papp = 1/P + di/D + do/D
References A. Finkelstein (1976) Water and nonelectrolyte permeability of lipid bilayer membranes, J. Gen. Physiol. 68, 127-135. A. Finkelstein and A. Cass (1968) Permeability and electrical properties of thin lipid membranes, J. Gen. Physiol.52, 145s-172s. A.R. Koch (1970) Transport equations and criteria for active transport, Am. Zool. 10, 331346. S. G. Schultz (1980) Basic Principles of Membrane Transport, Cambridge University Press, Cambridge. A. Walter and J. Gutknecht (1984) Monocarboxylic acid permeation through lipid bilayer membranes, J. Membrane Biol. 77, 255-264.
Page 6.7

CHAPTER 7: PERMEABILITY AND CONDUCTANCE OF ELECTROLYTES
D. Njus, 2000
CHAPTER 7: PERMEABILITY AND CONDUCTANCE OF ELECTROLYTES
Section 7.1. Permeation of Electrolytes The permeation of electrolytes may be analyzed using the same approach as is used for nonelectrolytes (Section 6.3). The flux J is assumed to be proportional to the thermodynamic driving force, the derivative of the electrochemical potential: (7.1) J = -
( )
C Nf
d dx
[ o + RT ln C + zF ] = -
RT Nf
dC dx +
zFC RT
) ]
d dx
As before, we must integrate this from one side of the membrane to the other. In this case, however, we have two parameters, C and , that will vary as we cross the membrane. We may use the steady-state assumption to define one of these parameters in terms of the other, but we will need another equation to define both. We will return to this problem later. First, note that (7.2) d dx
[Ce
(zF /RT)
] [ dx
= dC
zFC d RT dx
] e(zF
/RT)
A comparison of equations 7.1 and 7.2 reveals that exp (zF/RT) may be used as an integrating factor so that (7.3) J exp (zF/RT) = - (RT/Nf) d/dx[C exp (zF/RT)]
If we make the steady-state assumption (J is constant across the membrane), then we may partially integrate equation 7.3: (7.4) J 0 e
d
zF/RT
zFmi/RT
zFmo/RT
dx = - (RT/Nf) [Cmi e
- Cmo e
Cmi and Cmo are the concentrations in the membrane at d and 0 respectively (Figure 7.1). They are related to the concentrations Ci and Co in the bulk aqueous phases by the membrane:water partition coefficient (Kp) and by the surface potential: (7.5) (7.6) Therefore, Cmi = Ci Kp exp[zF(i- mi)/RT] Cmo = Co Kp exp[zF(o- mo)/RT] zF /RT
RTK p ( C e zF i /RT - C e zF o /RT ) dx = o i Nf 0 e If we define the electrical potential as zero on the outside (o = 0), then (7.7) J
d
Page 7.1
(7.8)
J =
RTKp Nfd
d exp (zF/RT) dx 0
d
Co - C i e
zF m /RT
where m is the membrane potential. We may note that RTKp/Nfd is the permeability coefficient P (equations 6.19 and 6.20). Therefore, (7.9) J = PQ[Co - Ci exp (zFm/RT)]
where Q is defined as (7.10) Q = d/0 exp (zF/RT) dx Membrane Inside C mi Ci C mo Co o

mo
d
Outside
mi
Figure 7.1. Concentration profile for steady-state electrolyte flow The problem now is to evaluate Q. To integrate exp (zF/RT), we need to define (x). We will use the constant field approximation first proposed by Goldman. A second possibility is the assumption of electrical neutrality in the membrane, an approach first explored by Planck. Physically, the two approaches are similar. A constant field within the membrane implies that there must be electrical neutrality. In terms of formalism, however, the Goldman approach is simpler. The equations obtained by assuming a constant field (or a linear gradient in electrical potential) are simpler than those obtained by summing anion and cation concentrations within the membrane and setting the total charge equal to zero. The constant field assumption states that the electric field (E = -d/dx) is constant throughout the membrane. Therefore, (7.11) (x) = - Ex + (0)
If we assume that surface potentials are negligible, then (0) = 0 and m = (d) = - Ed. Equation 7.10 may then be integrated to give
Page 7.2
(7.12)
Q =
exp (zF /RT) - 1

m
zF m/RT
Introducing equation 7.12 into equation 7.9 yields a final expression for the flow J: (7.13) J = P
zF m /RT exp (zF m /RT) - 1
) (Co - C i e
zF m /RT
Equations 7.9 and 7.13 provide us with alternative expressions for determining the flow of electrolytes across biological membranes with the latter equation including the assumption of a constant field. These expressions allow us to calculate the flow of electrolyte driven across the membrane by a membrane potential, by a concentration gradient, or by a combination of these two forces. In Figure 7.2, the membrane potential and the concentration gradient are used as the axes of a two-dimensional graph. Equations 7.9 and 7.13, therefore, allow us to calculate the flow at any point on this graph. Let us examine some special cases. First, note that both equations reduce to the Nernst equation if the flow J is zero: (7.14) Ci/Co = exp (-zFm/RT)
Second, if m is zero, Q = 1 and equation 7.9 reduces to Fick's Law: (7.15) J = P (Co - Ci)
Finally, if there is no concentration gradient (Ci/Co = 1), equation 7.13 reduces to: (7.16) J = - PCzFm/RT
Since I = -JFz, equation 7.16 is equivalent to Ohm's Law (I = g m) where the conductance g is PCz2F2/RT.
TABLE I. Permeability Coefficients of Ions H2O Na+ K+ H+ Na+ and K+ Cl2 x 10-5 - 2 x 10-2 cm/sec 8 x 10-9 cm/sec 6 x 10-7 cm/sec 10-5 cm/sec 10-10 - 10-11 cm/sec 10-10 cm/sec
(squid axon) (squid axon) (chromaffin vesicle) (lipid bilayer) (phospholipid vesicle)
Page 7.3
Ohm's Law Fick's Law
Ohm's Law Fick's Law
C i /C o Nernst Equation
ln C i /C o
Nernst Equation 0 1 0
Figure 7.2. Regions of validity of the permeation and conductance equations
The above analysis of electrolyte permeation was developed using the electrochemical potential. We may also analyze the permeation of electrolytes using the transition state approach. Let us imagine that the energy of the electrolyte is Eo on the outside of the membrane, Ei on the inside, and Em in the membrane (Figure 7.3). The unidirectional flux in the inward direction will be (7.17) roi = Co exp [-(Em- Eo)/RT] x constant
The unidirectional flux in the outward direction will be (7.18) rio = Ci exp [-(Em- Ei)/RT] x constant
The net flow J will be the difference between these two fluxes: (7.19) J = roi - rio = {Co exp [-(Em- Eo)/RT] - Ci exp [-(Em- Ei)/RT]} x constant
Page 7.4
Energy Em
Outside
Membrane
Inside
Eo Ei Figure 7.3. Energy barrier for permeation of electrolytes Following our usual convention, we will define the electrical potential as zero on the outside. Then Eo will simply be the standard free energy of the electrolyte in water (Eo = w) and Ei will differ from this by the membrane potential (Ei = w + zFm). Upon introducing these values for the energies, equation 7.19 reduces to (7.20) J = exp [-(Em- w)/RT] [Co - Ci exp (zFm/RT)] x constant
If we let PQ = exp [-(Em- w)/RT] x constant, then equation 7.20 is the same as the flux equation derived using the electrochemical potential approach (equation 7.9). Section 7.2. The Born Charging Equation Several factors determine the energy Em of an ion in a membrane. These include 1) hydrophobic interactions, 2) electrostatic potentials (both surface and dipole), 3) short range forces (steric effects), and 4) the Born charging energy. Hydrophobic interactions and short range forces apply to non-electrolytes as well. Surface and dipole potential effects were considered earlier. In this section, we will consider the effect of the Born charging energy. The Born charging energy is the energy required to assemble a given amount of charge on a particle of a given size. Because this energy is lower in a medium with a high dielectric constant, the Born charging energy is much smaller for an ion in water than for an ion in a hydrocarbon medium. This means that an ion requires much more energy to enter a hydrocarbon phase than to enter an aqueous phase. The Born charging energy, therefore, accounts for the insolubility of ions in hydrocarbon phases and for the impermeability of biological membranes to ions. Because the Born charging energy is greater for a localized charge than for an equivalent delocalized charge, ions with delocalized charge will permeate through biological membranes more easily.
Page 7.5
dq x a
Figure 7.4. Charging a conducting sphere Imagine that an ion is a conducting sphere of radius a (Figure 7.4). If q is the charge placed on the sphere and is the dielectric constant of the medium, the Born charging energy is (7.21) W = q2/8oa
This equation can be derived as follows. Outside of a conducting sphere, the electric field created by the sphere is the same as the electric field created by a point charge (of equal charge) located at the center of the sphere. Therefore, the force between a sphere of charge q' and a charge dq' is given by Coulomb's Law: (7.22) F = q'dq'/4ox2
The work required to move the charge dq onto the sphere from an infinite distance away is (7.23) dW = - F dx = - (q' dq'/4ox2) dx = q' dq'/4oa
a a
The work required to place the entire charge q on the sphere is then (7.24) W = W = 0 q' dq'/4oa = q2/8oa
q
The change in the charging energy upon moving the ion from water into a hydrocarbon phase is (7.25) W = (q2/8oa) (1/hc - 1/w)
The membrane is not an infinite hydrocarbon phase, but is a thin layer of hydrocarbon with water on both sides. Therefore, the change in charging energy upon moving the ion from water into a membrane is somewhat smaller than the change shown in equation 7.25. The work done in moving a charge q a distance x into a membrane of thickness d has been approximated by Flewelling and Hubbell (1986):
Page 7.6
(7.26) W =
q 8a o
)(
1 hc
)[
a - 1.2 1 - 2x
( )( ) ]
a d x d 2
Section 7.3. The Goldman-Hodgkin-Katz Equation If ions are in equilibrium across a membrane, then the membrane potential will be given by the Nernst equation. This is rarely the case, however. Generally, ions are in constant flux (transport and permeation) and the capacitative charge is determined by the steady-state distribution of ions. The membrane potential can nevertheless be determined from this steady-state distribution using the Goldman-Hodgkin-Katz equation. At steadystate, the net charge flux will be zero. (7.27) 0 = cations zjFJj + anions zjFJj The fluxes Jj are defined by equation 7.13. If we impose the simplifying assumption that all of the ions are univalent, then (7.28) 0 = cationsFPj{(Fm/RT)/[exp(Fm/RT)-1]}{Coj - Cijexp(Fm/RT)} - anionsFPj{(-Fm/RT)/[exp(-Fm/RT)-1]}{Coj - Cijexp(-Fm/RT)} Dividing by Fm/RT and rearranging the exponentials in the anion term yields (7.29) 0 = cations FPj{1/[exp (Fm/RT) - 1] }{ Coj - Cij exp (Fm/RT) } - anions FPj{ (-1/[1 - exp (Fm/RT)] }{ Coj exp (Fm/RT) - Cij }
Upon dividing by F{1/[exp (Fm/RT) - 1] }, we obtain (7.30) 0 = cations Pj{Coj - Cij exp (Fm/RT)} - anions Pj{Coj exp (Fm/RT) - Cij}
Then solving for the exponential term gives (7.31) exp (F /RT) =
m
cations P jC oj + anions P jC ij cations P jC ij + anions P C oj j
Therefore, the membrane potential is defined by the ion concentrations and permeabilities as follows: = (RT/F) ln
m
(7.32)
cations P jC oj + anions P jC ij cations P jC ij + anions P C oj j
This is the Goldman-Hodgkin-Katz equation.
Page 7.7
References R.F. Flewelling and W.L. Hubbell (1986) Hydrophobic ion interactions within membranes, Biophys. J. 49, 531-540. R.F. Flewelling and W.L. Hubbell (1986) The membrane dipole potential in a total membrane potential model, Biophys. J. 49, 541-552. A.L. Hodgkin and A.F. Huxley (1952) A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol. 117, 500-544. A.R. Koch (1970) Transport equations and criteria for active transport, Am. Zoologist 10, 331-346. R.I. Macey (1978) Mathematical models of membrane transport processes, in Membrane Physiology (T.E. Andreoli, J.F. Hoffman, and D.D. Fanestil, eds.), Plenum, New York, pp. 125-146. A. Parsegian (1969) Energy of an ion crossing a low dielectric membrane: Solutions to four relevant electrostatic problems, Nature 221, 844-846.
Page 7.8

CHAPTER 8: CHANNELS AND EXCITABLE MEMBRANES
D. Njus, 2000
CHAPTER 8: CHANNELS AND EXCITABLE MEMBRANES Section 8.1. Channel-forming Antibiotics Ion channels are needed to conduct ions across biological membranes because ions, particularly cations, do not permeate readily across the lipid bilayer. The structural simplicity required to create an ion-conducting channel across a biological membrane is exemplified by channel-forming antibiotics, such as gramicidin and amphotericin B. These antibiotics form ungated channels, so they dissipate the ion gradients needed for proper membrane function and cause cells to spend energy in futile ion pumping. Since these ungated channels are lethal, regulation of channel opening or gating is obviously an important feature of natural ion channels. Gramicidin is a pentadecapeptide consisting of alternating L and D amino acids: HCO-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-TrpD-Leu-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-NHCH2CH2OH The conductance through gramicidin channels varies as the square of the gramicidin concentration indicating that the compound functions as a dimer. Indeed, two peptides can be linked at the formyl groups on their amino terminal ends, and the coupled structure will function as a channel. The channel formed by gramicidin is not very selective and has a unitary (single channel) conductance of 5 pS. It is thought that gramicidin forms a helix with the hydrophobic side chains on the outside and the carbonyl oxygens oriented to the inside. This forms a channel 2 in diameter. Amphotericin B forms a larger channel and increases the permeability of lipid bilayers to water and small electrolytes as well as ions. The conductance depends on the 4th-12th power of the concentration suggesting that a number of molecules are required to form the channel. Because amphotericin B is an elongated molecule with a hydrophobic side and a hydrophilic side, it is thought to line the sides of the channel like the staves on a barrel. Amphotericin B requires a sterol for activity, and thus makes channels in membranes that contain cholesterol. Section 8.2. Voltage-Gated Channels In recent years, molecular biological techniques have yielded a wealth of information about the membrane-spanning proteins that form ion channels. A generalization that may be emerging is that channels with similar gating mechanisms have similar structures. The voltage-gated channels, in particular, have common structural features despite the fact that they have different ion selectivities and conductances. Voltage-gated channels comprise the S4 superfamily, so named because the proteins function as tetramers having either four subunits or four homologous segments. The K+ channel, for example, is a tetramer with six membrane-spanning regions in each subunit. The Na+ channel is a single large peptide (~260 kDa), but that peptide has four homologous segments with 6 membrane-spanning regions in each. Looking down at the membrane, the four segments are arranged at the corners of a square with the ion channel itself passing down through the center between them. As mentioned, the core of the Na+ channel is formed by a single large subunit. In the eel electroplax, that is the only subunit. The sodium channel from mammalian skeletal
Page 8.1
muscle also contains a 1 subunit (38 kDa), while the sodium channel in mammalian brain contains both a 1 (36 kDa) and a 2 subunit (33 kDa) along with the subunit. Voltage-gated potassium channels include the delayed rectifier (DR) channel, which functions in actions potentials in excitable membranes, and the CaK channel, which is activated by Ca2+ as well as by depolarization. Both are blocked by barium. The CaK channel has a very high unitary conductance and is specifically blocked by charybdotoxin. Calcium channels have been classified into a variety of types based on functional characteristics and pharmacology. The well characterized L-type channel is a highthreshold, slow inactivating channel, which is blocked by dihydropyridines. Lowthreshold, fast inactivating Ca2+ channels are classed as T-type. Two other high-threshold channels (N and P) are distinguished by their sensitivity to peptide toxins. N channels are blocked by -conotoxin GVIA while P channels are blocked by -agatoxin IVA. Structurally, these channels are thought to be similar. The L-type Ca2+ channel from skeletal muscle has five subunits: 1 (170 kDa), 2 (175 kDa), (52 kDa) and (32 kDa). The 1 subunit contains binding sites for Ca2+ channel antagonists and is thought to be the subunit forming the functional Ca2+ channel. The 2 piece consists of a large 2 subunit linked to the subunit by disulfide bonds. Section 8.3. Ligand-Gated Channels Extracellularly activated ligand-gated ion channels seem to fall into three major groups based on the number of subunits. The nicotinicoid group, represented by the nicotinic acetylcholine receptor, has five homologous subunits arranged in a pentagonal structure around a central channel. This group includes the cation-conducting nicotinic and serotonin (5HT) receptors and the anion-conducting GABAA, GABAC and glycine receptors. The best characterized member of the nicotinicoid receptor group is the nicotinic acetylcholine receptor. Structurally, the acetylcholine receptor consists of five homologous subunits: 2, 1, 1 and 1 . Each subunit has at least four transmembrane segments. The acetylcholine binding sites are located on the subunits. The second group of extracellularly activated ligand-gated ion channels, the glutamate-activated cation channels, has four homologous subunits. This group includes the AMPA, Kainate and NMDA receptors named for ligands (agonists) that specifically activate each type. The third group of ligand-gated ion channels, the ATP-gated channels, have three homologous subunits and include the ATP2x and ATP2z receptors. As ion channels, the ligand-gated channels seem to exhibit less specificity than the voltage-gated channels. The cation channels do not discriminate between Na+ and K+, so they drive the membrane potential toward zero (midway between the Na+ and K+ equilibrium potentials). Because this depolarizes the membrane, these receptors are often called excitatory. The anion channels drive the membrane potential toward the Clequilibrium potential (negative inside). Thus, they tend to restore the resting membrane potential and are often called inhibitory. Section 8.4 Ryanodine and Inositol Tris Phosphate Receptors The ryanodine and inositol trisphosphate receptors are related proteins that form Ca2+ channels in intracellular membranes. The ryanodine receptor is a very large protein (565 kDa) and is responsible for releasing Ca2+ from the sarcoplasmic reticulum (SR) in muscle. Most of this protein (the amino-terminal 80%) is cytoplasmic and constitutes a Page 8.2
"foot" structure. The remaining 20% on the carboxyl end includes 4 to 10 transmembrane segments and presumably creates the ion channel structure. Ryanodine, a plant product, opens this channel. In vivo, however, the channel is gated by cytoplasmic Ca2+. Thus, when L-channels in the T-tubule membranes open and allow Ca2+ to enter the muscle cell, the ryanodine receptor channel in the sarcoplasmic reticulum membrane responds by releasing more Ca2+ from the SR. Thus, the ryanodine receptor mediates Ca2+-induced Ca2+ release. A related protein, the inositol-1,4,5-trisphosphate receptor releases Ca2+ from the endoplasmic reticulum in response to the intracellular messenger inositol-1,4,5trisphosphate (IP3). It has a molecular mass of 260 kDa and, like the ryanodine receptor, consists of a large amino-terminal foot and a smaller carboxyl portion containing 8-10 transmembrane segments. Both proteins appear to associate into homotetramers. TABLE 8.1. Characteristics of Channels Type Na+ K+ (DR) K+ (Ca2+) Ca2+ (L) (slow inact.) 2+ Ca (T) (fast inact.) 2+ Ca (N) Ca2+ (P) Nicotinic Receptor Ryanodine Receptor InsP3 Receptor Gating Depolarization Depolarization Depolarization/Ca2+ High-threshold depolarization Low-threshold depolarization High-threshold Depolarization Depolarization Acetylcholine Ca2+ Inositol trisphosphate Unitary Conductance 10 pS 55 pS 240 pS 9 pS (Ca2+) 25 pS (Ba2+) 8 pS (Ba2+) 8 pS (Ca2+) 12 pS (Ba2+) Blockers Tetrodotoxin Ba2+ Charybdotoxin/Ba2+ Dihydropyridines
-conotoxin GVIA -agatoxin IVA Bungarotoxin
90 pS
Section 8.5. Channel Conductance As described in Section 7.1, the flow of an electrolyte across a membrane (expressed as a current I = -JFz) depends on the membrane potential and the concentration gradient as described by equation 8.1: (8.1) I = -FzP
zF m /RT exp (z Fm /RT) - 1
)(
Co - C i e
zF m /RT
The current carried by ion channels is commonly expressed using a variation on Ohm's Law: (8.2) Ii = gi (m - i)
Page 8.3
Ii is the current carried by ion i, gi is the conductance of the membrane to that ion, m is the membrane potential and i is the equilibrium potential for that ion as defined by the Nernst equation (equation 6.3). Adjusting the membrane potential by subtracting i insures that there will be no current when the membrane potential equals the ions equilibrium potential. Equation 8.2 predicts that a plot of current (Ii) against voltage (m), also known as an IV curve, will be linear. The slope is the conductance gi and the X-intercept is the equilibrium potential i. If a channel is perfectly selective for a particular ion, that channel will exhibit an IV curve as defined by equation 8.2. Often, however, a channel is not absolutely selective and will pass different kinds of ions with different conductances. In this case, the current through the channel must be summed over the different ions: (8.3) I = [gi (m - i)] = m gi - (gi i) = gi {m - (gi i) / gi }
For a non-selective channel, Ohm's Law still holds, but the conductance is the sum of the conductances for all of the ions, and the X-intercept is a weighted average of the equilibrium potentials. The X-intercept is called the reversal potential because the current through the channel reverses direction as the membrane potential crosses this value. If the channel is highly selective for a particular ion, the reversal potential will be close to the equilibrium potential for that ion. Thus, the reversal potential is an indicator of a channel's ion selectivity. Note that the reversal potential of a channel depends on the channel's selectivity and also on the equilibrium potentials (concentration gradients) of the ions it conducts. The functioning of individual channels has been illuminated in recent years by the development of the patch clamp technique. This technique, which permits the observation of currents through single channels, has revealed the opening and closing times for different channel types. It has also enabled measurement of unitary conductances. The unitary conductance is important because it reflects the restriction imposed by the channel's selectivity filter. For example, the sodium channel, which has a relatively low unitary conductance, probably has a relatively long narrow tunnel. It is estimated that this is a pore 3 x 5 in cross section and 10-12 in length. The high conductance K channel, by contrast, probably has a relatively short narrow tunnel. In both cases, it is imagined that the channel has large vestibules on one or both sides of the selectivity filter. This permits free (and rapid) diffusion through most of the membrane and limits conductance only to the extent necessary to maintain selectivity. Section 8.6. Channel Selectivity An important property of ion channels is that they exhibit selectivity for particular ions over other closely related ions. Eisenman examined the five monovalent metal cations (Cs+, Rb+, K+, Na+, Li+) and noted that, although there are 120 possible selectivity sequences, only 11 sequences are observed. He rationalized this by arranging the eleven sequences in order from low field strength to high field strength:
Page 8.4
TABLE 8.2. The Eisenman Selectivity Series for Monovalent Cations I II III IV V VI VII VIII IX X XI Cs Rb Rb K K K Na Na Na Na Li > > > > > > > > > > > Rb Cs K Rb Rb Na K K K Li Na > > > > > > > > > > > K K Cs Cs Na Rb Rb Rb Li K K > > > > > > > > > > > Na Na Na Na Cs Cs Cs Li Rb Rb Rb > > > > > > > > > > > Li Li Li Li Li Li Li Cs Cs Cs Cs Low field strength
High field strength
To cross through a channel, an ion must shed its water of hydration and pass a selectivity site in the channel. This implies that the ion must interact more favorably with the selectivity site than with its water of hydration. At low field strength, the selectivity of the channel is dominated by the dehydration energy of the ion. Ions that are easily dehydrated pass through the channel more readily than do ions that interact strongly with water. At low field strength, therefore, larger ions are favored over smaller ions and the order is in the direction of decreasing atomic weight. At high field strength, the selectivity of the channel is dominated by the attraction of the ion for the selectivity site. Those ions that bind well will be favored over those that bind poorly. At high field strength, therefore, Intermediate-field-strength site
-G
High-field-strength site Hydration energy
Low-field-strength site
Cs+ Rb+ K+ 1/r
Na+
Li +
Figure 8.1. Competition between hydration and selectivity-site binding in ion channel selectivity.
Page 8.5
smaller ions are favored over larger ions and the order is in the direction of increasing atomic weight. The sodium channel has selectivity characteristics typical of a high field strength. This high field strength is attributed to one or more carboxylic acid groups that may line the tunnel and interact with the passing cations. TABLE 8.3. Properties of Ions Ion Li+ Na+ K+ Rb+ Cs+ Atomic Weight 6.94 23.00 39.10 85.47 132.91 Ionic Crystal Radius () 0.68 0.98 1.33 1.48 1.67 Hhydration (kcal/mole) 121 95 76 69 62
TABLE 8.4. Selectivity of Channels Type ACh Receptor Na+ K+ (DR) K+ (Ca2+) Ca2+ (L) Ca2+ (T) Selectivity NH4+ > Cs+ > Rb+ > Na+ Na+,Li+ > K+ > Rb+ > Cs+ Tl+ > K+ > Rb+ > NH4+ > Na+ > Li+ >> Cs+ Tl+ > K+ > Rb+ > NH4+ > Na+, Li+, Cs+ Ca2+ > Sr2+ > Ba2+ > Li+ > Na+ > K+ > Cs+ > Mg2+ Ca2+ Ba2+
Section 8.7. Excitability As defined by the Goldman-Hodgkin-Katz equation (Equation 7.32), the membrane potential is determined by the relative permeability of the membrane to different ions. Because animal cell membranes are relatively more permeable to K+ than to Na+, the resting membrane potential is normally close to the K+ equilibrium potential and is negative (inside relative to outside). When a nerve or muscle fiber is stimulated and conducts and action potential, the membrane depolarizes (the membrane potential becomes less negative and then positive) and then repolarizes to the resting membrane potential. This happens because Na+ channels open first and then K+ channels open. When Na+ channels open, the membrane becomes more permeable to Na+ than to K+ and the membrane potential shifts toward the Na+ equilibrium potential (or more accurately, the reversal potential of the Na+ channel). When the K+ channels open, the membrane again becomes more permeable to K+ than to Na+ and the membrane potential shifts back toward the reversal potential of the K+ channel. Na+ and K+ channels open and close in the course of an action potential because their opening (gating) depends on the membrane potential. Of course, the membrane potential in turn depends on the opening of the channels making the action potential an autocatalytic event. To analyze this, recognize that the currents across the membrane all contribute to a change in the capacitative charge: (8.4) dqc/dt = - ( IK + INa + Il ) Page 8.6
Equation 8.4 includes currents of K+ and Na+ as well as a small residual current (mostly Cl), which is commonly known as the leakage current. Equation 8.4 may be expanded knowing that the membrane potential is related to the capacitative charge by the capacitance (Equation 4.11), and the currents are given by Ohms Law (Equation 8.2): (8.5) C (dm/dt) = - gK(m-K) gNa(m-Na) gl (m-l) Equation 8.5 makes it clear that the change in the membrane potential m depends on the conductances of the sodium and potassium channels. The sodium and potassium channels in turn are gated by voltage, so their conductances change depending on the membrane potential. To separate the interdependence of channel conductance and membrane potential, Hodgkin and Huxley used the voltage-clamp technique, in which the membrane potential is fixed or clamped so that Na+ and K+ currents can be recorded at that constant membrane potential. They found that Na+ and K+ channels remain closed at the resting membrane potential but their probability of opening increases when the membrane potential is raised. When the membrane is depolarized, Na+ channels open quickly and then inactivate or close. K+ channels open more slowly and remain open. This means that depolarizing a membrane will cause Na+ channels to open first. The inward Na+ current will drive the membrane potential in the positive direction toward the Na+ equilibrium potential. This accelerates the opening of Na+ channels ensuring a strong depolarization. After a brief time, however, the Na+ channels inactivate and the Na+ current stops. Concurrently, K+ channels open, and the outward K+ current drives the membrane potential back down toward the K+ equilibrium potential. This brings the membrane potential back to the resting value and also causes the K+ channels to close terminating the action potential. From data gathered in their voltage clamp experiments, Hodgkin and Huxley found empirical relations that express the changes in conductances as functions of the membrane potential. These functions are quite complicated and for our purposes need only be represented as f1(gK, m) and f2(gNa, m). (8.6) dgK/dt = f1(gK, m) (8.7) dgNa/dt = f2(gNa, m) Knowing how the membrane potential depends on conductances (equation 8.5) and how the conductances depend on membrane potential (equations 8.6 and 8.7), it is possible to reconstruct the action potential. A membrane action potential is an action potential in which the membrane potential changes along the whole length of the nerve fiber at the same time. Thus, the entire nerve fiber depolarizes and repolarizes simultaneously. A membrane action potential can be simulated by numerically integrating equations 8.5, 8.6 and 8.7. Assume that the membrane potential begins at a value somewhat above the resting potential. Given this membrane potential, calculate the change in gNa, gK and m expected to occur in a brief interval of time (say 10 sec). After changing the values of gNa, gK and m accordingly, calculate the changes expected in the next brief time interval by repeating the process. If the initial membrane potential was set to a value above
Page 8.7
threshold, then the membrane potential will rise up in an action potential and then return to rest. If the initial membrane potential was set to a value below threshold, then the membrane potential will simply drift back down to its resting value. The membrane action potential is easy to calculate, but normally an action potential does not occur along an entire nerve fiber simultaneously. Rather, it travels from one end to the other. To analyze this propagating action potential, recognize that the action potential travels because charge diffuses down the nerve fiber, depolarizing the membrane ahead of the action potential, and causing the action potential to move forward along the fiber. The current along the length of the nerve fiber is given by Ohms Law: (8.8) Ilong = - (1/r ) (dm/dx) The longitudinal current is proportional to the change in potential (dm/dx) and inversely related to the resistance of the nerve fiber r. The current across the membrane now must equal the change in the current along the membrane. (8.9) Imem = (-1/D) dIlong/dx D is the diameter of the nerve fiber. Therefore, (8.10) Imem = (1/Dr)(d2m/dx2) = (1/Dr) (d2m/dt2) (dt/dx)2 Recognize that dx/dt is the conduction velocity of the action potential (). Then, (8.11) Imem = (1/ Dr2) (d2m/dt2) = C (dm/dt) + gK (m - K) + gNa (m - Na + gl(m - l) Equation 8.11 may be numerically integrated as for the membrane action potential if the correct value for the conduction velocity is chosen. References T. Begenisich (1987) Molecular properties of ion permeation through sodium channels, Ann. Rev. Biophys. Biophys. Chem. 16, 247-263. W.A. Catterall (1986) Molecular properties of voltage-sensitive sodium channels, Ann. Rev. Biochem. 55, 953-985. W.A. Catterall (1988) Structure and function of voltage-sensitive channels, Science 242, 50-61. G. Eisenman and J.A. Dani (1987) An introduction to molecular architecture and permeability of ion channels, Ann. Rev. Biophys. Biophys. Chem. 16, 205-226. A.L. Hodgkin and A.F. Huxley (1952) A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol. 117, 500-544. A.F. Huxley (1959) Ion movements during nerve activity, Ann. N.Y. Acad. Sci. 81, 221246. Page 8.8
H.A. Lester (1992) The permeation pathway of neurotransmitter-gated ion channels, Ann. Rev. Biophys. Biomol. Struct. 21, 267-292. P.S. McPherson and K.P. Campbell (1993) The ryanodine receptor/Ca2+ release channel, J. Biol. Chem. 268, 13765-13768. R.J. Miller (1992) Voltage-sensitive Ca2+ channels, J. Biol. Chem. 267, 1403-1406. M. Mishina, T. Kurosaki, T. Tobimatsu, Y. Morimoto, M. Noda, T. Yamamoto, M. Terao, J. Lindstrom, T. Takahashi, M. Kuno and S. Numa (1984) Expression of functional acetylcholine receptor from cloned cDNA, Nature 307, 604-608. M. Noda, T. Ikeda, T. Kayano, H. Suzuki, H. Takeshima, M. Kurasaki, H. Takahashi, and S. Numa (1986) Existence of distinct sodium channel messenger RNAs in rat brain, Nature 320, 188-192. M. Noda, S. Shimizu, T. Tanabe, T. Takai, T. Kayano, T. Ikeda, H. Takahashi, H. Nakayama, Y. Kanaoka, N. Minamino, K. Kangawa, H. Matsuo, M.A. Raftery, T. Hirose, S. Inayama, H. Hayashid, T. Miyata and S. Numa (1984) Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence, Nature 312, 121-127. M. Noda, H. Takahashi, T. Tanabe, M. Toyosato, S. Kikyotani, Y. Furutani, T. Hirose, H. Takashima, S. Inayama, T. Miyata and S. Numa, (1983) Structural homology of Torpedo californica acetylcholine receptor subunits, Nature 302, 528-532. B.M. Olivera, G. Miljanich, J. Ramachandran, and M.E. Adams (1994) Calcium channel diversity and neurotransmitter release: The -conotoxins and -agatoxins, Ann. Rev. Biochem. 63, 823-867. R.W. Tsien, P. Hess, E.W. McCleskey and R.L. Rosenberg (1987) Calcium channels: Mechanisms of selectivity, permeation, and block, Ann. Rev. Biophys. Biophys. Chem. 16, 265-290. G. Yellen (1987) Permeation in potassium channels: Implications for channel structure, Ann. Rev. Biophys. Biophys. Chem. 16, 227-246. C.D. Ferris, and S.H. Snyder (1992) Inositol 1,4,5-trisphosphate activated calcium channels, Ann. Rev. Physiol. 54,
Page 8.9

CHAPTER 9: ACTIVE TRANSPORT
D. Njus, 2000
CHAPTER 9: ACTIVE TRANSPORT Active transport is usually defined as transport of molecules or ions from a region of lower to a region of higher concentration (i.e., transport against a concentration gradient). Here, we will define it in a stricter sense as transport which requires chemical or photochemical energy. By this definition, only a small number of enzymes are capable of catalyzing active transport. With few exceptions, these enzymes transport inorganic cations: Na+, K+, Ca2+ and H+. Active transporters may be divided into four general classes: 1) ion-translocating ATPases, 2) H+-transporting electron transfer chains, 3) group-translocating enzymes, and 4) photochemically-driven transporters such as bacteriorhodopsin. It should be recognized that these enzymes do not consume energy; rather they transduce chemical energy into electrochemical potential gradients. Section 9.1. Ion-translocating ATPases Ion-translocating ATPases derive energy from the hydrolysis of ATP and use this energy to move ions across the membrane against concentration gradients. The ATPases fall into two broad classes: the P (or E1E2) type and the F/V/A type. The P-type include the widely studied Na+/K+ ATPase found in the plasma membrane of animal cells, the Ca2+ ATPases found in sarcoplasmic and endoplasmic reticulum (SERCA) and in the plasma membrane (PMCA), the H+/K+ ATPase which is involved in acid extrusion, and an H+ ATPase which is found in the plasma membranes of plant and fungal cells. The P-type ATPases are all inhibited by vanadate, a characteristic which seems to be shared by ATPases that are phosphorylated in the course of their catalytic cycle. The phosphate group hydrolyzed from ATP is transiently attached to an aspartyl residue on the enzyme. The P-type ATPases characteristically have a single, large polypeptide chain, which mediates both ion transport and ATP hydrolysis. In general, the catalytic segment appears to lie near the middle of this chain with 4 membrane-spanning domains on the amino end and four to six membrane-spanning domains on the carboxyl end. Thus, the amino terminus and the catalytic site both lie on the cytoplasmic side of the cell membrane. The P-type ATPases hydrolyze cytosolic ATP and transport the substrate out of the cytosol (out of the cell or into the sarcoplasmic or endoplasmic reticulum). The Na+/K+ and H+/K+ ATPases are exceptions in that they mediate an exchange of ionic substrates. Both enzymes bring K+ into the cytosol as the other ion (Na+ or H+) is transported out. These two enzymes are also unusual in having a second () subunit. They are thus heterodimers, and the subunit is thought to function in K+ transport. In the catalytic cycle of the Na+/K+ ATPase, 3 Na+ ions bind from the cytosolic side of the enzyme. ATP hydrolysis phosphorylates the enzyme and initiates a conformational change exposing the ion binding sites to the external side of the membrane. The Na+ ions dissociate and are replaced by 2 K+ ions. Then, the enzyme dephosphorylates and returns to the original conformation releasing the K+ ions to the inside. It is important that ATP hydrolysis not occur until 3 Na+ ions are bound and that dephosphorylation not occur unless 2 K+ ions are bound. Otherwise, ATP hydrolysis and ion transport would not be properly coupled, and ATP would be spent inefficiently on suboptimal transport cycles. The F/V/A-type ATPases include the F (F1FO) ATPases found in mitochondria, chloroplasts and bacterial cells, the vacuolar-type ATPases found in Golgi-derived organelles including lysosomes and secretory vesicles in animal cells and vacuoles and tonoplasts in plant and fungal cells, and the A-type ATPases found in archaebacteria. Page 9.1
These ATPases transport H+ by a rotary mechanism and have a two-part structure including a membrane stalk and a large head. They differ from the plasma membrane ATPases in being insensitive to vanadate and in lacking a stable phosphorylated intermediate. The subunit composition of the F, V and A ATPases is also much more complex than that of the P-type ATPases. They are characteristically sensitive to N,N'dicyclohexylcarbodiimide (DCCD), which reacts with carboxyl groups and typically inhibits proton-translocating enzymes. The F or F1/FO class of H+-ATPases are found in mitochondria, chloroplasts and bacterial cells and are, of course, responsible for ATP synthesis rather than ATP hydrolysis. Nevertheless, they should be considered here because they are functionally similar to the V and A type ATPases, and they can catalyze active transport of H+ when the free energy of the reaction favors that direction. These enzymes have a complex subunit composition including a hydrophilic F1 component which carries the catalytic site and a hydrophobic FO component which is thought to be the proton channel. The F1 component includes five kinds of subunits (, , , , ) in a stoichiometry of 3:3:1:1:1. The nucleotide binding sites lie between the and subunits. At any given time, these sites are in the tight, loose or open state depending on the position of the subunit. The FO component includes three kinds of subunits (a, b, c) in a stoichiometry of 1:2:~12. DCCD inhibits the F1FO ATPases by reacting with a glutamate residue in the c subunit. The c subunits are relatively small (2 transmembrane segments) and the cluster of c subunits together with the subunit form an axle around which the 33 head rotates. In the ATP hydrolysis mode, the F1FO ATPase is thought to function as follows: ATP hydrolysis drives the rotation of the 33 head group relative to the subunit and the c cluster attached at the base. This rotation causes the three nucleotide binding sites to step alternately through the loose, tight and open conformations. In the process, ATP is bound, hydrolyzed and the products released. Concomitantly, 3 H+ are pumped through the FO component for each ATP hydrolyzed. The V or vacuolar ATPases are similar in structure to the F ATPases, although their normal function is H+ transport driven by ATP hydrolysis rather than ATP synthesis driven by H+ movement. The V ATPases differ from the F ATPases in that they are inhibited by the antibiotic bafilomycin A1 and by N-ethylmaleimide. ATPases couple a chemical reaction to vectorial movement of an ion. For this to work, three criteria must be met. 1) The energetics must be such that the chemical reaction releases more free energy than is required for ion translocation. 2) The chemical reaction and ion translocation must be obligatorily coupled processes. One cannot be allowed to occur in the absence of the other. 3) The enzyme must change its affinity for the ions during the translocation cycle. First, let us consider the energetics. For ion transport, the change in the electrochemical potential of the transported ion is: (9.1) j = j,final - j,initial = j + RT ln Cj,final + zjFfinal - j - RT ln Cj,initial - zjFinitial = RT ln (Cj,final/Cj,initial) + zjF(final- initial)
Page 9.2
The total free energy change required for transport is the sum of the electrochemical potential differences (j) for each of the transported ions multiplied by the stoichiometry nj for that ion: (9.2) Gions = j nj j
If we define nj as positive for ions transported from outside to inside and negative for ions transported from inside to outside, then (9.3) j = RT ln (Cj,in/Cj,out) + zjF(in- out) This convention simplifies the bookkeeping of directionality and allows us to use the membrane potential (in- out) without worrying about the sign. Now, for the chemical reaction, the free energy change is (9.4) GATP = GATP + RT ln ([ADP][Pi]/[ATP])
where GATP = -7.3 kcal/mol. Of course, ion transport will proceed as long as (9.5) GATP + Gions 0
Equation 9.5 emphasizes that the ATPases catalyze coupled reactions. It would not do for either ATP hydrolysis or ion movement to occur independently. If ATP hydrolysis occurred independently of ion movement, then the free energy released by ATP hydrolysis would be wasted. If ion movement were allowed to proceed in the absence of ATP hydrolysis, then the ions would move in the wrong direction: from higher concentration to lower concentration. To catalyze ion movement against a concentration gradient, the enzyme must take the ion from a solution in which the ion is present at a lower concentration and release the ion into a solution in which the ion is present at a higher concentration. Thus, in addition to moving the ion, the enzyme must change its affinity for the ion in order to function efficiently. The ATPase must have a high affinity for the ion on the low-concentration side and a low affinity for the ion on the high-concentration side. Part of the energy released by ATP hydrolysis, therefore, must go into changing the enzyme's affinity for the ion. Section 9.2. Electron-transfer chains In mitochondria, chloroplasts and bacterial cells, substrate oxidation is used to create an H+ gradient. This was first suggested by Peter Mitchell in 1961 as the celebrated "Chemiosmotic Hypothesis." We will discuss some of the details of this energy coupling later. Here, let us just review the energetics of redox-driven H+-translocation. In electron transfer reactions, an electron is passed from an electron donor to an electron acceptor. Donorred + Acceptorox Donorox + Acceptorred The free energy change associated with this reaction may be expressed in the usual way:
Page 9.3
(9.6)
Gredox = G + RT ln
( [Donor]
[Donor]ox[Acceptor]red
red
[Acceptor ]ox
Rather than tabulate G values for all combinations of electron carriers, however, electron affinities are catalogued by considering each half- reaction separately: Donorred Donorox + n eAcceptorox + n e- Acceptorred The reduction potential E of the donor is defined as (9.7) E = E + (RT/nF) ln {[Donor]ox/[Donor]red}
where n is the number of electrons transferred in the reaction and E is the midpoint or standard reduction potential for the electron donor. A similar equation defines the reduction potential of the electron acceptor. The reduction potentials are related to the free energy change as (9.8) Gredox = nF Edonor - nF Eacceptor = nF (Edonor -Eacceptor) + RT ln
[Donor]ox[Acceptor]red [Donor]red [Acceptor ]ox
Since E is measured in volts, G will be in units of joules/mole. The best characterized redox-driven proton-pump is cytochrome oxidase, the enzyme catalyzing the transfer of electrons from cytochrome c to oxygen. Mitchell originally suggested that cytochrome oxidase accepted electrons from cytochrome c externally and that it carried the electrons across the membrane to O2. Since the reduction of O2 to H2O takes up two protons, Mitchell hypothesized that this enzyme contributed to proton translocation simply by removing H+ from the mitochondrial matrix. Since E for cytochrome c is +0.24 volts and E for O2/H2O is +0.816 volts, this electron transfer step releases a lot of energy and one might expect it to be captured to a greater extent. In 1978, Krab and Wikstrom showed that this is the case; cytochrome oxidase not only takes up protons because of the reduction of oxygen but it also physically transports protons across the membrane. Cytochrome oxidase contains two hemes (a and a3) and two copper atoms. One Cu and heme a are on the cytoplasmic side of the inner mitochondrial membrane and accept electrons from cytochrome c. The other Cu and heme a3 are on the matrix side of the membrane and react with O2. Cytochrome oxidase from bovine heart consists of eight subunits. Three (I, II and III) are coded by the mitochondrial DNA and the rest are coded by nuclear DNA.
Page 9.4
Section 9.4. Bacteriorhodopsin Bacteriorhodopsin is a light-driven proton pump. It is found in Halobacterium halobium, a bacterium found growing in hot salt springs. In the presence of oxygen, Halobacterium uses respiration as a source of metabolic energy. Under anaerobic conditions, however, Halobacterium uses light energy to produce ATP directly. Halobacterium is not photosynthetic since it does not split H2O (produce O2) or exhibit light-driven electron flow. Instead, it produces a "purple membrane" which is essentially pure bacteriorhodopsin: 25% lipid and 75% bacteriorhodopsin. Bacteriorhodopsin pumps H+ out of the cell in the presence of light. This sets up an H+ gradient, just as respiration would, and the proton gradient can be used to make ATP in the usual chemiosmotic way. Bacteriorhodopsin is a particularly well characterized ion pump, because it can be obtained in large quantity and essentially pure. Moreover, it is a rather simple protein having a molecular weight of 26,000. The amino acid sequence has been determined (Ovchinnikov et al., 1979) and its three-dimensional structure elucidated (Henderson and Unwin, 1975). Bacteriorhodopsin consists of seven membrane-spanning domains. A cofactor, retinal, is responsible for light absorption. Retinal is covalently bound to the amino of a lysine residue via a Schiff's base linkage. The energy of a quantum of light depends on the wavelength (): (9.10) Elight = h = hc/
where h is Planck's constant and c is the speed of light. For an einstein (mole) of quanta, the energy is (9.11) Glight = hcN/
The value of hcN is 28,592 nm.kcal/einstein. Section 9.5. Group Translocation Group translocation is a transport mechanism in which the substrate is metabolized as it is transported. The energy released by that metabolism is used to drive transport. For example, glucose is brought into some bacterial cells as glucose 6-phosphate. Phosphoenolpyruvate serves as the source of both the energy and the phosphate. There are three proteins involved: Enzyme I, Enzyme II, and HPr. Enzyme I is a soluble protein that catalyzes the phosphorylation of HPr using phosphoenolpyruvate. P-HPr then donates the phosphate to glucose bound to enzyme II. Enzyme II is specific for the sugar. This system, studied by Saul Roseman, is found in E. coli, Salmonella and Staphylococcus. References J.P. Abrahams, A.G.W. Leslie, R. Lutter, and J.E. Walker (1994) Structure at 2.8A resolution of F1- ATPase from bovine heart mitochondria, Nature 370, 621-628. R. Addison, (1986) Primary structure of the Neurospora plasma membrane H+-ATPase deduced from the gene sequence, J. Biol. Chem. 261, 14896-14901.
Page 9.5
K.B. Axelsen and M.G. Palmgren (1998) Evolution of substrate specificities in the P-type ATPase superfamily, J. Mol. Evol. 46, 84-101. P.D. Boyer, (1997) The ATP Synthase - a splendid molecular machine, Ann. Rev. Biochem. 66, 717T. Elston, H. Wang and G. Oster, G. (1998) Energy transduction in ATP synthase, Nature 391, 510-513. S.P.A. Fodor, J.B. Ames, R. Gebhard, E.M.M. van den Berg, W. Stoeckenius, J. Lugtenburg and R.A. Mathies (1988) Chromophore structure in bacteriorhodopsin's N intermediate: Implications for the proton-pumping mechanism, Biochemistry 27, 7097-7101. M. Forgac (1999) Structure and properties of the vacuolar (H+)-ATPases, J. Biol. Chem. 274, 12951-12954. K.M. Hager, S.M. Mandala, J.W. Davenport, D.W. Speicher, E.J. Benz, Jr. and C.W. Slayman (1986) Amino acid sequence of the plasma membrane ATPase of Neurospora crassa: Deduction from genomic and cDNA sequences, Proc. Natl. Acad. Sci. USA 83, 7693-7697. P. Henderson and P.N.T. Unwin (1975) Three-dimensional model for purple membrane obtained by electron microscopy, Nature 257, 28-32. E. Hilario and J.P. Gogarten (1998) The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid suunits, J. Mol. Evol. 46, 703-715. P.L. Jorgensen, J.M. Nielsen, J.H. Rasmussen, and P.A. Pedersen (1998) Structurefunction relationships of E1-E2 transitions and cation binding in Na, K-pump protein, Biochim, Biophys. Acta 1365, 65-70. P.M. Kane (1999) Vacuolar ATPases: structure, function, assembly and biosynthesis, J. Bioenerg. Biomembr. 31, 1-83. K. Kinosita, Jr., R. Yasuda, H. Noji, S. Ishiwata and M. Yoshida (1998) F1-ATPase: a rotary motor made of a single molecule, Cell 93, 21-24. K. Krab and M. Wikstrm (1987) Principles of coupling between electron transfer and proton translocation with special reference to proton-translocation mechanisms in cytochrome oxidase, Biochim. Biophys. Acta 895, 25-39. S. Lutsenko and J.H. Kaplan (1995) Organization of P-type ATPases: Significance of structural diversity, Biochemistry 34, 15607-15613. D.H. MacLennan, C.J. Brandl, B. Korczak and N.M. Green (1985) Amino-acid sequence of a Ca2++Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence, Nature 316, 696-700. D.H. MacLennan, W.J. Rice and N.M. Green (1997) The mechanism of Ca2+ transport by sarco(endo)plasmic reticulum Ca2+-ATPases, J. Biol. Chem. 272, 28815-28818. M. Mohraz, M.V. Simpson, and P.R. Smith (1987) The three-dimensional structure of the Na, K-ATPase from electron microscopy, J. Cell Biol. 105, 1-8. J.V. Moller, B. Juul and M. LeMaire (1996) Structural organization, ion transport and energy transduction of P-type ATPases, Biochim. Biophys. Acta 1286, 1-51. V. Muller, C. Ruppert and T. Lemker (1999) Structure and function of the A1A0-ATPases from methanogenic archaea, J. Bioenerg. 31, 15-27. N. Nelson and W.R. Harvey (1999) Vacuolar and plasma membrane protonadenosinetriphosphatases, Physiol. Rev. 79, 361-385.
Page 9.6
D. Njus, P.M. Kelley, and G.J. Harnadek (1986) Bioenergetics of secretory vesicles, Biochim. Biophys. Acta 853, 237-265. H. Noji, R. Yasuda, M. Yoshida and K. Kinosita, Jr. (1997) Direct observation of the rotation of F1-ATPase, Nature 386, 299-302. Y.A. Ovchinnikov, N.G. Abdulaev, M.Y. Feigina, A.V. Kiselev, and N.A. Lobanov (1979) The structural basis of the functioning of bacteriorhodopsin: An overview, FEBS Lett. 100, 219-224. M.G. Palmgren and K.B. Axelsen (1998) Evolution of P-type ATPases, Biochim. Biophys. Acta 1365, 37-45. G. Rudnick (1986) ATP-driven H+ pumping into intracellular organelles, Ann. Rev. Physiol. 48, 403-413. Y. Sambongi, Y. Iko, M. Tanabe, H. Omote, A. Iwamota-Kihara, I. Ueda, T. Yanagida, Y. Wada and M. Futai, M. (1999) Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation, Science 286, 1722-1724. B. Schulenberg, R. Aggeler, J. Murray and R.A. Capaldi (1999) The !"-c subunit interface in the ATP synthase of Escherichia coli: cross linking of the " subunits to the c subunit ring does not impair enzyme function, that of ! to c subunits leads to uncoupling, J. Biol. Chem. 274, 34233-24237. R. Serrano, M.C. Kielland-Brandt and G.R. Fink (1986) Yeast plasma membrane ATPase is essential for growth and has homology with (Na++K+), K+- and Ca2+-ATPases, Nature 319, 689-693. G.E. Shull and J.B. Lingrell (1986) Molecular cloning of the rat stomach (H++K+)-ATPase, J. Biol. Chem. 261, 16788-16791. G.E. Shull, L.K. Lane and J.B. Lingrel (1986) Amino-acid sequence of the beta-subunit of the (Na++K+)ATPase deduced from a cDNA, Nature 321, 429-431. G.E. Shull, A. Schwartz and J.B. Lingrel (1985) Amino-acid sequence of the catalytic subunit of the (Na++K+)ATPase deduced from a complementary DNA, Nature 316, 691-695. D. Stock, A.G.W. Leslie, and J.E. Walker (1999) Molecular architecture of the rotary motor in ATP synthase, Science 286, 1700-1705. C. Toyoshima, M. Nakasako, H. Nomura and N. Ogawa (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 resolution, Nature 405, 647-655. J. Weber and A.E. Senior (1997) Catalytic mechanism of F1-ATPase, Biochim. Biophys. Acta 1319, 19-58.
Page 9.7

CHAPTER 10: FACILITATED DIFFUSION
D. Njus, 2000
CHAPTER 10: FACILITATED DIFFUSION As we have discussed, some molecules and ions cross biological membranes by simple diffusion through the hydrophobic milieu and some pass through specific channels. The passage of other molecules and ions across biological membranes is facilitated by means of transporters. Except in those few cases in which the transporters draw upon chemical or electromagnetic energy to transport ions against concentration gradients, this facilitated transport is known as facilitated diffusion. Section 10.1. Ionophores Of the agents which mediate facilitated diffusion, the simplest are antibiotics known as ionophores, hydrophobic compounds which can complex an ion and carry it across a lipid bilayer. Ionophores are traditionally classified in two categories: neutral ionophores such as valinomycin and carboxylic ionophores such as nigericin. To these two categories, we should add a third: those agents that carry hydrogen ions across biological membranes. H+ carriers are commonly called uncouplers of oxidative phosphorylation (or simply uncouplers) because of their effect on mitochondrial respiration. Because their effect is more precisely to transport protons across membranes, we will refer to them here as protonophores. The protonophores are weak acids capable of permeating the lipid bilayer in their
OH NO2
N C C N N C NH OCF3
NO2 2,4-Dinitrophenol (DNP) Cl O C NH H3 C C OH CH 3 CH3 Cl NO2
Carbonylcyanide p-trifluoromethoxyphenyl hydrazone (FCCP)
N C C N N C NH
Cl
5-Chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13)
Carbonylcyanide m-chlorophenyl hydrazone (CCCP)
Figure 10.1. Structures of some protonophores
anionic form. Of course, they can also pass across the membrane in their uncharged (protonated) form, so they carry H+ across the membrane and bring H+ concentrations on Page 10.1
the two sides of the membrane to electrochemical equilibrium. In terms of structure, the protonophores are generally aromatic compounds that are both hydrophobic and capable of distributing the negative charge over a number of atoms in the molecule. Structures of some protonophores are shown in Figure 10.1. Neutral ionophores generally have a cyclic structure so they are effectively hydrophobic doughnuts capable of complexing a cation in the hole. Valinomycin, for example, is a ring composed of three units each of D-hydroxyisovalerate, L-valine, Llactate and D-valine linked by alternating peptide and ester bonds (Figure 10.2). Valinomycin complexes K+, Rb+ or Cs+, but Na+ and Li+ are too small to be effectively bound. The selectivity for K+ is 10,000 times greater than the selectivity for Na+. Valinomycin will cross the membrane either with or without a bound ion so it carries charge across the membrane. Ion transport mediated by valinomycin, therefore, depends upon the membrane potential. Moreover, valinomycin will create a membrane potential by transporting capacitative charge.
O N O N O N O O O
N O O O O N O Dicyclohexyl-18-crown-6 Enniatin B N O N O N O O O O O O O O O N O O O O O
O O O
O O O
Valinomycin
Figure 10.2. Some neutral ionophores
Unlike the neutral ionophores, the carboxylic ionophores have a linear structure with a carboxyl group on one end and one or two hydroxyls on the other (Figure 10.3). They cyclize by head-to-tail hydrogen bonding and will cross the membrane with the carboxyl group either protonated or complexed to an ion. Nigericin, for example, will cross the membrane carrying either H+ or K+. It functions, therefore, as a K+/H+ exchanger. Because nigericin does not carry a net charge across the membrane, transport is not affected by the membrane potential nor does it contribute to the creation of a membrane potential.
Page 10.2
Section 10.2. Transporters Integral membrane proteins which catalyze facilitated diffusion differ from ionophores in some important ways. First, the proteins span the membrane creating a pathway for facilitated diffusion. They do not diffuse across the membrane as do ionophores. Although transporters are sometimes called carriers, the latter term suggests a protein that binds to a substrate and moves with it through a medium, not a protein through which the substrate moves. Therefore, the term carrier may be applied to ionophores but should not be used for transporter proteins. A second important characteristic of transporters is that they are inserted into the membrane with a fixed orientation. Therefore, whereas the binding process for ionophores is the same on both sides of the membrane, transporters may be asymmetric. In particular, the binding constants observed on the two sides of the membrane may be quite different. In fact, for transporters to achieve maximum efficiency, the transporter should have a binding constant higher than the expected substrate concentration on one side of the membrane and lower than the expected concentration on the other side. This will allow the transporter to bind the substrate on one side and release it on the other. Finally, transporters customarily facilitate coupled transport of two or more substrates. That is, like nigericin, transporters facilitate either exchange diffusion of two or more substrates or codiffusion of two or more substrates. For example, the sodium/hexose transporter, commonly found in the plasma membrane of animal cells, mediates codiffusion of Na+ and a hexose molecule (glucose) into the cell (Figure 10.4). As we shall see, this coupled transport is perhaps the most important mechanism for transporting substances across biological membranes.
Page 10.3
Outside Membrane Na +
Inside
Figure 10.4. Codiffusion mediated by the sodium/hexose transporter
Section 10.3. Equilibria of Facilitated Diffusion The ionophores and transporters mediating facilitated diffusion will reach equilibrium when the free energy change for the processes they mediate is zero. For transport processes (10.1) G = i ni i = ni (iin - iout) = i ni [RT ln (Cin/Cout) + ziF m] ni is the stoichiometry for transport of substrate i and is positive if i is transported inward and negative if i is transported outward. For protonophores, which carry only H+, (10.2) G = RT ln ([H+]in/[H+]out) + F m
We obtain the equilibrium reached by protonophores by setting G = 0. Not surprisingly, this yields the Nernst equation: (10.3) m = (RT/F) ln ([H+]out/[H+]in)
For nigericin, which exchanges K+ and H+, (10.4) G = RT ln ([H+]in/[H+]out) + Fm - RT ln ([K+]in/[K+]out) - Fm By setting G = 0, we obtain
Page 10.4
(10.5)
[H+]in/[H+]out = [K+]in/[K+]out
Nigericin will reach equilibrium when the [H+] and [K+] gradients are proportional. For the sodium/hexose transporter, (10.6) G = RT ln ([H]in/[H]out) + RT ln ([Na+]in/[Na+]out) + F m
Upon setting G = 0, we obtain the equilibrium condition: (10.7) [H]in/[H]out = [Na+]out/[Na+]in exp (-F m /RT)
Section 10.4. Kinetics of Facilitated Diffusion The kinetics of facilitated diffusion may be analyzed by considering the simple system illustrated in Figure 10.5. Let us imagine that this facilitated diffusion is mediated by an ionophore so that we may make the following assumptions: 1. The rate constants for transmembrane movement of the liganded ionophore (k1) are the same in both directions. 2. The rate constants for transmembrane movement of the empty ionophore (k2) are the same in both directions. 3. The binding constants (K) on both sides of the membrane are the same. Finally, we will make the important assumption of rapid equilibrium. That is, we will assume that the rate limiting steps are the ones involving transmembrane movement and that the binding processes on the two surfaces of the membrane occur relatively quickly. This allows us to assume that the binding reactions are always at equilibrium. Consequently, (10.8) K = To Co/TCo = Ti Ci/TCi
The rate at which the total internal transporter concentration changes is (10.9) d(TCi+Ti)/dt = k1TCo + k2To - k1TCi - k2Ti
If we assume that the transporter distribution reaches a steady-state, then d(TCi+Ti)/dt = 0, so (10.10) 0 = TCo (k1 + k2K/Co) - TCi (k1 + k2K/Ci)
Multiplying both sides of equation 10.10 by KCi + 2CiCo + KCo gives (10.11) 0 = TCo[(k1Co + k2K)(Ci+K) + (K+Co)(k1Ci + k2KCi/Co)] - TCi[(k1Ci + k2K)(Co+K) + (K+Ci)(k1Co + k2KCo/Ci)] If we let T be the total amount of transporter in the membrane, then (10.12) T = TCo + To + TCi + Ti = TCo (1 + K/Co) + TCi (1 + K/Ci) Page 10.5
Multiplying both sides of equation 10.12 by k2K(Co - Ci) gives (10.13) k2KT(Co-Ci) = TCo(k2K-k2KCi/Co)(K+Co) - TCi(k2K-k2KCo/Ci)(Ci+K) Upon adding equations 10.11 and 10.13, we obtain (10.14) k2KT(Co-Ci) = (TCo-TCi)[(k1Co + k2K)(Ci+K)+(K+Co)(k1Ci+k2K)] Note that the flow of substrate J = k1 (TCo-TCi) so k1k2KT(Co-Ci) (10.15) J = (k1Co + k2K)(Ci+K) + (k1Ci + k2K)(Co+K)
This expression gives us the steady-state flux in terms of three parameters: k1T, K, and the ratio k1/k2. If we restrict our consideration to the initial velocity of transport (Ci = 0), then equation 10.15 reduces to (10.16) J = (k1k2CoT)/(k1Co + 2k2K + k2Co)
Equation 10.16 may be rewritten as (10.17) If we define (10.18) (10.19) then (10.20) 1/J = 1/Jmax + Km/JmaxCo Jmax = k1k2T/(k1 + k2) Km = 2Kk2/(k1 + k2) 1/J = (k1+k2)/k1k2T + 2K/k1CoT
Initial velocity of transport, therefore, can be described by only two parameters, Jmax and Km. As in enzyme kinetics, Jmax is the velocity at infinite substrate concentration; Km is the substrate concentration giving one-half the maximum velocity. Note that the Km measured for initial velocity of transport is equal to K, the binding constant for the substrate, only if the rate constants (k1 and k2) are about equal.
Page 10.6
Outside TC o
Membrane TC i
Inside
Co To Ti
Ci
Figure 10.5. A simple model of facilitated diffusion Section 10.5. Facilitated Diffusion Mediated by Ionophores McLaughlin and his colleagues have analyzed the kinetics of H+ transport mediated by the ionophores FCCP and CCCP (Benz and McLaughlin, 1983; Kasianowicz et al., 1984). In the absence of a membrane potential, the model illustrated in Figure 10.5 applies at least in the short term. Values for the rate constants k1 and k2 and the binding constant (expressed as pK) are given below: FCCP 10,000 sec-1 700 sec-1 6.0 CCCP 12,000 sec-1 175 sec-1 5.9
k1 k2 pK
First, note that protonation reactions occur on a time scale of 1011 sec-1 so the transmembrane rates (k1 and k2) are clearly limiting. Second, note that the rate constants for FCCP are larger than those for CCCP consistent with the more potent protonophoric activity of FCCP. The simple model shown in Figure 10.5 applies to protonophores with two restrictions. First, it applies only in the absence of a membrane potential since a membrane potential will change the rate constants for the movement of the anionic species. In fact, because the membrane potential will facilitate the movement of the anionic species in one direction and slow its movement in the other, k2 will not be the same in both directions. This introduces an additional parameter. Second, the model applies only for short time periods. Because the ionophore slowly partitions between the aqueous and membrane phases, the membrane-bound concentration may not remain constant over longer time periods. References R. Benz and S. McLaughlin (1983) The molecular mechanism of action of the proton ionophore FCCP (carbonylcyanide p-trifluoromethoxy phenylhydrazone), Biophys. J. 41, 381-398.
Page 10.7
J. Kasianowicz, R. Benz and S. McLaughlin (1984) The kinetic mechanism by which CCCP (carbonyl cyanide m-chlorophenylhydrazone) transports protons across membranes, J. Membrane Biol. 82, 179-190. R.I. Macey (1978) Mathematical models of membrane transport processes, in Membrane Physiology (T.E. Andreoli, J.F. Hoffman, and D.D. Fanestil, eds.), Plenum, New York, pp. 125-146. S.G.A. McLaughlin and J.P. Dilger (1980) Transport of protons across membranes by weak acids, Physiol. Rev. 60, 825-863. B.C. Pressman (1973) Properties of ionophores with broad range cation selectivity, Fed. Proc. 32, 1698-1703. B.C. Pressman (1976) Biological applications of ionophores, Ann. Rev. Biochem. 45, 501530. B.C. Pressman and M. Fahim (1982) Pharmacology and toxicology of the monovalent carboxylic ionophores, Ann. Rev. Pharmacol. Toxicol. 22, 465-490.
Page 10.8

CHAPTER 11: COUPLED TRANSPORT
D. Njus, 2000
CHAPTER 11: COUPLED TRANSPORT In 1961, Peter Mitchell (1961) proposed that the H+ concentration gradient drives the synthesis of ATP. In the same year, Crane (1965) showed that the sodium concentration gradient drives sugar and amino acid transport across epithelial cell plasma membranes. In the years since, it has become evident that ion concentration gradients are an important energy store in all cells. Ion pumps are therefore crucial transducers that transform chemical-bond energy into electrochemical potential energy. Coupled transport, in turn, uses the electrochemical potential of one substrate to drive the transport of a second. Section 11.1. The Master Pump Concept It is obviously possible to imagine all sorts of combinations of coupled transport. Fortunately, this picture may be greatly simplified by applying the concept of a master pump. According to this concept, each cellular membrane possesses one master pump which uses chemical energy to transport one or two inorganic ions. This pump creates transmembrane gradients in the electrochemical potential of the transported ions across the membrane in which it is located. Other ions and molecules are transported across that membrane by coupling their movement to the movement of the transported ion(s). It is evident that a given pump can store electrochemical potential energy only across the membrane in which it is located. Thus, the master pump must be the predominant active transport enzyme in the membrane. For example, the Na+/K+ ATPase, which is located in the plasma membrane of animal cells, can store energy only across the plasmalemma. Accordingly, transport of other ions and molecules across animal cell plasma membranes is likely to be coupled to Na+ or K+. Similarly, transport across bacterial cell, chloroplast thylakoid and mitochondrial inner membranes is likely to be coupled to the H+ gradients generated by the redox chains in those membranes. In Golgiderived organelles in both plant and animal cells, the master pump is the V-type H+translocating ATPase, while, in plant and fungal cell membranes, the master pump appears to be the P-type H+-translocating ATPase. A master pump must have three attributes: 1) high capacity, 2) high efficiency, and 3) low dissipation. Low dissipation is probably the reason that pumps almost exclusively transport the relatively impermeant inorganic cations. The exception is H+ which, because of its small size, is relatively permeant. The saving feature of H+ is that the rate of dissipation (leakage current) is proportional to the concentration and H+ is present at exceedingly low concentrations. Thus, because of low concentration, H+ gradients exhibit a low dissipation. High capacity is the requirement that the ion gradient involve concentrations that are relatively large compared to the concentrations of the compounds that are to be transported. Thus, if the master pump is to drive uptake of glucose and other metabolites present at millimolar concentrations, the ion gradient established by the pump should be an order of magnitude or two greater than this so the ion gradient is not greatly perturbed by the secondary transport systems. For example, the Na+ concentration outside animal cells is usually on the order of 100 mM while that inside is on the order of 10 mM. Consequently, the Na+ gradient has a high capacity and is suitable for the transport of millimolar concentrations of metabolites. By contrast, the Ca2+ concentration is on the order of 5 mM outside animal cells and 10-7 M inside. While the concentration gradient is
Page 11.1
large, the capacity of the system is low. Ca2+ concentration gradients would be greatly perturbed by the transport of millimolar concentrations of metabolites. The H+ ion is anomalous because it is normally present intracellularly at very low concentrations (10-7 M). Although this is necessary to reduce dissipation of the gradient, it might be expected to adversely affect capacity. H+, however, is unusual in that its concentration is buffered and the capacity of the system is determined not by the absolute H+ concentration but by the buffering capacity. Because the cytosol is well buffered, the capacity of the H+ gradient is in the millimolar range eventhough the H+ concentration is micromolar or less. The concept of a master pump is significant because it suggests a likely mechanism for all transport systems in a given membrane once the master pump has been identified. The master pump concept raises some interesting questions. First, why is transport coupled to a single master pump? A number of possible reasons can be imagined. Coupled transport may be more efficient than independent pumps. Ion gradients generally store smaller packets of energy than ATP. By choosing the proper coupling stoichiometry, the energy required for transport can be matched more precisely, thereby using energy more efficiently. Coupled transporters may be inherently more efficient than pumps. An indication that this may be so is provided by bacteria. Under anaerobic conditions, some cells produce ATPases for transporting amino acids. However, under aerobic conditions, the cells dispense with the amino acid pumps and transport amino acids by coupling transport to the H+ gradient. Coupling transport to a single master pump may also serve a control function. This is particularly evident in the case of sequential coupled transport (tertiary and quaternary transport). For example, the mitochondrion does not need to import citrate as a substrate for the TCA cycle if no phosphate is available for phosphorylation. Because citrate transport is coupled to the phosphate gradient, citrate transport automatically stops when phosphate is depleted. There are two reasons why a transport system might not be coupled to the master pump. First, if the transport system has a high capacity itself, it may adversely affect the ion gradients established by the master pump. Second, if the transported substrate serves a regulatory function, then it may be desirable to control its concentration separately. Exceptions to the master pump concept may exist for both of these reasons. The K+/H+ ATPase is a possible example of an exception for the reason of high capacity. The ATPase must transport large quantities of H+ into the lumen of the gut and kidney. If H+ movement were coupled to the Na+/K+ ATPase, it might radically affect the magnitude of the Na+ gradient. The Ca2+ ATPase may be an exception for the reason of independent regulation. The Ca2+ concentration serves a regulatory function in animal cells, so its concentration should not be dependent on the size of the Na+ gradient. Ion pumps may catalyze a net transport of charge. Because this increases charge separation across the membrane, it is termed electrogenic. Coupled transport may also produce a net transfer of charge (non-neutral exchanges). These transport systems are normally driven by the membrane potential, however. Therefore, these transporters tend to dissipate rather than create a membrane potential. For that reason, they should be termed electromotive or electrically dissipative rather than electrogenic. Section 11.2. Animal Cell Plasma Membrane Animal cells, which find themselves in a medium that is high in sodium, can use the sodium gradient for transport. Since no other membrane can rely on a high sodium capacity, it is not surprising that the Na+/K+ ATPase is limited to the plasma membrane of
Page 11.2
animal cells. The Na+/K+ ATPase transports both Na+ and K+ and, in principle, the K+ gradient could also be used for transport. In this regard, it is significant that the membrane potential in animal cells is usually negative inside. This electrical potential adds to the chemical potential gradient for Na+ but nearly cancels the potential gradient for K+. Thus the Na+/K+ ATPase puts the chemical energy into the Na+ gradient rather than the K+ gradient. Since K+ ions can contribute little energy for transport, it is not surprising that K+ is usually not involved in coupled transport across the plasma membrane. For example, in the squid axon, the membrane potential is -90 mV, the K+ equilibrium potential is -102 mV, and the Na+ equilibrium potential is +50 mV. A K+ ion can contribute only 12 mV of energy to transport whereas a Na+ ion can contribute +140 mV. This illustrates how the energy stored by the Na+/K+ ATPase is put mainly into the Na+ ions and not into K+.
ATP 3 Na+
Na+
+ 2K
Glucose ADP + Pi
Figure 11.1. Coupled transport across the animal cell plasma membrane. In animal cell plasma membranes, a glucose transporter uses the Na+ gradient to bring glucose into the cell. It does this by mediating the cotransport of 1 glucose molecule and 1 Na+ ion. The energetics of this coupled transport may be analyzed by applying the same logic used earlier. The total free energy change is the sum of the electrochemical potential changes of all transported species (equation 10.1): (11.1) G = i ni i = ni (iin - iout) = i ni [RT ln (Cin/Cout) + ziF m] Both glucose (G) and Na+ are transported from outside to inside, so they may both be assigned a stoichiometry of +1. Then, at equilibrium, (11.2) G = RT ln ([G]in/[G]out) + RT ln ([Na+]in/[Na+]out) + F m = 0
Consequently, the glucose concentration gradient will depend on the sodium gradient and on the membrane potential as (11.3) [G]in/[G]out = {[Na+]out/[Na+]in } exp (- F m/RT)
Page 11.3
Section 11.3. Inner Mitochondrial Membrane The electron transfer chain in the mitochondrial inner membrane uses energy derived from substrate oxidation to pump H+ out across the membrane. This gradient, of course, is used to drive the synthesis of ATP. It is also used to transport many metabolites across the inner mitochondrial membrane. ATP and ADP cross the inner mitochondrial membrane via a transporter catalyzing ATP/ADP exchange. ATP carries one more negative charge than ADP, so the exchange is electrically dissipative. The membrane potential (inside negative) generated by H+ transport, will drive ATP/ADP exchange in the direction of ATP efflux and ADP influx. (11.4) = RT/F ln { [ATP]out[ADP]in/[ATP]in[ADP]out } PO4- enters mitochondria via PO4-/OH- exchange. This is an electroneutral exchange but it will respond to the H+ concentration gradient by virtue of the inverse relationship between H+ and OH- concentrations. Consequently, PO4- will accumulate in mitochondria because of the higher internal OH- concentration. (11.5) [PO4-]in/[PO4-]out = [OH-]in/[OH-]out = [H+]out/[H+]in The electrochemical potential of one proton (H+) is (11.6) H+ = + RT/F ln {[H+]in/[H+]out} From equations 11.4 - 11.6, it should be apparent that the electrochemical potential of one proton drives the uptake of ADP and phosphate into the mitochrondrial matrix and the export of ATP from the matrix. The electrical part of H+ drives ADP uptake and ATP export, while PO4- uptake is driven by the chemical part of H+. P
i
H ++ 1/2 O2 NADH H+ NAD + + H 2O
OH ATP 4-
ADP
3-
Figure 11.2. Coupled transport across the inner mitochondrial membrane.
Page 11.4
Section 11.4. Secretory Vesicle Membranes Secretory vesicle membranes have a vacuolar ATPase that pumps H+ into the intravesicular space. Consequently, we would expect transport across secretory vesicle membranes to be coupled to the H+ gradient. Catecholamines (epinephrine, norepinephrine and dopamine) are indeed transported across the secretory vesicle membrane via a catecholamine/proton exchange. The stoichiometry of this exchange is 2 protons per protonated amine so the equilibrium catecholamine gradient is (11.7) [RNH3+]in/[RNH3+]out = {[H+]in/H+]out}2 exp (F/RT) The equilibrium gradient depends on the square of the H+ gradient because two protons are exchanged for each amine. Because the charge on the amine cancels the charge on one of the two protons, there is a net movement of only one charge in the exchange. For this reason, the catecholamine gradient depends on the membrane potential only to the first power. ATP H+ 2 H+ ADP + Pi Figure 11.3. Coupled transport across secretory vesicle membranes. Section 11.5. Some Coupled Transporters Transporter Sodium/glucose transporter Vesicular monoamine transporter (Major facilitator superfamily) Serotonin transporter Anion exchange (Band 3) protein Sodium/proton antiporter Ca2+/Na+ antiporter Mitochondrial nucleotide transporter Cation/chloride symporter Substrates Hexose/Na+ cotransport monoamine:H+ exchange Serotonin/Na+ cotransport Cl-/HCO3- exchange Na+/H+ exchange 3 Na+/Ca2+ exchange ATP4-/ADP3- exchange Na+/K+/2Cl- cotransport Inhibitors Reserpine Fluoxetine Stilbene disulfonates Amiloride Atractyloside Furosemide
+ RNH 3
For a much longer listing, go to www-biology.ucsd.edu/~msaier/transport
Page 11.5
References Bell, G.I., C.F. Burant, J. Takeda, and G.W. Gould (1993) Structure and function of mammalian facilitative sugar transporters, J. Biol. Chem. 268, 19161-19164. Crane, R.K., Miller, D., and Bihler, I. (1961) in Symposium on Membrane Transport and Metabolism (A. Kleinzeller and A. Kotyk, ed.), Academic Press, London, p. 439. Crane, R.K. (1965) Na+-dependent transport in the intestine and other animal tissues, Fed. Proc. 24, 1000-1006. Mitchell, P. (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism, Nature 191, 144-148. Schuldiner, S. (1994) A molecular glimpse of vesicular monoamine transporters, J. Neurochem. 62, 2067-2078. West, I.C. (1980) Energy coupling in secondary active transport, Biochim. Biophys. Acta 604, 91-126.
Page 11.6

CHAPTER 12: ENERGY COUPLING
D. Njus, 2000
CHAPTER 12: ENERGY COUPLING Section 12.1. Thermodynamic Efficiency Biological systems, like other machinery, often use the energy released by one process to drive another. Maximizing the efficiency of this energy coupling is obviously of great importance. The thermodynamic definition of efficiency is (12.1) = [output power]/[input power] = - [JoutputXoutput]/]JinputXinput] where J is the flow and X is the force driving that flow. Conservation of energy requires that output power not exceed input power: (12.2) JinXin - JoutXout
Consequently, 1 0. The flows are related by the stoichiometry of coupling n: (12.3) n = Jout/Jin
Combining equations 12.1 and 12.3 gives (12.4) = - n[Xout/Xin]
Maximum efficiency is attained when = 1, but this is the equilibrium condition (JinXin = JoutXout). Therefore, no net flow will be produced. Consequently, productive energy coupling with maximum efficiency requires that be close to but not equal to one, and that the coupled flows be close to but not at equilibrium. Section 12.2. Characteristics of Biological Redox Reactions Among the most important phenomena mediated by biological membranes are transport processes that are linked to redox reactions. These participate in numerous cellular phenomena including the vital bioenergetic functions of mitochondria and chloroplasts. To understand these processes, it is necessary to consider both the equilibria and the kinetics of redox reactions. Redox reactions involve the transfer of n reducing equivalents (either e- or H) from a donor to an acceptor: Donorred + Acceptorox Donorox + Acceptorred As discussed in Chapter 9 (equation 9.8), the free energy change for this reaction is (12.5) Gredox = nF Edonor - nF Eacceptor = nF (Edonor -Eacceptor) + RT ln
[Donor]ox[Acceptor]red [Donor]red [Acceptor ]ox
Page 12.1
The free energy change will be less than zero when Edonor < Eacceptor. Therefore, reducing equivalents will be transferred from carriers with lower reduction potentials to carriers with higher reduction potentials and this will generally be in the direction of increasing E. At equilibrium, Gredox = 0 and the ratio of products to reactants is equal to the equilibrium constant K12. Therefore, (12.6) K12 = exp{(Eacceptor - Edonor)(nF/RT)}
The mechanisms of biological redox reactions have significant implications, because biology uses both hydrogen atom carriers and electron carriers and exploits the difference between them. Although it is common to speak of electron transfer chains and electron transfer reactions in biology, typical outer-sphere electron transfer reactions are rare. Outer-sphere electron transfer reactions occur spontaneously at rates determined only by the relative reduction potentials of the donor and acceptor and by the intrinsic reactivity of each reactant. The Marcus theory for electron transfer reactions in solution defines the rate constant for electron transfer from compound 1 to compound 2 (k12) in terms of selfexchange rate constants for each compound (k11 and k22) and the equilibrium constant for the reaction: (12.7) k12 = {k11k22K12f12}1/2 f12 is a collision factor which may be defined as (12.8) log (f12) = (log K12)2/(4 log {k11k22/Z2}) where Z = 1011 M-1s-1. In practice, f12 is usually about equal to 1. A significant implication of the Marcus theory is that electron transfer is an intrinsically non-specific process and will occur most rapidly between the lowest-potential electron donor and the highestpotential electron acceptor. Electron transfer, therefore, is not compatible with the sequential redox reactions of a redox chain. In fact, electron carriers tend to be avoided in biological systems because their reactions are uncontrolled. Instead, biological redox reactions employ organic compounds that donate or accept hydrogen atoms. These compounds do not react spontaneously so their redox reactions can be controlled by enzymes. The enzymes that react with these organic hydrogen-atom carriers, however, are generally metalloproteins. The metal ligand will accept and donate only electrons, not hydrogen atoms. The active site of the metalloenzyme, therefore, must provide its substrate (the hydrogen-atom carrier) with a way to exchange a proton as well as an electron. We will call this phenomenon concerted proton/electron transfer. Concerted proton/electron transfer implies that biological redox reactions will naturally release or consume H+. The generation of proton gradients, therefore, is a natural consequence of the vectorial organization of these reactions. Because electron transfer reactions are avoided in biology, terminology such as electron transfer chain is misleading. Instead, we will refer to the respiratory chain in mitochondria or to redox chains.
Page 12.2
Section 12.3. The Secretory-Vesicle Ascorbic Acid-Regenerating Chain A good, simple example of a biological redox chain is the ascorbic acidregenerating system in secretory vesicles. The function of this system is to provide reducing equivalents for redox reactions occurring inside the secretory vesicles. Two reactions, those catalyzed by dopamine -monooxygenase (DM) and peptidylglycine amidating monooxygenase (PAM), are especially significant. DM converts dopamine to norepinephrine and mediates catecholamine biosynthesis in adrenal chromaffin cells, in peripheral sympathetic nerve endings and in noradrenergic neurons in the central nervous system. PAM and an associated lyase lead to amidation of the carboxyl termini of many peptide hormones including vasopressin, oxytocin, VIP, neuropeptide Y, -MSH, substance P, calcitonin and gastrin. The monooxygenases both incorporate one atom from O2 into the substrate and reduce the second oxygen atom to H2O. The reducing equivalents are provided by intravesicular ascorbic acid (vitamin C) which in turn is oxidized to the radical anion, semidehydroascorbate. The intravesicular ascorbate is recycled by importing reducing equivalents across the vesicle membrane through cytochrome b561 (Figure 12.1). Cytochrome b561, which spans the secretory-vesicle membrane, is reduced in turn by cytosolic ascorbic acid. This transport of electrons into the vesicles is driven by both the membrane potential (interior positive) and the pH gradient (interior acidic) generated by a V-type H+-translocating ATPase in the vesicle membrane. The pH-gradient favors inward electron flow because the midpoint potential of ascorbate is pH-dependent being higher at the lower internal pH. Thus, contrasting with the mitochondrial respiratory chain in which H+ is transported using energy from redox reactions, the secretory-vesicle system drives the redox reaction using energy from the proton gradient.
Figure 12.1. Mechanism of ascorbic acid regeneration in secretory vesicles. 1) semidehydroascorbate reductase; 2) cytochrome b561, 3) dopamine -monooxygenase, 4) H+-translocating ATPase.
Page 12.3
H2 COH H COH O H HO OH
H2 COH
H2 COH
pK1
O -H+ +H+
H COH O H HO O-
pK2
O -H+ +H+
H COH O H
-
O O-
AH2
E1
AH+H
+e-e-
A=
-H
+e-e-
E2
H2 COH H COH O H HO
H2 COH
pKr
O -H+ +H+ O
H COH O
. AH .
H O
. A+e-
-e-
E3
H HO H H O
KH
O O OH -H2O +H2O
H2 COH H COH O H O O
OH OH
AH2O
Figure 12.2. Interconversion of ascorbate species. Protonation reactions are shown horizontally and electron-transfer reactions are shown vertically.
Page 12.4
To understand how this system works, it is important to understand that ascorbic acid functions as a donor of single hydrogen atoms. At physiological pH, ascorbic acid exists predominantly as a monoanion (AH-). Similarly, the oxidized form occurs as a radical anion (A-) because this form is stabilized by its capacity to distribute the unpaired electron over a number of atoms. The fully oxidized form, dehydroascorbate, is not normally formed because that requires formation of a very unfavorable reaction intermediate (A). Consequently, cytochrome b561 must react with ascorbic acid and its radical anion by exchanging the equivalent of a single hydrogen atom. Because the metal ligand (Fe) will only accept an electron, there must be a mechanism to facilitate ascorbate deprotonation. This implies that cytochrome b561 reacts with ascorbate/semidehydroascorbate by concerted proton/electron transfer. Because the reaction involves only one reducing equivalent and causes no other chemical changes, it is an especially simple example of this kind of redox reaction. The distinction between concerted H+/e- transfer and e- transfer is illustrated by comparing cytochrome b561 with cytochrome c. The rate at which ascorbate reduces cytochrome c is relatively slow at neutral pH, strongly pH-dependent, and does not saturate at high ascorbate concentrations. By contrast, the rate of cytochrome b561 reduction is faster, only weakly pH dependent, and saturates at ascorbate concentrations over about 1 mM. This implies that cytochrome c is reduced by the ascorbate dianion (A=) by outer sphere electron transfer whereas cytochrome b561 is reduced by the ascorbate monoanion by concerted H+/e- transfer. A probable but unproven implication of concerted H+/e- transfer is that the substrate binding site is sufficient to make the substrate reactive. By creating a mechanism for proton transfer, the binding site:substrate complex becomes capable of engaging in electron transfer reactions. This means that metalloproteins can be thought of as two (or more) separate electron transfer centers: the metal and the bound substrate(s). The bound substrate will normally exchange electrons with the metal because that pathway is available, but the bound substrate may also react with other redox centers that may happen to be in the vicinity. Consequently electrons may occasionally go astray, for example, reducing O2 to superoxide (O2-). Cytochrome b561 reacts with ascorbate on both sides of the membrane and therefore must possess two ascorbate binding sites. The site on the cytoplasmic side probably contains a histidine residue, because histidine modification inhibits reduction of cytochrome b561 by external ascorbate. Cytochrome b561 has recently been cloned and sequenced and has a molecular weight of 30,061. Hydropathy analysis suggests that the protein has six transmembrane domains with both amino and carboxyl termini being on the cytoplasmic side of the membrane. The protein contains seven histidine residues, two of which act as ligands to the heme. The other five are candidates for the ascorbate binding sites which mediate concerted H+/e- transfer. Section 12.4. The Mitochondrial Respiratory Chain In mitochondria, a pair of reducing equivalents are transferred from NADH or succinate through the respiratory chain to molecular oxygen. Peter Mitchell championed the hypothesis that the flow of reducing equivalents down the respiratory chain resulted in the transfer of protons out across the inner mitochondrial membrane and that the proton gradient generated thereby provided the energy for ATP synthesis. To analyze the respiratory chain and the manner in which it transports H+, it is convenient to divide the
Page 12.5
chain into three segments: the NADH oxidase/CoQ reductase, CoQ oxidase/cytochrome c reductase, and cytochrome c oxidase. Let us consider each of these components in turn.
TABLE 12.1. Electron Carriers in the Mitochondrial Electron Transfer Chain Redox Pair NADPH/NADP+ NAPH/NAD+ FMNH2/FMN FADH2/FAD Fe/S centers Succinate/Fumarate Ubiquinone/Ubihydroquinone Ubiquinone/Ubisemihydroquinone Ubisemihydroquinone/Ubihydroquinone Cytochrome b566 Cytochrome b562 Cytochrome c1 Cytochrome c Cytochrome a/CuA Cytochrome a3/CuB 2 H2O/O2 n 2 2 2 2 1/center 2 2 1 1 1 1 1 1 1 1 4 E (mV) -324 -320 -219 -219 -30 to -300 -31 +9 -240 +258 -30 +30 +230 +260 +280 to +350 +260 to +900 +816
NADH oxidase/CoQ reductase oxidizes NADH and reduces coenzyme Q (ubiquinone). Thus it takes the reducing equivalents from a reduction potential of -320 mV through a series of iron-sulfur centers to a potential of about -30 mV. In the process, each pair of reducing equivalents causes 4 H+ to be transferred across the inner mitochondrial membrane. The H+/e- stoichiometry was measured in a clever experiment by Rottenberg and Gutman (1977). A proton gradient can drive a reverse flow of reducing equivalents from succinate to NAD+ if the flow of reducing equivalents down the respiratory chain is blocked. Rottenberg and Gutman inhibited the flow of reducing equivalents through cytochrome oxidase using cyanide, and used ATP to generate a proton gradient via the F1Fo ATPase. The free energy derived from ATP hydrolysis is (12.9) GATP = GATP + 1.38 log {[ADP][Pi]/[ATP]} The free energy required to drive reverse redox flow is (12.10) Gox = Gox + 1.38 log {[NADH][fumarate]/[NAD+][succinate]} If the two processes are allowed to come to equilibrium, Gox + n GATP = 0, where n is the stoichiometry of coupling. By measuring the equilibrium concentrations of the various reactants, Rottenberg and Gutman could calculate Gox and GATP and then determine n. They found a stoichiometry of 4/3. Since the F1Fo ATPase transports 3H+/ATP, this segment of the respiratory chain must transport 4 H+ per pair of reducing equivalents. Page 12.6
The CoQ oxidase/cytochrome c reductase segment of the respiratory chain transports H+ across the inner mitochondrial membrane through a process that Peter Mitchell termed the Q Cycle. This mechanism is diagrammed in Figure 12.3. It depends upon two reactions occurring on opposite sides of the inner mitochondrial membrane. On the matrix side, ubiquinone is reduced by two reducing equivalents, one taken from cytochrome b562 and the other taken from an iron sulfur center: Q + cyt b562 red +FeS red + 2 H+ QH2 + cyt b562 ox + FeS ox The reduced quinone is uncharged and hydrophobic and will diffuse to the other side of the membrane where it reduces cytochrome c1 and cytochrome b566: QH2 + cyt c1 ox + cyt b566 ox Q + cyt c1 red + cyt b566 red + 2 H+
Intermembrane Space Q 2 H+ Cyt b566 Cyt b562 Q
FeS
Matrix
2 H+
QH 2
QH 2
Cyt c 1 Cyt c
Figure 12.3. The Q Cycle
Page 12.7
OH CH3O CH3O OH CH3 R
pK1
-H+ +H+ CH3O CH3O
OCH3 R OH
pK2
-H
+
OCH3O CH3O OCH3 R
+H+
QH2
E1
QH+e-e-
Q=
+e-e-
E2
O CH3O CH3O OH
pKr
CH3 R -H+ +H
+
O CH3O CH3O
. QH .
CH3 R
. Q+e-e-
E3
O CH3O CH3O O CH3 R
Q
Figure 12.4. Interconversion of quinone species. Protonation reactions are shown horizontally and electron-transfer reactions are shown vertically.
Page 12.8
This results in the net transfer of 2 H+ across the membrane. Because one of the two reducing equivalents is recycled by cytochromes b562 and b566, a pair of reducing equivalents passing completely through the Q cycle will cause 4 H+ to be pumped out across the mitochondrial inner membrane. Note that the quinone is a carrier of two hydrogen atoms and would be expected to react with the metalloproteins on both sides of the membrane by concerted proton/electron transfer. The requirement for a specific active site for each reaction is indicated by the fact that the reactions are blocked by different compounds; myxothiazol inhibits quinone oxidation on the outside and antimycin inhibits quinone reduction on the inside.
Figure 12.5. Dioxygen reactions of cytochrome oxidase. Cytochrome oxidase is the final segment in the mitochondrial respiratory chain. It is linked to cytochrome c1 by cytochrome c, a soluble protein found in the space between the inner and outer mitochrondrial membranes. Because cytochrome c is soluble, it introduces an experimentally accessible break in the respiratory chain and isolates the cytochrome oxidase segment. Cytochrome oxidase is a multicenter protein with two heme groups and two copper atoms. Heme a and CuA are located on the cytosolic side of the membrane and act as the primary acceptors of electrons from cytochrome c. Heme a3 and CuB lie on the matrix side of the membrane and function in the four-equivalent reaction by Page 12.9
which O2 is reduced to 2 H2O. Cytochrome oxidase catalyzes a series of electron transfer reactions and only the reduction of O2 involves H+. Consequently, the cytochrome oxidase redox reactions do not naturally lead to formation of a proton gradient. Instead, cytochrome oxidase actually functions as a proton pump. Redox energy is captured and used to transport H+ across the inner mitochondrial membrane. H+ transport is believed to be linked specifically to the flow of electrons through the heme a/CuA region of cytochrome oxidase. Having now considered the entire mitochondrial respiratory chain, it is possible to assess the theoretical energy yield of oxidative phosphorylation. Assuming that the ATP synthase has a stoichiometry of 3 H+/ATP and that 1 H+/ATP is consumed for transport of ATP, ADP, and inorganic phosphate, the following stoichiometries hold: Segment of electron transfer chain NADH/succinate Succinate/cytochrome c Cytochrome c/O2 H+/2e4 4 2-4 P/2e1.0 1.0 0.5-1.0
The passage of two reducing equivalents down the entire mitochondrial respiratory chain will cause 1 atom of O to be reduced to H2O. The theoretical P/O ratio (molecules of ATP formed per atom of O reduced) is 2.5 - 3.0. According to long-held dogma, the theoretical P/O ratio is 3 but this was established before it was recognized that the ratio did not have to be an integer. The observed P/O ratio is typically between 2 and 3. The observed P/O ratio is expected to be less than the theoretical value because of H+ leakage or poor coupling. The degree of coupling is indicated by the phenomenon of respiratory control. In the absence of ADP, mitochondria consume O2 at a slow but measurable rate. This so-called state 4 rate is the rate at which reducing equivalents may be transferred down the respiratory chain in the absence of ATP synthesis. Presumably, this redox flow generates a large H+ and the energy needed to pump H+ against this substantial gradient slows the flow of reducing equivalents. Another way to look at this is to recognize that, in the steady state, electron flow may pump protons only as fast as the protons come back across the membrane. When ADP is added, the rate of O2 consumption increases dramatically. This so-called state 3 rate is faster because H+ is dissipated by ATP synthesis. Thus, electron flow may pump protons as fast as H+ are consumed by the ATP synthase (F1Fo ATPase). The respiratory control ratio is the ratio of the rate of O2 consumption in state 3 to the rate of O2 consumption in state 4. Generally this ratio lies between 3 and 15. A high ratio indicates tight coupling between electron flow and ATP synthesis. A low ratio signifies poor coupling. Complete uncoupling (for example, by adding an uncoupler) will accelerate state 4 respiration to the rate of state 3 respiration and will cause the respiratory control ratio to drop to 1. Section 12.5. The Redox Chain of Photosynthetic Bacteria The redox chain in purple photosynthetic bacteria is interesting for a number of reasons. First, it combines features of the mitochondrial respiratory chain and of photosystem II of the photosynthetic redox chain. As such, it provides some insight into the evolutionary origins of biological redox chains. Second, the reaction center of Rhodopseudomonas has been crystallized and its three-dimensional structure has been Page 12.10
solved. It was one of the first membrane structures for which this was accomplished, and for it Diesenhofer and Michel shared the 1988 Nobel Prize. The redox chain incorporates a reaction center which takes reducing equivalents from cytochrome c2 (a soluble protein), invests energy from light, and then reduces ubiquinone. Reducing equivalents from ubiquinone return to cytochrome c2 by passing through b and c cytochromes. In the process, protons are pumped across the membrane. The proton gradient can be used to make ATP as in mitochondria or chloroplasts. Alternatively, it can be used to drive reverse electron flow from succinate to NADH. Cytochrome c2 can pass reducing equivalents to the reaction center or to a cytochrome oxidase which reduces O2. Thus, the redox chain can create a proton gradient by a cyclic photosynthetic pathway in the light or it can create a proton gradient by oxidizing succinate or NADH aerobically in the dark. The photosynthetic reaction center incorporates four molecules of bacteriochlorophyll, two of pheophytin and two of ubiquinone. The structure has two fold symmetry. A bacteriochlorophyll dimer near the outer surface of the membrane donates an electron upon absorbing light. The electron passes crosses the membrane via a bacteriochlorophyll and pheophytin molecule on one side of the reaction center. The pigment molecules on the other side apparently do not function in the photosynthetic process. The electron finally passes to a quinone on the inside of the membrane. This quinone (QA) is reduced to the semiquinone but apparently does not become fully reduced. QA passes the electron to QB, accepts a second electron from the reaction center, and passes it to QB to form the fully reduced dihydroquinone. The dihydroquinone (QH2B) then dissociates from the reaction center and is replaced by an oxidized quinone from the quinone pool. The quinone reduction results in proton uptake and the nature of the proton uptake mechanism has received some attention. Note that the oxidized quinone Q may be reduced to the semiquinone by electron transfer, because the radical species have a pK near neutrality so both Q- and QH. are formed readily. The second reduction step must form QH2 because the pK for ionization of the species is greater than 12. Consequently, we might imagine that QH2 will form by reduction of QH. by concerted proton/electron transfer.
Page 12.11
Rhodopseudomonas Redox Chain Q = Ubiquinone BChl = bacteriochlorophyll Y
+ NAD Succinate
Q Cyt b
Cyt c 1 Cyt c 2
BChl
Cyt a/a 3 1/2 O 2
Mitochondrial Respiratory Chain NADH FeS Q Cyt b Cyt c 1 Cyt c Cyt a/a 3 1/2 O 2 Photosynthetic Redox Chain PQ = plastoquinone PC = plastocyanin Fd = ferredoxin PQ Cyt b559 h H 2O P 680 Photosystem II Cyt f PC P700 Photosystem I h Succinate
X Fd NADP+
Page 12.12
Section 12.6 The Photosynthetic Redox Chain The thylakoid membranes in chloroplasts accomplish the following so-called light reactions of photosynthesis: H2O + NADP+ + h 1/2 O2 + NADPH + H+ 2 ADP + 2 Pi 2 ATP The free energy of NADP+ reduction is about 40 kcal/mole and that of ATP synthesis is about 10 kcal/mole. The energy obtainable from a single photon of red light is about 40 kcal/mole. The efficiency of energy conversion in photosynthesis is about 40%, but this means that the energy from four photons of light are needed to drive one pair of reducing equivalents through the redox chain. Each electron must receive the energy from two photons and this is accomplished by placing two photosystems in the redox chain. The light energy is collected by antenna pigments in the chloroplast and transferred to the reaction centers. To make photosynthesis proceed efficiently, it is important that light be distributed evenly between the two photosystems so that they run at the same speed. Plants accomplish this by using the redox state of the quinone pool to regulate the distribution of light energy between the two photosystems. If the quinone pool is too reduced, then PS I is operating too slowly and more light needs to be diverted to it. Conversely, if the quinone pool is too oxidized, then more light must be shunted to PS II. This is accomplished by phosphorylation control of light harvesting protein (Allen et al., 1981). If the quinone pool is reduced, a kinase is activated increasing phosphorylation of the LHCP. This directs more of the light energy to PS I. If the quinone pool becomes too oxidized, the kinase is inactivated and the LHCP is dephosphorylated by a phosphatase. A greater fraction of the absorbed quanta are then shunted to PS II. Photosystem II is quite similar to the reaction center of bacterial photosynthesis in Rhodopseudomonas. Consequently, the solution of the structure of the bacterial reaction center has also illuminated the mechanism of photosystem II.
References J.F. Allen, J. Bennett, K.E. Steinback and C.J. Arntzen (1981) Chloroplast protein phosphorylation couples plastoquinone redox state to distribution of excitation energy between photosystems, Nature 291, 25-29. P. Brzezinski (1996) Internal electron-transfer reactions in cytochrome c oxidase, Biochemistry 35, 5611-5615. S.I. Chan and P.M. Li (1990) Cytochrome c oxidase: Understanding Nature's design of a proton pump, Biochemistry 29, 1-12. K. Krab and M. Wikstrom (1987) Principles of coupling between electron transfer and proton translocation with special reference to proton translocation mechanisms in cytochrome oxidase, Biochim. Biophys. Acta 895, 25-39. R.A. Marcus and N. Sutin (1985) Electron transfers in chemistry and biology, Biochim. Biophys. Acta 811, 265-322. H. Michel and J. Deisenhofer (1988) Relevance of the photosynthetic reaction center from purple bacteria to the structure of photosystem II, Biochemistry 27, 1-7.
Page 12.13
P. Mitchell (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism, Nature 191, 144-148. P. Mitchell (1975) Protonmotive redox mechanism of the cytochrome b-c1 complex in the respiratory chain: Protonmotive ubiquinone cycle, FEBS Lett. 56, 1-6. P. Mitchell (1976) Possible molecular mechanisms of the protonmotive function of cytochrome systems, J. Theoret. Biol. 62, 327-367. P. Mitchell (1979) Keilin's respiratory chain concept and its chemiosmotic consequences, Science 206, 1148-1159. D. Njus and P.M. Kelley (1993) The secretory-vesicle ascorbate-regenerating system: A chain of concerted H+/e- transfer reactions, Biochim. Biophys. Acta 1144, 235-248. M.Y. Okamura and G. Feher (1992) Proton transfer in reaction centers from photosynthetic bacteria, Ann. Rev. Biochem. 61, 861-896. H. Rottenberg and M. Gutman (1977) Control of the rate of reverse electron transport in submitochondrial particles by the free energy, Biochemistry 16, 3220-3227. B.L. Trumpower and R.B. Gennis (1994) Energy transduction by cytochrome complexes in mitochondrial and bacterial respiration: The enzymology of coupling electron transfer reactions to transmembrane proton translocation, Ann. Rev. Biochem. 63, 675-716. I.C. West, P. Mitchell and P.R. Rich (1988) Electron conduction between b cytochromes of the mitochondrial respiratory chain in the presence of antimycin plus myxothiazol, Biochim. Biophys. Acta 933, 35-41. J.R. Winkler, B.G. Malmstrom, and H.B. Gray (1995) Rapid electron injection into multisite metalloproteins: intramolecular electron transfer in cytochrome oxidase, Biophys. Chem. 54, 199-209.
Page 12.14

CHAPTER 13: EPITHELIAL TRANSPORT
D. Njus, 2000
CHAPTER 13: EPITHELIAL TRANSPORT
Section 13.1. Topology and General Principles of Epithelia Epithelia, such as the lining of the gastrointestinal tract and the nephrons of the kidney, are layers of cells across which ions and metabolites must be transported. Transport across epithelial layers follows a common pattern with different functions achieved using different combinations of transporters. It is possible, therefore, to consider a general mechanism and to establish conventions for defining the various parts and parameters of the system. The epithelium separates two compartments: the serosal side (blood) and the mucosal side (lumen). The epithelial cells are polarized, so membranes with different characteristics face each side. The basolateral membrane faces the serosal side and the apical (or brush border) membrane faces the mucosal side. The Na+/K+ ATPase is found in the basolateral membrane but not in the apical membrane. Usually, the basolateral membrane is permeable to K+ (it has a barium-sensitive K+ channel) but not to Na+. The apical membrane, by contrast, is permeable to Na+ but not to K+. The K+ current creates a membrane potential (negative inside) across the basolateral membrane. The inward Na+ current dissipates the membrane potential across the apical membrane, so the potential on the serosal side is generally positive relative to the mucosal side. This transepithelial potential (tep)drives current through the paracellular space. The transepithelial potential is a measure of the tightness of the epithelium. Tight epithelia have a greater resistance and therefore can have a greater transepithelial potential. Examples are the frog skin, urinary bladder, colon and distal nephron. Leaky epithelia have a lesser resistance and a smaller transepithelial potential. Epithelia of the small intestine and gall bladder are examples. Many kinds of transporters are found in epithelial cells, but those involved in transport of the principal ions (Na+, K+, Cl-, H+, Ca2+) are common and relatively few in number. A listing of these and their diagnostic inhibitors follows. Table 13.1. Common Ion Transport Activities in Cells Transporter Na+/K+ ATPase K+ channel Na+/Ca2+ exchanger Na+/H+ exchanger Na+/K+/2Cl- cotransporter Cl-/HCO3- exchanger Cl- channel Membrane Basolateral Either Basolateral Apical Either Apical Apical Inhibitors Ouabain Barium Amiloride Furosemide, Bumetanide SITS, DIDS
Section 13.2. Analysis of a Tight Epithelium The frog skin is the classical example of a tight epithelium. It transports Na+ inward across the skin. This is achieved by an apical membrane that is permeable only to Na+ and a basolateral membrane that is permeable only to K+. To analyze the electrical properties of this system, we define the lumen as the reference potential. The intracellular potential api is then the
1
Page 13.1
membrane potential across the apical membrane. The potential on the serosal side is the transepithelial potential (tep). The membrane potential across the basolateral membrane is then (13.1) baso = api - tep Serosal (Blood) I baso K+ ATP 2 K + I api ADP + Pi Mucosal (Lumen)
3 Na
Na +
I para Basolateral Membrane Figure 13.1. Frog skin The apical membrane is permeable to Na+ but not K+, so the current across the apical membrane is (13.2) Iapi = (api - Na)/RNa Na is the Na+ equilibrium potential across the apical membrane, and RNa is the resistance to the Na+ current across the apical membrane. The basolateral membrane is permeable to K+ but not Na+, so the current across the basolateral membrane is (13.3) Ibaso = - (baso - K)/RK = - (api - tep - K)/RK K is the basolateral K+ equilibrium potential and RK is the basolateral resistance to the K+ current. The current through the paracellular space is
2
tep
api
=0 Apical Membrane
Page 13.2
(13.4) Ipara = tep/Rpara At steady state, these currents must all be equal, so (13.5) Iapi = Ibaso = - Ipara Upon solving this series of equations, it is apparent that (13.6) api = Na - (Na - K)RNa/(Rpara+RNa+RK) (13.7) tep = (Na - K)Rpara/(Rpara+RNa+RK) This shows that api lies between the apical Na+ equilibrium potential (Na) and the basolateral K+ equilibrium potential (K). If RNa = 0, then Na+ will be in equilibrium across the apical membrane and api = Na. At the other extreme, if RK = Rpara = 0, then K+ will be in equilibrium across the basolateral membrane, and baso = K. The transepithelial potential will be zero, so api = baso = K. The transepithelial potential (tep) is a little smaller than the difference between the apical + Na and basolateral K+ equilibrium potentials (Na - K). Section 13.3. GI Epithelial Transport The small intestine functions to transfer nutrients and other important metabolites into the blood. The intestinal epithelium is leaky, so the transepithelial potential is small. This maximizes the membrane potential across the apical membrane (the brush border membrane) and thereby optimizes the energy that Na+ ions can contribute to coupled transport across that membrane. As an example of transport across the epithelium of the small intestine, hexoses (such as glucose) are carried across the apical membrane into the epithelial cells by Na+-linked transporters. Glucose transport across the basolateral membrane occurs by facilitated transport and is driven only by the sugar's concentration gradient. Note that a Na+-linked transporter would not function to transport glucose out of the cell into the blood because the Na+ gradient opposes transport in that direction.
Page 13.3
Serosal (Blood) K+ ATP 2 K +
Mucosal (Lumen)
Hexose
3 Na+
ADP + Pi Hexose
Na +
+10 mV Basolateral Membrane
-40 mV
0 mV Apical Membrane
Figure 13.2. Sugar transport in the small intestine The oxyntic cells of the gastric mucosa are responsible for secreting HCl to acidify gastric juice in the stomach. Because gastric juice is extremely acidic, H+ and Cl- are moved against large concentration gradients, and this requires considerable energy. Estimating the H+ and Clconcentrations in gastric juice and blood as shown below, the transepithelial for H+ and Clmay be calculated: H+ ClSerosal Concentration 3.4 x 10-8 M (pH 7.4) 50 mM Mucosal Concentration 150 mM 150 mM 8.4 kcal/equiv 1.1 kcal/equiv
The energy for H+ and Cl- transport is satisfied by a K+/H+ ATPase located in the apical membrane. This enzyme actively pumps H+ against the large H+ concentration gradient across the apical membrane. The concomitant K+ influx neutralizes charge movement. The K+ gradient is then used to drive Cl- into the lumen via an electroneutral K+/Cl- cotransport. Cl- influx across the basolateral membrane is driven by the Na+ gradient generated by the Na+/K+ ATPase. Na+ influx is coupled to Cl- influx via parallel transporters: a Cl-/HCO3- exchanger and a Na+/H+ exchanger.
Page 13.4
Serosal (Blood) K+ ATP 2 K + K

+
Mucosal (Lumen) ATP
ADP + Pi 3 Na+ ADP + Pi Na +
H Cl -
Cl -
H2 O
Cl -
K+
CO 2
HCO3 Basolateral Membrane Apical Membrane
Figure 13.3. Gastric mucosa
Section 13.4. Renal Epithelial Transport In the kidney, the proximal tubule is responsible for reabsorption of nutrients and other important metabolites. Consequently, like the small intestine, the renal proximal tubule is a leaky epithelium. This maximizes the energy stored in the Na+ gradient across the apical membrane and optimizes the efficiency of Na+-linked uptake of amino acids, sugars, phosphate and sulfate. The proximal tubule also functions in the reabsorption of Na+ and Cl-. This is accomplished by a Na+/H+ exchanger and an anion exchanger in the apical membrane. Acidification of the lumen by Na+/H+ exchange facilitates reabsorption of HCO3- as CO2. Na+ and Cl- uptake causes H2O to be reabsorbed by osmosis. Amiloride, which blocks the Na+/H+ exchanger, inhibits reabsorption of Na+ and HCO3-. It also blocks the concomitant uptake of water and consequently functions as a diuretic.
Page 13.5
Serosal (Blood) K+ Cl -
Mucosal (Lumen)
ATP 2 K +
HCO 3 H+ H 2O Na
+
3 Na+
ADP + Pi
CO 2
Basolateral Membrane Figure 13.4. Renal proximal tubule Serosal (Blood) Cl ATP 2 K +
Apical Membrane
Mucosal (Lumen) K+ Na + K+ 2 Cl -
3 Na+
ADP + Pi
Basolateral Membrane
Apical Membrane
Figure 13.5. Thick ascending limb - Loop of Henle In the thick ascending limb of the loop of Henle, NaCl reabsorption must be achieved without loss of water by osmosis. Na+ and Cl- are taken up from the lumen by the Na+/K+/2ClPage 13.6
6
cotransporter which is driven by the Na+ and Cl- gradients. The Na+ concentration in the epithelial cells is kept low by the Na+/K+ ATPase. The Cl- concentration is kept low by a Clchannel in the basolateral membrane. Cl- efflux through this channel is driven by the membrane potential. A K+ channel is located in the apical membrane rather than in the basolateral membrane. This reduces the K+ gradient across the apical membrane and prevents it from interfering with the proper functioning of the cotransporter. The K+ channel can also cause the transepithelial potential to be positive on the lumen side. This arrangement results in the active transport of NaCl across the epithelium and can make the urine hypotonic relative to the blood. The loop diuretic furosemide (Lasix) inhibits NaCl reabsorption by blocking the Na+/K+/2Clcotransporter. Because it blocks K+ reuptake from the lumen, furosemide leads to K+ loss. Amiloride, which blocks water reabsorption by acting on Na+/H+ exchange, does not do this and is known as a K+-sparing diuretic.
Section 13.5. Airway Epithelia In airways, mucus production in the lumen depends on H2O secretion by the epithelium. H2O secretion occurs by osmosis consequent to Cl- secretion. Cl- secretion occurs via a Na+/K+/2Cl- cotransporter in the basolateral membrane and a Cl- channel in the apical membrane. Serosal (Blood) K+ ATP 2 K + Mucosal (Lumen)
3 Na +
ADP + Pi K+ 2 Cl Na +
Cl -
Basolateral Membrane Figure 13.6. Airway Epithelium
Apical Membrane
The Na+/K+ ATPase drives Cl- influx across the basolateral membrane, and Cl- flux across the apical membrane then occurs passively. The symptoms of cystic fibrosis are caused by a defect
7
Page 13.7
in regulation of the Cl- channel resulting in inadequate H2O secretion making secretions characteristically salty and too viscous. Airway epithelia and the epithelia in the nephrons of the kidney are structured in similar ways, yet airway epithelia function to secrete Cl- while the epithelia in the kidney take up Cl-. This illustrates how the general structure of an epithelium can be adapted to achieve different functions simply by incorporating appropriate combinations of transporters in the apical or basolateral membranes.
References W.B. Reeves and T.E. Andreoli (1992) Renal epithelial chloride channels, Ann. Rev. Physiol. 54, 29J.R. Riordan (1993) The cystic fibrosis transmembrane conductance regulator, Ann. Rev. Physiol. 55, 609-630. B. Thorens (1993) Facilitated glucose transporters in epithelial cells, Ann. Rev. Physiol. 55, 591608. E.J. Weinman and S. Shenolikar, (1993) Regulation of the renal brush border membrane Na+exchanger, Ann. Rev. Physiol. 55, 289-304. M.J. Welsh (1987) Electrolyte transport by airway epithelia, Physiol. Rev. 67,1143-1184. M.J. Welsh, M.P. Anderson, D.P. Rich, H.A. Berger, G.M. Denning, L.S. Ostedgaard, D.N. Sheppard, S.H. Cheng, R.J. Gregory and A.E. Smith, (1992) Cystic fibrosis transmembrane conductance regulator: A chloride channel with novel regulation, Neuron 8, 821-829. C.S. Wingo and B.D. Cain (1993) The renal H-K-ATPase: Physiological significance and role in potassium homeostasis, Ann. Rev. Physiol. 55,323-347. E.M. Wright (1993) The intestinal Na+/glucose cotransporter, Ann. Rev. Physiol. 55, 575-589.
Page 13.8

Membrane Biophysics

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Membrane Biophysics

Uploaded by

Copyright:

Available Formats

Fundamental Principles of Membrane Biophysics

David Njus Department of Biological Sciences Wayne State University

kilo centi milli micro nano pico femto atto

A = 2rh A = 4r2 V = r2h V = (4/3) r3

Fundamental Principles of Membrane Biophysics

David Njus Department of Biological Sciences Wayne State University

CHAPTER 1: BIOLOGICAL MEMBRANES

Fundamental Principles of Membrane Biophysics

David Njus Department of Biological Sciences Wayne State University

CHAPTER 2: THERMODYNAMICS OF MICELLE FORMATION

Alternatively, equation 2.11 can be solved for ln Xm (2.13) ln Xm = -(m/RT)(mic,m - w) + m ln Xw + m ln fw + ln m

0 -2 -4 RT ln cmc -6 -8 50 larger micelles Um - w 75 A min 2 smaller micelles 100 U mic,m - w

W m (low ionic strength) 0 Wm (high ionic strength) U mic,m - w

Lauryl Sulfate (Dodecyl sulfate)

Cetyltrimethylammonium (CTAB) CH 3 H3C N+ CH 3 CH 2 (CH2 )14 CH 3

Cholic Acid: X = OH Deoxycholic Acid: X = H CH 3 OH CH 3

X Fig 2.6. Some ionic detergents

Triton X-100 (Octylphenoxypolyoxyethanol)

(CH2 -CH2 -O)10

Lubrol W HO (CH2 -CH2 -O)7 (CH2 )15 CH3

Fig. 2.7. Some nonionic detergents

Fundamental Principles of Membrane Biophysics

David Njus Department of Biological Sciences Wayne State University

CHAPTER 3: THE FLUID MOSAIC MEMBRANE

Phosphat idylet hanolamine (PE) NH3 + CH2 CH2

Phosphat idic A cid (PA ) OHO P O O

Fundamental Principles of Membrane Biophysics

David Njus Department of Biological Sciences Wayne State University

CHAPTER 4: MEMBRANE ELECTROSTATICS

0 Single plane of charge Parallel plate capacitor

Figure 4.1. Electric fields associated with planes of charge

dipole potential (outside) surface potential (outside) dipole potential (inside)

potential created by capacitative charge

membrane potential surface potential (inside)

Figure 4.2. Components of the membrane potential

We define () = 0, so (4.19) qs = o (do/dx)

2 = (F2/RTo) i Cio zi2

Fundamental Principles of Membrane Biophysics

David Njus Department of Biological Sciences Wayne State University

CHAPTER 5: SPECIFIC AND NON-SPECIFIC BINDING

Lmax Lmax Lm Lm Lmax 2 [L] K

At equilibrium, G = 0 and [LM]/([L][M]) = 1/K. Therefore, (5.8) G = RT ln K

Fundamental Principles of Membrane Biophysics

David Njus Department of Biological Sciences Wayne State University

CHAPTER 6: PERMEABILITY AND CONDUCTANCE

Figure 6.1. Transition-state energy for membrane permeation.

Figure 6.2. Permeation of a weak base.

For a non-electrolyte, d/dx = d(RT ln C)/dx, so (6.13) J = (-C/Nf)(RT/C)(dC/dx) = -(RT/Nf)(dC/dx)

Fundamental Principles of Membrane Biophysics

David Njus Department of Biological Sciences Wayne State University

CHAPTER 7: PERMEABILITY AND CONDUCTANCE OF ELECTROLYTES

where Q is defined as (7.10) Q = d/0 exp (zF/RT) dx Membrane Inside C mi Ci C mo Co o

exp (zF /RT) - 1

zF m /RT exp (zF m /RT) - 1

Ohm's Law Fick's Law

Ohm's Law Fick's Law

Figure 7.2. Regions of validity of the permeation and conductance equations

cations P jC oj + anions P jC ij cations P jC ij + anions P C oj j

cations P jC oj + anions P jC ij cations P jC ij + anions P C oj j

This is the Goldman-Hodgkin-Katz equation.

Fundamental Principles of Membrane Biophysics

David Njus Department of Biological Sciences Wayne State University

-conotoxin GVIA -agatoxin IVA Bungarotoxin

zF m /RT exp (z Fm /RT) - 1