Membrane Structure

The Fluid Mosaic Model:

- The plasma membrane is primarily composed of 2 components: lipids and
proteins.
o Contributes to its fluidity. The phospholipids contribute fluidity to the
membrane. Protein structures attribute to the membrane.
o Lipids also play a large role in signalling. They are important in the
fluidity of the membrane, but also the function.
- Lipids give the membrane its basic “fluid” structure, and provide a relatively
impermeable barrier for the cell.
o A membrane can’t be permeable to everything. The cell is very
exclusive, it only lets in certain molecules and ions.
o Example, water and gases cross easily. There are transporters that are
specific of transporting water, in the absence of protons.
o Membrane is selectively permeable.
o Once we start adding receptors, we can transport other ions and
substances.
o By way of this complex of proteins this allows certain agents access to
the cell.
- Proteins give the membrane its mosaic characteristics, and mediate nearly all
other functions of the membrane.
o Membranes have fluidity. This is very important because proteins
must undergo conformational changes. For example during apoptosis,
Bac proteins must aggregate for phosphorylation to occur.
 They dimerize and the come together via ligand binding.
 When they are brought close together there is this auto-
phosphorylation of a kinase that activates other kinases.
o Membrane fluidity is very important for receptor proteins to move
and change their form. If a protein changes its structure it will change
its function. Fore example phosphorylation.
o The Mosaic characteristic of the membrane is caused by proteins. A
complement of these proteins are specific to the cell type. It could be
related to other cells in the tissue.

Phospholipids:
- Most abundant lipids in the membrane bilayer
o There are many different types of proteins.
- 1 polar head, 2 hydrocarbon tails
- Amphipathic
o They have polar and non polar regions.
o Hydrophilic head, points out towards the aqueous environment.
o Hydrophobic tails, they arrange on the inside of the bi-layer
o This nature gives the membrane its structure and behaviour.
 - Presence of unsaturated double bond procedures “kink” and influences
fluidity of the membrane.
o Many phospholipids have these kinks in the hydrocarbon chain.
o The kink is an unsaturated hydrocarbon. This allows for some more
space between adjacent lipids.
o More space= more allowance for fluidity.
- There chemical structure helps determine the fluidity of the membrane.
The Fluid Bilayer
- Phospholipid molecules are quite mobile within their monolayer
- Lateral diffusion occurs rapidly (107 s-1)
o Mobility of phospholipids in the membrane.
o Later diffusion= the lateral movement of the phospholipid
o It occurs easily and constantly.
- Translocation from one layer to the other is rare and requires ATP hydrolysis.
o Flip-Flop movement occurs rarely.
o This is an energetic process.
o Necessary for the maintenance of asymmetry.
- Necessary for maintenance of asymmetric bilayer.
o We may find specific phospholipids expressed on the inner leaflet
compared to the outer leaflet.
o The asymmetric property is important in cell signalling. There are
some phospholipids involved in signalling. These phospholipids have
to be present on the inner leaflet. If they are expressed on the wrong
side they will not be able to perform their function.
o Example during apoptosis, phosphatidylserine is usually on the inner
leaflet. It will become exposed to the outside, and macrophages will
come and perform phagocytosis.
- How is this asymmetry formed?
o Phospholipids are inserted into the membrane in the ER and brought
to the Golgi.
o They go through the secretory pathway the same way as proteins do.
- Pathway Diagram:
o We have red polar heads facing the cytosol, and yellow
polar heads facing the cell exterior.
o Along comes a patch of membrane. This path of
membrane was delivered the plasma membrane by a
vesicle that comes out of the Golgi apparatus.
o SNARE complexes have formed, fusion has occurred,
and all the proteins and lipids are dropped off into the
membrane.
o Insertion into the membrane. But not necessarily in
their final orientation.
o There is an enzyme called flipase that mediates the
flip-flop.
  It takes these phospholipids so that they can maintain their
asymmetry.
o We end up with all the red phospholipids on one side and all the
yellow phospholipids on the other.
o There are some cells that perform specific functions, that express
certain factors in the cytoplasm, where phospholipids in the
membrane must work together, so they have to be properly oriented.
- When these proteins and lipids arrive at the plasma membrane they are
ordered properly.
- Therefore this idea of membrane asymmetry is very important.

The Fluid Bilayer

- Several types of phospholipids found in eukaryotic cells:
o Phosphatidylcholine (i.e. lecithin)
o Phosphatidylethanolamine (minor, mostly in mitochondria)
o Phosphatidylserine (-) (e.g. phagocytosis of apoptotic cells)
o Shingomyelin (e.g. lipid rafts)
o Inositol phospholipids (e.g. cell signalling)
- Asymmetry
- Cholesterol helps manage deformability and fluidity of membranes.
o Maintains fluidity and the structure of the membrane at certain
temperatures.
o It is also involved in plasticity.
o They reside between phospholipid molecules and they interact with
hydrocarbon chain.
 They help maintain deformability and fluidity.
o Allows membrane to function at a variety of temperatures and allow
for conformational changes.
- Interacts with hydrocarbon chain of phospholipid.

Lipid Rafts
- The idea of lipid rafts comes largely from artificial bilayers
- Sphingolipids and cholesterol form highly ordered microdomains or rafts.
- Sphingolipid hydrocarbon tails are longer and saturate (i.e. not kinked)
o The lipids are longer, and there will be slight greater thickness
because of the nature of the hydrocarbon tails.
o Cholesterol is expressed: helps to maintain fluidity and deformability.
o The centering of the hydrocarbon tails allow for these phospholipids
to interact with each other.
o The thicker membranes can accommodate for large inter-membrane
proteins.
 Very large, and specialized to be inserted into the lipid rafts.
This is important in a signalling mechanism.
- Rafts are produced in the ER and sent to the plasma membrane.
 o It is brought through the secretory pathway to the plasma membrane.
- Rafts can accommodate proteins with long transmembrane domains
o The rafts sail along the secretory pathway as a functional
microdomain.
o Some where along the line they are inserted earlier in the secretory
pathway so that the specific trans-membrane proteins with other
lipids so that they can be in the same domain and function in cell
signalling.
o The entire lipid raft will be delivered to the plasma membrane.
- Organize and cluster proteins to function together (e.g. for receptor-mediates
signalling)
- GPI-anchored proteins may mediate cell adhesion, or may be enzymes
(GPI=glycosyl phosphatidyl inositol).
- Glycolipids (lipids+sugar) are important for interactions with cell
surroundings, and cell-cell adhesion (when bound to lectins)
o Important for the interaction with the extracellular environment.
o Cytosolic is always cytosolic.

Roles of Phospholipids in Cell Signalling

- Example A: A proximal signalling system sets everything off. There is an
extracellular signal (ex. survival factor) that
binds to an enzyme linked receptor. Fluidity
of the membrane allows for the two dimers to
move closer together and cross
phosphorylate.
o PI 3-kinase (phosphatidylinositol 3-
kinase) is a protein kinase that
phosphorylates inositol
phospholipids.
  PI3 kinase phosphorylates the phospholipid. It will become
phosphorylate in the presence of ex. a growth factor.
o Phosphorylation of polar head group leads to recruitment of
intracellular signalling proteins.
 A phospholipid becomes phosphorylated. Once it becomes
phosphorylated it can recruit protein kinases to broadcast the
signals.
 Once phosphorylated at the polar head region this recruits
other proteins (kinases) that are going to broadcast the signal.
 Example Protein Kinase B and TOR.
o Signal transduction and the role of a nearby phospholipids in sending
this signal further on so it can have some effect on the cell.

- Example B:
o Phospholipase C cleaves inositol phospholipid to produce 2 fragments
(PIP21P3 + DAG).
 It cuts the phospholipid, cleaving the
polar head and leaving the hydrocarbon
tail.
 It is also being phosphorylate so there
will be an event cleavage.
 An extracellular signal activates the
receptor that produces 2 fragments.
o Polar head (IP3) may release Ca2+ from ER
 IP3 becomes soluble in the cytosol.
 IP3 is important for binding to receptors in the ER membrane
to release Ca.
 IP3 leaves the membrane and becomes an important
messenger to release calcium from stores (ER, mito, outside)
o Tail (DAG) remains in membrane and may activate PKC.
 DAG is what is left after IP3 is cleaved away.
 DAG remains and may activated kinase C.
- The same inositol phospholipid involved in these two cell signalling systems.
It is important for physiology and not just the structure.

3 Types of Membrane Proteins

- Integral proteins: ion channels, transporters, receptors
o Have alpha helices.
o They are integrated into the membrane. They are stabilized and have
been folded such that they are stable and they will be maintain in the
membrane.
- Peripheral proteins: Spectrin, ankyrin
 o They are associated with proteins that are integrated into the
membrane.
o Stabilized by their association.
- Lipid-anchored proteins: GPI, prenyl or fatty acid-anchored.
o GPI is attached to the polar head group of a phospholipid molecule.
o The last two categories have in common that they are both proteins
that are indirectly associated with the membrane.

Recall: Many Membrane Proteins Originate in the ER

- Recall insertion of polypeptide chains into the ER membrane (i.e. single,
double, multi-pass).
- Integral proteins carried in membrane material through constitutive
secretory pathway.
- Cytosolic end of protein will always be cytosolic (i.e. at ER or plasma
membrane)
o The ER lumen is always topologically equivalent to the outside surface
of the cell.

Integral Membrane Proteins

- Association with the Lipid Bilayer
o There is going to be some modification in their secondary structure.
- Single alpha helix: single pass polypeptide chain.
o They can be modified as an alpha helix.
- Double ad multiple a helices: Composed of double or multi-pass polypeptide
chains.
- B-barrel (a rolled up B sheet)
o Utilized alpha helices that are formed in more of a sheet.
o Very common in mitochondrial membranes.
- What is the reason for this secondary structure of transmembrane regions?
o Because of H-bonding interactions between hydrophobic or
hydrophilic amino acids, we can get different secondary structures.
o Think about the role of the secondary structure. Secondary structure
is very important for their stability in the membranes.
A Primer on Proteins
- Structural Organization
- Primary structure: amino acid sequence
- Secondary structure: formation of polypeptide chains into an alpha helix or
B-sheet
- Tertiary structure: a fully folded protein.
- Quaternary structure: more than one polypeptide chain (folded as a protein)
involved.
- A polypeptide back bone is hydrophilic
o The sequence of amino acids is being formed in the cytosol. The
backbone must be hydrophilic.
- The lipid rich environment inside the plasma membrane is hydrophobic
o If we are going to insert proteins inside we have to do something
about the secondary structure of the proteins.
- Formation of secondary structure creates H-bonding between N-H and C=O
groups, thus forming a shield of nonpolar amino acid side chains to protect
the polypeptide from the hydrophobic environment.
o It orients the amino acid residues outward. It protects the hydrophilic
backbone from the interior of the membrane.
- Such H-bonding is maximized in alpha helices and B sheets.
o H-bonds are actually weak interactions. They are strong enough to
have an effect on the secondary structure of proteins.
o As the proteins are produced, they are translated in such a way that
allows for the regions to accumulate bonding interactions.
o Allow for the natural folding of the proteins.

Alpha Helix:
- Amino acid side chains are oriented outward from the backbone.
- Amino acids orient outward because they are better of at interacting with the
hydrophobic environment and it maintains a hydrophilic region inside.
- H-bonding leads to a twisting of the sequence and will lead to the formation
of the alpha helix.
- Important for the stability of the protein.
B-sheet:
- Amino acids are going to help stabilize the protein in the hydrophobic medium.

Alpha Helices and Beta Sheets Compared

- Alpha helices:
o Secondary structures of most transmembrane proteins of the
eukaryotic plasma membrane.
o Flexible structure allows conformational changes.
 And provide stability.
o E.g. ion channels.
 Can also help create aqueous pores to allow for the movement
of ions through the membranes.
- Beta Sheets:
o Secondary structure of many transmembrane proteins of
mitochondria and bacterial membranes.
o Relatively rigid structure.
 Undergo few conformational changes. They don’t have the
same flexibility as alpha helices.
o E.g. porins, transporters.
 Aquaporins that transport water. B-barrels are important.

Peripheral Proteins
- E.g. found in red blood cells where they are bound to the inner surface of the
membrane.
o Not in the membrane. But they interact with integral membrane
proteins.
- Form a meshwork with the cytoskeletal proteins (actin) to provide elasticity
and support (e.g. squeezing through a capillary).
o Eg. A red blood cell that need to pass through tiny regions.
- Spectrin is a cross-linking peripheral protein and is the major structural
component of this meshwork in RBCs
- Ankyrin and band 4.1 are also peripheral proteins that bind to
transmembrane proteins and mediate spectrin biding.
o Help form the meshwork in red blood cells.
- Figure: There is a phosphorylated head. The peripheral membrane proteins
spectrin, ankyrin and band 4.1. There is also actin involved. This an example
of who the peripheral proteins are helpful in the structure of the membrane.
Lipid Anchored Proteins
- 2 types
- 1) GPI anchored proteins
- 2) Proteins attached to lipids by fatty acid or prenyl groups.

GPI-Anchored Protein
- In the ER after co-translational translocation is complete, the signal sequence
is cleaved away, exposing the free C-terminus of the protein.
- The C-terminus is re-attached to a preassembled GPI molecule.
o A GPI molecules is going to take over binding of that protein.
o It binds to the C-terminus of the protein that was just translocated.
- This tethered protein will end up on the extracellular side
o It is associated to the membrane through the GPI and the
phospholipids.
o If the protein is in the lumen, it ends up on the extracellular side.
- Cell adhesion.

Fatty Acid and Prenyl-Attached Proteins

- These proteins are directed to the plasma membrane from the cytosol.
- Attached only to cytosolic monolayer
 - Bound by covalent attachment (fatty acid or prenyl
group)
o All the same class of protein associated with
the membrane.
- Such proteins will remain intracellular
o Proteins that are not attached to the
membrane at the ER.
o Protein finds a way to associate indirectly to
the membrane.
- Prenylated proteins involved in control of cell
growth and division (e.g. Ras protein GTPase).
o Important in cell signalling.
o Example G-proteins are tethered to the membrane in this way.

Things to Consider:
- Though lipids primarily provide structure and fluidity to the membrane
bilayer, they also play functional roles.
- Be familiar with the different classes of membrane proteins.
 Transport and Ion Channels I

Transport Across and Impermeable Barrier

- Transport of ions and small molecules is important for homeostasis and
function of the cell.
o Mechanisms that contribute to maintaining the cell in its normal state.
 May involved maintaining pH, or level of O2, Ca, CO2
o If any of these factors are dis-regulated then the cell begins to loose its
equilibrium.
o There are a number of small ions and molecules (ex. glucose) that are
important for cellular homeostasis.
- Multi-pass transmembrane proteins were inserted into the membrane back
in the ER, and sorted by the constitutive secretory pathway.
o Many transporters and ion channels have certain domains.
o Multi-pass for example 6 times. If we have a 4 subunits complex each
with the domain crossing the membrane 6 times than we have a total
of 24 times that the complex protein is passing the membrane.
- These proteins act as either transporters or channels.
- Create hydrophilic and microenvironments.
o The inside of the bilayer is very hydrophobic. Proteins are stabilized
by creating alpha helices and B-barrels. How do soluble ions cross the
membrane?
- They provide a continuous pathway across the plasma membrane to allow
solutes to cross the lipid bilayer without coming into contact with the
hydrophobic environment.
o Proteins form aqueous paths through the membrane. The protein
complexes form a pore in the middle that cross all the way across the
membrane.
o Inside the pore is a hydrophilic environment. The nature of the
protein complex is organized so that the ions are fooled into thinking
that they are still in an aqueous medium.
- Within these complexes are hydrophilic microenvironments. It is water
loving but not necessarily aqueous. Microenvironments produce a continuous
path across the membrane so solutes can cross.

First, A Few General Principles

- Relative permeability of molecules
o O2 is a gas so it diffuses quite readily across the membrane.
o Charge ions do not cross the membrane easily. They have very low
permeability coefficients. Since they are charged, they have difficulties
crossing the membrane.
o A variety of ions are important in the functioning of the cells.
- How are these charged ions transported across the membrane?
- Concentration gradients vs. electrochemical gradients.
 o We have to take into account the charge of the ions as well as the
membrane potential.
o Cells are not neutrally charged, they are very negatively charged.
o mV: We measure membrane potential in terms of mV.
o Many cells have a resting potential of -70mV.
o If we have a separation of charge, we can determine which direction
the ions will flow.
o Electrochemical gradient: we have an electrical and a chemical
gradient.
- Gradients store potential energy.
o Changes in voltage are actually potential energy.
o Very important because this is going to explain the energy that drives
Na ions through the Na channels.
o Energy is stored in the gradients. What stores this energy are active
transporters and passive transporters, which allow for the flow of
ions.

Ion Concentrations.
- How are these concentration differences maintained and what is their
purpose?
Component Intracellular Extracellular
Concentration mM Concentration mM
Na 5-15 145
K 140 5
Mg 0.5 1-2
Ca 10 -4
1-2
H 7x10-5 (pH 7.2) 4x10-5 (pH 7.4)
Cl 5-15 110
- Because H concentration we end up with differences in pH.
o Cells are more acidic inside then they are outside.
- The Transporters, the pumps, utilize ATP, or not,

Mechanisms of Transport
- Simple Diffusion (e.g. gases, water)
o Have a high permeability.
o They are non-charged, non-polar, and get across the membrane easily.
 - Passive Transport (driven by a gradient and facilitated by membrane
proteins, e.g. ions, water)
o Channel mediated transports. Example Na and Ca channels.
o Then energy stored in gradients allow for the movement of ions.
- Active Transport (primary and secondary), involves membrane proteins and
ATP or ion gradient, e.g. ions, glucose.
o Involves utilization of ATP.
o Energy that is transduced in the mitochondria is then transferred to
the cytosol as ATP.
o ATP is used to power these pumps.
o Move ions against its electrochemical gradient.
o Secondary transport involves an indirect utilization of ATP. It might
rely on a gradient of sodium ions.
 Secondary because it was ATP that put the Na there in the first
place.
o Primary transport ex is a sodium-potassium pump.

Carrier Mediated Transport

- Generally, a carrier proteins has one or more specific binding site
o They are protein complexes that alternate between 2 different states.
- 2 conformational states occur, and the process is reversible
o Some sort of conformational change occurs when bound by the
substrate that is to be transported.
o Specific for certain ions.
- Net movement of transported solute is dependent on concentration (or
electrochemical) gradient.
o The solutes gradient dictates that it is going to move in a certain
direction.
Active Transport
- Primary active transport: ATP driven pumps, energy from ATP hydrolysis
o We have an electrochemical gradient. The solute or the particle being
pumped opposes the electrochemical gradient.
o This occurs because of utilization of ATP.
- Secondary Active Transport: coupled carriers; energy for transport derived
from gradient of solute (e.g. Na+), indirectly from ATP hydrolysis.
o There could be an addition gradient that is powering the movement of
the ions.
o The two together bind. They yellow is being transported.
o Can be divided into symport or antiport.
o The transported molecule could be moving in the opposite direction
as the energy source.

Na+ Glucose Symport

 - Binding of Na+ in state A promotes binding of glucose.
o Both molecules are moving across the membrane,
but the one that is being transported is the glucose.
o Glucose is being transported across the membrane
against its concentration gradient.
o It does so because of co-transport with Na+. Na+ is
powering this transport because Na has energy.
 The driving source of Na+ was built up by
primary transporters that pump Na out of
the cell.
o State A: The system is open to the outside.
- Binding of both Na+ and glucose induces conformational
change in membrane protein to state B
o Binding of Na promotes binding of glucose.
o Once all the binding sites are tied up, this induces a conformational
change.
- Na+ and glucose are released to the cytosol.
o Dissociation of the particles into the cytosol.
- Na+ gradient drives glucose transport.
o This is how the transported glucose is delivered.
o The energy behind the Na gradient is driving glucose transport.
o Na and glucose move in the same direction.
o Therefore this is a symport mechanism.
- E.g. intestinal and kidney epithelial cells where absorption is important.

H+ Exchangers Regulate Intracellular pH

- Ion gradients (e.g. Na+) can be used to transport H+
o Transport mechanisms mostly drive the transport of H ions.
o Utilization of existing electrochemical gradients to allow the exchange
for protons.
- These exchangers are electroneutral, yet pH is regulated
o pH is regulated within a very tight window.
o Electroneutral: there is an exchange of ions but there is no change in
membrane potential. There are more negative charges inside the cell,
so this creates a separation of charge leading to membrane potential.
 Charges balance each other out.
 We are not changing the membrane potential
o If ions are moving in and out of the cell the charge is becoming more
negative or positive.
- Important for many biochemical reactions.
o This helps cells regulate pH. The inside of the cell should be more
acidic then the intercellular space.
- Loss of a bicarbonate ion at the same time helps to regulate pH.
 o If we wanted to inhibit one of these exchangers we could reduce the
concentration of extracellular sodium. It would then shut down the
extrusion of protons.
o The ions that power these transporters are important in this exchange
mechanism.

Transport ATPases (Pumps)

Class of ATPase Solute Location Function
P-type ATPase Na+ and K+ Plasma membrane Pumps Na out and
K into the cytosol
Ca Plasma membrane Pumps Ca out of
cell
Ca SR Pumps Ca into SR
V-type ATPase H Membrane of Pumps H into
secretory vesicles vesicle lumen
F-type ATPase H Mito IM H gradient drives
ATP synthesis
ABC transports Many (ions, sugars, Plasma membrane Nutrient uptake
amino acids, protein export
proteins
- Summary of the different types of transporters.
- The transporters all have inherent enzymatic activity. They are protein
complexes that hydrolyse ATP.
- They allow transport of solutes against their electrochemical gradients.

Na+/K+ ATPase
- P (phosphorylation)-type ATPase
- Function dependent on phosphorylation of intracellular aspartic acid residue
and ATP hydrolysis.
 o Important for conformational changes that
help to run the system.
- Establishes Na+ and K+ gradients in almost all
animal cells.
o Pumps 3 Na out and 2 K in. There is always
this 3:2 ratio in this type of transport.
o Both move against their respective
electrochemical gradients.
o This is what creates that storage of energy.
- Uses up to 2/3 cells ATP.
o We are utilizing ATP and pumping ions against their electrochemical
gradients.
o Uses a lot of the cells ATP supply.
o Neurons readily use Na ions to underlie action potentials.
o Na/K pumps store the energy that will then power the flow of Na ions
through channels that create propagation of potentials.
o Any cell that is metabolically active uses up a lot of the cells energy for
building up these gradients.
- Net flux-electrogenic.
o Tribute to a change in membrane potential of the cell
- Model: Na+/K+ ATPase
- Step 1: Binding of 3 Na ions to the open transport complex on the cytosolic
side.
o When the complex is opened to the intracellular side of the
membrane, Na will bind.
o ATP is the hydrolysed
- Step 2: The phosphate will phosphorylate the aspartic acid residue.
o Phosphate associates with the intracellular side of the complex.
o Conformational change occurs.
- Step 3: Conformational change occurred. Release of 3 Na ions to the outside
of the cell.
o The transport is now open to the outside of the cell.
o What occurs then is an exchange. Na is lost, but K binds.
- Step 4: 2 K ions bind to the transport.
o Binding of K to its binding site induces a loss of the phosphate.
o Lead to the opening of the transport on the cytosolic side of the
membrane
- Step 5: The extracellular side is closed. The cytosolic side is opened. 2 K is
released to the cytosol.
o The cycle can then repeat.
- It is really a cycle of the two conformational changes. By repeating stages of
hydrolysis and phosphorylation, dephosphorylation and conformational
change and specific binding at certain times it can allow for the dissociation
of ions into an environment against its concentration gradient.
 - It is the energy from ATP that is moving the transport between the two
conformational states.

Ca2+ ATPase
- P-type
o Phosphorylation type mechanism.
- External [Ca2+] = 0.001M, Cytosolic [Ca2+]= 10-7M
o Cytosolic Ca must be kept at really low levels.
o It doesn’t take much for there to be a vast change in electrochemical
gradient.
- Thus, there is a large Ca2+ gradient between extracellular space and cytosol
and between cytosol and ER
o The concentration differences are built up by Ca ATPases
o Very important cells.
- 2 mechanisms by which this is achieved is by a Ca2+ pump in the plasma and
ER membrane.

SR Ca2+ ATPase
- Ca2+ ATPase of the sarcoplasmic reticulum.
o The calcium binding domain is the membrane spanning segment.
o It will bind and release calcium.
 o There will be a conformational change such that Ca will get inside this
complex.
o Another movement of the complex will allow Ca to be dissociated.
- Contains 10 alpha helices
- 3 of these line a channel that spans membrane
o They allow for binding of Ca ions which are soluble in water.
- 2 believed to interact directly with Ca2+
- 6 major steps of pumping Ca2+ from cytosol to SR.
- Module:
o The transporter contains different domains. All of them are important
because it allows for a number of different conformational changes.

SR Ca2+ ATPase Steps: 6 Different steps are needed to pump calcium in.
- 1. 2 Ca2+ bind to alpha helices on cytosolic side of transmembrane protein
and induce conformational change on nucleotide-biding and activator
domains.
o Bind to the cytosolic side of the protein.
o Two large structures close in. Conformational change after Ca ion bind
o Note: Protons are also transported. Important in the mechanism.
o Still not open to the lumen of the ER.
o Hydrolysis of ATP.
- 2. ATP bound to nucleotide binding domain and phosphorylation of aspartic
acid close access to cytosol.
o ATP bound is hydrolysed.
o This closes access to the cytosol. Must close before it can re-open, or
else it will become an open pore.
 Ca would leak out.
- 3. Exchange of ADP for ATP induces major conformational change of cytosolic
domains and transmembrane helices.
o ADP is lost and ATP is gained.
o Another conformational change occurs.
o The ntd binding domain and the activator domain now separate
- 4. Ca2+ ions dissociate and are released into SR. Entry of 3 H+
 o We’ve had this conformational change.
o We have opening to the lumen.
o They are being released to an area already with a higher concentration
of calcium
- 5. Lumen side of transporter is closed
- 6. Dephosphorylation of aspartic acid resets transporter.
o So it is a cycle
o Loss of a phosphate and we return to the open state on the cytosolic
side.
- P-type transporters can involve the movement of H ions.

H+ ATPases
- E.g. F-type
o Runs in a direction that produces ATP.
- Located in the inner mitochondria membrane
o The proton gradient allows it to produce ATP instead of utilize it.
o There are more protons in the intermembrane space
- Phosphorylation not required.
- These are also called ATP synthases because they often run in reverse to
synthesize ATP
o The produce ATP if going in the forward direction.
- H+ gradient generated during oxidative phosphorylation powers the turbine
like F0F1 ATPase
Mitochondrial F0F1 ATPase
- Resembles a turbine like structure with a rotor, which spins around.
- The electron transport chain in-oxidases phosphorylation
o There is a pass of electrons and a shuttling of protons into the
intermembrane space.
o This creates a gradient.
o The movement of H ions down their electrochemical gradient back
into the matrix of the mitochondria powers the circular motion of the
rotor.
o The motion of the rotor against the stator that generates forces
generates ATP.
- This process can be reversed experimentally.
- F0: Is the transmembrane carrier. This is the channel aspect.
- F1: Is the ATPase. This is the enzyme component.

V-types vs F-type ATPases

- V-Type ATPases resemble in some ways the mitochondrial F-type but they
utilize ATP. They do not generate it.
o Used in the membrane of secretory vesicles.
o Pump protons in to acidify the space.
- F-Type: For every 3-4 protons that pass through, one molecule of ATP is
produced.
o This translates to 100 molecules of ATP per second.
o There are many F-type ATPases, so mitochondria actually produce
way more ATP than 100.
o Take protons and puts them in the intermembrane space.
- They generally have similar structures.

ATP-binding Cassettes
- ATP binding cassettes are transporters
o They transport small molecules by utilizing ATP.
- 2 domains of 6 transmembrane helices from the structure through which
small molecules pass through the membrane.
o Each domain has 6 transmembrane helices.
o In total there is 12 transmembrane segments,
 - 2 intracellular domains for ATP binding
- ATP binding causes dimerization of these domains; hydrolysis induces
release
o The transport is not open to the outside of the cell until ATP binds.
o It will produce a dimerization of the two domains.
 Brings the two cassettes together.
- Dimerization induces conformational changes to allow for transport.
o Binding energy that changes the conformation outside the cell.
o There is a reaction on the inside of the cell. The change in
conformation is translated to the outer part of the transport facing the
extracellular space.
o This allows dissociation of the soluble unit.
o Hydrolysis of ATP will allow it to return to its normal conformation.
- Export amino acids, sugars, inorganic ions, and proteins.

Aquaporins:
- Not all transporters transport solutes
- A group of transmembrane proteins called aquaporins were discovered that
selectively transport H2O
o They are a special type of transport that transports water.
o They selectively transport water.
- Initially discovered in red blood cells, these allow passive movement of water
into or out of cells.
- Also physiologically important in kidney function, and have been implicated
in kidney disease.
o Important in water transport.
- Transmembrane protein with several hydrophilic alpha helices
o Helps for stability and create hydrophilic pores.
- 4 identical subunits, each with a central water channel.
- Structure:
o Water molecules for example are moving downward. Water will go
down its concentration gradient.
o Asparagine residues inside the pores of the channels that change the
alignment of water molecules as they move through.
o It changes the way H-bonding occurs.
  Instead of allowing water molecules to H-bond with each other,
water will form H-bonds with the amino acid residues.
- Water molecules traverse membrane in single file through hydrophilic
channels.
o They allow water molecules to pass to a point where they exclude H
ions.
o They can transfer water without changes in pH.
o Will occur in the selectivity region of the channel, were single file is
seen.
o The Asparagine residues re-orient water molecules in such a way to
prevent H-bonding between waters.
- Selectivity excludes even H+
o They way that H ions move is that they hop from one water molecule
to another. Temporarily forms H3O+
o If H-bonding between water occurs, we will get transport of water and
H ions from these interactions.
o The amino acid residues prevent H ions from moving.
- (+) Charged residues attract O atoms and prevent H-bonds from forming
between waters.
o When H2O molecules arrive at these residues is they become re-
oriented so they can’t form H-bonds.
o H ions will not be able to hop through.
o Selectively excludes protons.
- H+ cannot jump between water molecules and are excluded.

Things to Consider
- Be familiar with the physiological roles that each transporter can perform
- As well, you should know the energy source behind each transporter (e.g.
ATP, phosphorylation, concentration gradient, electrochemical gradient)
-
 Transporters and Ion Channels II

Ion Channels and Transporters Compared

- Like some transporters, ion channels allow for the passage of ions down their
electrochemical gradient via a passive mechanism (ATP not directly required)
o They allow for the passage of ions.
o We have to consider the charge of the particle and the charge across
the membrane of the cell
- However, many ion channels exhibit high selectivity for specific ions at very
high rates.
o Shows the mechanism of selectivity.
o How ion channels can selectively allow single species of ions through
the hydrophilic pore.
 Not all channels have this selectivity.
- 10 -10 ions s-1 for channels; 103 ions s-1 for transporters
7 8

o The transporters are much slower than the ion channels.

o Involves 2 different states.
 Conformational changes, where one state is open to the cytosol
and another where it is open to the lumen or the extracellular
space.
o Ion channels have a much faster mechanism in terms of the number of
ions they transport per second.
o The movement is approximately equal to the movement of ions in
solution.
o How are ion channels able to be so selective but still allow ions to
move through them at a rapid pace?
 We began to understand once we had the crystal structure of
the ion channels.
- Ion channels exhibit a hydrophilic pore
o Important for the selectivity and this high rate of transfer.
o The channels trick the ions into believing they are still in aqueous
medium.
- In addition, ion channels may be gated.
o There are many different ways an ion channel can be gated.
o The way that channels are turned on or off.

Types of Ion Channels

- There are many types of ion channels, too many to mention
- Generally, they can be divided into groups based on the ion that they conduct,
or allow to pass through their pore
o We divide them based on the ions that they transport.
- Most common are:
o K+ channels: regulate membrane potential, AP recovery
 Selective for potassium
  Responsible for setting the resting membrane potential.
 K repolarizes the membrane and lead to recovery following an
action potential.
o Na+ channels; underlie action potentials
 Found in neurons in specific regions along the axon.
 We find Na channels involved in saltatory conduction and the
propagation of action potentials along the axon.
 Allow for the influx of Na. Na depolarizes the membrane.
o Ca2+ channels; increase cytosolic [Ca2+] and induce secretion
 Ca important in muscle contraction and secretion
 Channels important in controlling the amount of Ca in the cell,
and turning off and on certain mechanisms that require Ca.
o Cl- channels; regulate ionic balance
o H+ channels; rapidly increase intracellular pH
 Exchange other ions for protons
 Allow for the movement of H depending on their
electrochemical gradient.
o Non-selective ion channels; many functions.
 Ex. The nicotinic receptor. They are ligand-gated channels that
have conducting pores.
 They are non selective; selective in the charge but allow for the
movement of Na and K.
 Some pores in the membrane don’t care what they conduct.
 It has to deal with the size of the pore and whether or
not there is a selectivity filter.
 Selective Filter: a sequence of amino acids that are part
of the primary structure of the protein that helps to
selectively regulate species of ions moving through the
channel.
- These can be opened or closed.
o What controls opening and closing of these ion channels?
o It all comes down to conformational changes.
o We can have one signal open a channel or the presence of a ligand to
bind or rely on a change in membrane potential

Some General Properties

- Ion channel pore is lined by polar regions of the protein and is hydrophilic.
o Alpha helices are formed to exploit H-bonding interactions.
o There are hydrophilic parts that face the inside of the pore, which
helps to create a hydrophilic environment.
 An environment that is conducive to an ion, which is soluble in
water.
- Hydrophobic amino acid sire chains interact with the membrane bilayer
 o Amino acid residues are faced towards the outside of the protein to
interact with hydrophobic environments in the membrane.
- Opening of an ion channel is produced by a conformational change in the
protein structure.
o The conformation change must occur. This is gating.
o In Na channels, there is a C-terminus and 1 domain of a protein that is
inserted into the membrane that functions as a gate or a little plug.
 This can move in and out of the pore.
o There are parts of the protein complexes that act as gates.
o They move in and out of position of a pore.
o Looking at large conformational changes.
o There is a whole series of chain reactions of conformational changes.
 Something binds to an extracellular part of a receptor leading
to a conformational change.
 Translated to another part of the protein.
 Makes its way down to the gate, which moves and allows or
closes access of the ions through the pores.
o Some sort of conformational change that allows the passage of ions or
particles through the ion channels.

Gating of Ion Channels

- Voltage-gated channels; opening occurs following a change in membrane
potential (i.e. depolarization or hyperpolarization). E.g. K+, Na+ and Ca2+
channels.
o Change the potential across the membrane and we can open or close
ion channels.
o There are amino acid residues, which respond to the very subtle
changes in membrane potential.
o Sensitive to changes in voltage, which allows for the movement of
ions.
- Ligand-gated (extracellular): opening occurs because of binding of an
extracellular ligand (NTs such as glutamate, Ach). E.g. glutamate or Ach
receptors.
o Relies on an extracellular ligand.
o Glutamate is the most important NT in the C.N.S
 o Receptors that receive NTs being release from pre-synaptic cells that
bind to the receptors on the post-synaptic cell.
o Ligand binding in the extracellular space.
o Leads to the opening of channels, and the passage of ions or signals.
- Ligand-gated (intracellular): channel gating is regulated by binding of and
intracellular factor (such as ATP, cAMP). E.g. K+ channels, cyclic nucleotide
gated (CNG) channels.
o Intracellular ligand.
o Ion channels binding to intracellular messengers.
o ATP, cAMP are ntds that can bind to the cytosolic side receptors.
o Lead to the movement of ions through the ion channel pore.
- Mechanically gated: a mechanical stress or force opens the channel. E.g.
cation channels in stereocilia of hair cells.
o The cells that are found in the inner ear
o Sensitive to forces on the head and sound.
o The movement of the membranes specializations can force open ion
channels.

K+ Channels
- K+ channels are highly conserved in prokaryotic and eukaryotic cells and
represent and excellent model for studying ion channels.
o Doesn’t mean that the whole channel is the same.
 There are some portions of the ion channels that codes for how
these channels are selective for K ions.
o It is conserved among species.
- The first structure of an ion channel was the KcsA K+ channel described from
the prokaryotic bacterium, Stremptomyces lividans
- Illuminated the basic structure and potential function of an ion channel (e.g.
ion selectively, rate of conduction).
o Helps us to see how ion channels are so selective yet maintain a high
rate of conduction.
o Rate of conduction is really close to rate of ion moving in solution.
 - Since K+ channels are well described physiologically and at the molecular
level, we will discuss these.
o Important in resting membrane potential.
o

KcsA K+ Channel
- Composed of 4 subunits (2 shown)
o 4 identical components.
- Each subunit has 2 transmembrane helices. M2 controls gating
o Open when the appropriate signal is received.
- And 1 pore-forming segment composed of a helix and a non-helical loop that
forms the selectivity filter.
o So there are 4 copies of this in total.
o A loop is a string of amino acids that connect the different parts of the
protein.
o We can take this protein and stretch it out, so we would have a helix,
loop, helix, all these subunits are connected together.
o Pore forming part of the structure.
o There will be a loop from each of the four subunits that will come
together and form a selectivity filter.
- K Ions
o K ions are hydrated with water before and after they enter the
channel.
o While they are inside the selectivity filter they are interacting with the
channel and are not hydrated.
o O=C, atoms forms bridges of stability along the selectivity filter.
o 4 structures that are contributing to the C=0 in the selectivity filter.
 Important for understanding of K ions interact with the filter.
 They create 4 regions of stability.
o There can be no more than 2 K ions bound at a time occupying the
selectivity filter.
o The forces of 2 like charged ions of the same size is too great so we
can’t put more than 2 K ions in the filter at any given time.
o This keeps the line of K ions moving.
o If there is a strong electrochemical gradient to push the K ions out of
the cell.
 There is energy moving K ions from the inside of the cell to the
outside through the filter.
 The like charges repelling each other will keep things moving
along inside the selectivity filter.
o There must actually be K ions inside the filter, or else the structure of
the filter will collapse.
 o If we remove K ions from the system and we use X-ray crystallography
to see the structure, the channel is collapsed.
o In the absence of the conducting ion the selectivity filter collapses.
 This is important because it is about the transfer of energy.
 The structure of the filter is dependent on the weak interaction
of K ions with the structure for it to maintain its state.
o There is energy imparted as K ions move through.
o This is used to make up the very structure of the channel.

The Selectivity Filter

- Structure of KcsA channel reveals high selectivity for K+
- Other ions (e.g. Na+) are excluded.
o They are excluded because they are too small.
o 1 in 1000 ions that make its way through the channel are Na.
 Very low rate of transfer of Na ions through k selective ion
channels.
- Basically, the lining of the pore contains 5 rings having a diameter of 3A (a K+
is 2.7A, while a Na+ is 1.9A) that are lined with O atoms.
o If we look down the barrel of the channel we would see subunits.
o Each subunit contributes an O atom.
 This will occur at different levels.
o K ion interacts with 4 O atoms from the subunits.
 Region of stability for K ions.
o K ions fit perfectly in the filter. It is a perfect fit.
o Na ions are smaller; they don’t interact comfortably along the rings.
 There is too much energy required to make this happen.
 Thermodynamically unfavourable.
- As the K+ ions enters the pore, it loses its hydration shell and interacts with O
atoms all the way through.
o K ions are hydrated before entering the filter and after they leave.
o K ions interact with the O molecules.
o Hydration involves continuous turn over of water molecules.
o 1 water molecules will interact with an ion, and then it will change
 There is a constant change in interaction with water molecules.
 There is this constant turn over or exchange with water
molecules.
o How do the K ions swap the water for the O atom?
 It will give up its coat of hydration because its going to
exchange the water for an O atom.
 Water has Oxygen.
 Because of the electrochemical gradient these ions will be
pushed into the filter.
 They will loose water and gain an O atom that is part of the
structure of the ion channel.
  K ion is now moving through the channel. It is not actually in an
aqueous environment, it just thinks it is.
 It behaves as though it is still hydrated.
- In this way, the pore is highly selective and allows rapid flow of K+
o The same idea works for Na channels and Ca channels.
o Filters are a little different in their structure but the idea is the same.
o We have a rapid flow of ions because they aren’t really binding.
o They are just swapping the water molecule for a O atom in the protein.
o All these interactions are weak interactions.
 This is how we observe ions moving so quickly in ion channels.
- This is how we get high selectivity and high rate of conduction.
- Diagram: This is a reversible process.
o It can easily transition from interacting with the water to interacting
with the O atom.
o Ex: in equilibrium concentrations of K, we might see this.
o As soon as we change the concentration of the chemical gradient then
we will see movement in one direction.
 We will see rectification of the ions (the preference of the
movement of ions in one direction or another).

Ion Basis of Membrane Potential

- Grey space is the membrane.
- Left: Case where there is no membrane potential.
o (+)(-) inside = (+)(-) outside.
o If there is a Na/K pump that starts pumping ions from the right to the
left we get the situation on the right.
- Right: mV differences in membrane potential.
o More (+) ions being pumped in the left direction.
o Na/K pump exchanges at a 3/2 ratio.
o Net electrogenic movement of 1 charge.
o Pump over a (+) ion and leave a counter ion (-)
o The –ive is something you measure.
 - This sets up a separation of charge. The membrane acts as a capacitor that
allows for the separation of charge.
- It builds up potential energy across the membrane.
- Na/K pumps store energy in this way. We now have a voltage difference
across the membrane.
- Energy is stored, passive transporters use these for their electrochemical
gradients.
- There are parts of the ion channel structure that detect these subtle changes
in membrane potential.

Voltage Sensitivity
- From a eukaryotic K+ channel
- Transmembrane segments S1-S4 from the voltage sensor, but S4 imparts
voltage sensitivity to the channel protein
o Formed from 4 separate subunits.
o The voltage sensing domain
o The segments are all connected by non-helical loops.
o The first 4 segments are involved in voltage sensing.
o S4 has a higher proportion of (+)ive charges.
- (+) Charged helix of S4 is the key component
o It senses the change in voltage.
- At rest when cell interior is (-), S4 is near cytosol and channel is closed.
- When cell is depolarized (i.e. more +), then S4 is pushed across the
membrane and the channel opens.
o When we change the inside of the cell from (-) to something more
(+)ive the sensor S4 is going to move.
o Movement of the S4 segment.
o If we create a (+)ive charge in the cell it is going to reorient the sensor
so it moves away from the inside of the cell.
o The S4 segment is pushed across the membrane when depolarized.
o The movement is translated to the gating mechanism.
 Channels will now open. Leads to gating.
Not All Ion channels are Gated by Voltage
- Background or leak K+ channels are not gated or dependent in membrane
potential (i.e. voltage)
o Channels that are opened all the time.
o They have no sensing mechanism for voltage.
o The K channels can help set the resting membrane potential.
 At rest the cell has a high permeability to K.
- These channels lack the voltage-sensitive transmembrane segment that
normally imparts voltage sensitivity on the channel protein
o Leak channels, they are the primary channels that set the resting
potential.
o Allow the high permeability to K ions.
- As a result, they are always open and set the resting membrane potential of
the cell.
o K ions will flow out of the channels and reset the membrane potential.
- The electrochemical gradient determines which direction the ions move, not
the channels.

Na+ Channels of Neurons

- Na+ channels move from a closed, to an open, to an inactivated state.
o Na channels are regulated by voltage.
o Channels that are inactivated are channels that are open but not
conducting any ions.
o There is another particle that acts to occlude the conducting pore of
the channel.
o Inactivation of Na channels is important in propagation of action
potentials.
- This permits rapid re-polarization of the membrane necessary for
propagation of an action potential along the nerve fibre.
o In order for the membrane in any region to recover and allow for
another signal to come along, that patch of membrane has to return to
a resting state.
 o The inactivation state allows returning to resting potential very
quickly.
Na+ Channels of Neurons: Diagram
- One single polypeptide that was inserted into the ER and brought to the
membrane.
- We can divide this up into different domains. They are repeating domains.
- We have an inactivation gate between D3 and D4: that occludes the
conducting pore during inactivation.
- We have the 4 segments. The voltage sensory and the S4 helix.
o The sensor with the most positive charges that detects subtle changes
in membrane potential.
o When membrane depolarizes this S4 will sense that.
- Green segments have loops that contribute to the core.
o All four of these domains will fold in together and form a channel.
o Come together to form the selectivity filter.

Model of Na+ Channel Inactivation

- There are 3 states: closed, open and inactivated
- Cell is at rest with a negative membrane potential. The channel is closed
- Cell is depolarized (more positive inside) and channel opens.
o There is a change in the sensors
that allows for the opening of the
pore.
- Eventually, a gate will plug the channel
pore, thereby inducing inactivation
o The part between D3 and D4 that
will inactivate the channel.
o This occurs as soon as we
depolarize the membrane.
o We increase the chance of
inactivation.
o Inactivation is a process that takes
time that begins as soon as we stimulated the membrane.
o The act of depolarization begins the process of inactivation.
  We can remove inactivation by having a very high membrane
potential like -100mV.
 If we have a larger voltage difference across the membrane we
have a very small chance of getting inactivation.
o We have a channel that is opened but it is occluded. (Inactivated)
- When the cell is re-polarized the channel will be fully closed again.
o The –ive charge inside the cell removes inactivation.
o When the cell returns to a normal resting potential, the channel closes.
- This is important in the recovery of an axon.

Na+ Channels of Neurons: Experiment

- A brief stimulus is given to a neuron
- Na+ channels open and cell depolarizes
- Na+ channels rapidly inactivate
o Inactivation takes a little longer. It is initiated when the cell
depolarizes.
o The peak, is due to inactivation. It is because the Na channels are
becoming inactivated.
- Membrane potential returns to resting values
o Na channels are inactivated. Can lead itself to the recovery of the
membrane potential.
OR
- Note, K+ channels also play a role in re-polarization. This traces shows only
change in Vm due to K+
o As soon as a cell is depolarized K goes in the opposite direction.
o The reason Na channels cause this depolarization is
because there is a lot of Na outside the cell.
o Each Na carries with it a (+) charge.
o They are all bringing in (+) charges into the cell and
leading to this strong repolarization.
o With K, as soon as you being to depolarize a cell, K
channels themselves begin to return the membrane to
more negative values.
 K channels move in the opposite direction.
 This occurs along the same time.
o Allow for the extrusion of (+) charge ions.
o Only a few ions are needed to create a change.

Things to Consider
- What sets ion channels apart from transporters?
- Explains how ion channels can maintain such high ion selectivity while
conducting ions at nearly the same rate as ion diffusion in solution.