CHAPTER 5: Transport of Solutes and Water

Intracellular Fluid

Fluid inside the cells and occupies intracellular space

High in K+ and low in Na+ and Cl-

All Body Fluids Have Approx Same Osmolality, Each fluid

has Equal Numbers of + and Charges

Extracellular Fluid

Fluid outside the cells and occupies extracellular space

Low in K+ and high in Na+ and Cl-

Osmolality
o
describes the total concentration of all particles
that are free in a solution
o
expressed as the number of osmotically active
particles per kilogram of water (290mosmol/kg
of H2O

Electroneutrality

All solutions must respect the principle bulk of

electroneutrality: The number of positive charges in the
overall solution must be the same as the number of
negative charges
Anion Gap = difference between anions and cations in
blood plasma

Hydrostatic Pressure = Vessel to intracellular fluid

Oncotic Pressure = H2O accumulates in cells
Solute Transport Across Cell Membranes
In Passive, Noncoupled transport across a permeable membrane,
a solute moves down its electrochemical gradient

Na-K Pump = utilized by cell to maintain the optimal ion contents

in and out of the cell
Two Major Constituents of the ECF

Plasma
Interstitial Fluid

Electrochemical Gradient
o
the driving force that determines the passive
transport of solutes across the membrane
o
difference between the actual membrane
potential and equilibrium potential for any
specific set of intra and extra coonc
o
includes the contribution from the
concentration gradient of the solute
o
Determinants of Electrical Potential Energy:

Concentration Gradient

Voltage Difference
Noncoupled Transport
o
Movement of a substance means the
movement of it across the membrane is not
directly coupled to the movement of other
solutes

Diff EC/IC Conc of Soln with No Voltage Diff = Concentration

Gradient as driving force
Same E/IC Conc of Soln WITH Voltage Diff = Electrical Gradient as
driving force
INFLUX movement of solute from outside to inside
EFFLUX movement of solute from inside to outside
Unidirectional movement across the membrane in one direction
Net Flux (Net Transport Rate sum of two unidirectional fluxes
No driving force = EQUILIBRIUM = No net transport
Steady State conditions related to a substance do not change
with time

Where:

Protein-Free Plasma = [Plasma Content in meq/L] / 0.93

Effect of Protein Charge

Non-charged Solute
o
(Such as GLUCOSE) Lipid and Protein volume is
the only correction because plasma proteins
are net negative charged
o
Retain cations in plasma
Cation Conc of Protein-Free Plasma = Lowered by 5%
Anion Conc = Higher by 5%

zx = valence of solute
xo = conc outside
xi = conc inside
F = Faradays Constant

x = net driving force (jou/m)

i o = membrane potenti
R = gas constant

If NET DRIVING FORCE is not ZERO = X not in equilibrium

Nerst Equation = describes the conditions when an ion is in
equilibrium across a membrane
In SIMPLE DIFFUSION, the flux of an uncharched substance
through membrane lipid is directly proportional to its
concentration gradient.
Integral Membrane Proteins
3 Types of Protein Pathways:

Pore
o
Always open
o
Ex. Porins in the outer membranes of
mitochondria, cytotoxic pore-forming proteins =

Perforin released by lymphocytes and

aquaporin channels

Channel
o
Alternately open and closed because it is
equipped with a movable barrier or gate
o
Process is called gating
o
Gated Pore not a Gated Channel (Ex. Ion
Channels)
Carriers
o
Surrounds a conduit that never offers a
continuous transmembrane path because it is
equipped with at least two gates that are never
open at the same time

Porins

Large-size pores found in outer membrane of gramnegative bacteria and mitochondria

o
Mitochondrial Porin allows solutes as large as
5kDa to diffuse passively from cytosol into the
mitochondrial membrane

Perforin pore-forming protein

released by cytotoxic T lymphocytes to
kill their target cells

Eg Complement Cascade monomer

of C9
o
Nuclear Pore Complex (NPC)

Regulates traffic into and out of the

nucleus via active transport

Can transport huge molecules

involving ATP hydrolysis

Has AQUAPORIN for passive transport

Ion-Gated Channels

Consists one or more polypeptide subunits with ahelical membrane spanning segments

Functional Components
o
Gate determines whether channel is open or
closed
o
Sensors respond to one of several signals

Change in membrane voltage

Second-messenger systems acts at

the cytoplasmic face of the membrane
protein

Ligands bind to extracellular face of

the membrane protein
o
Selectivity Filter determines the classes of
ions or the particular ions that have access to
the channel pore
o
Open Channel Pore provides continuous
pathway when it assumes open conformation
between two sides of membrane so ions can
flow through it passively by diffusion until the
channel closes again

Channels
o
Na Channels

Passive transport

Voltage-Gated Na Channels

Responsible in generating
action potential
o
K Channels

Fairly close to zero or somewhat

positive

Move outward the cell

Has a major role in generating a
resting membrane voltage that is
inside-negative

Has a key in excitability cells where

these channels help terminate action
potential
Ca Channels

Always strongly negative

Move into the cell

Rapidly enters the cell down a steep

electrochemical gradient

Plays a vital role in transmembrane

signaling for both excitable and nonexcitable cells and generating action
potential
Proton Channels

H+ driving force generally tends to

move H into cells if Hv1 are open

Normally closed and activates only

when the membrane depolarizes or
cytoplasm acidifies

Help mediate H extrusion from cell

during states of strong membrane
depolarization (during action potential)
or severe intracellular acidification
Anion Channels

Mostly negative electrochemical

driving force

Carrier Mediated Transport Systems

Transfers a broad range of ions and organic solutes

across the plasma membrane

Behave according to the general kinetic scheme

Can mediate only passively or downhill

Km = Affinity
o
Solute Carrier Superfamily

Do not either hydrolyze ATP or couple

to an electron transport chain

Differs in:

Molecular mechanism

Kinetic properties

Regulation

Sites of membrane targeting

Tissues which they are

expressed

Developmental stage at
which they are expressed

GLUT1 = glucose transporter on the

cell surface

GLUT4 = present in the intracellular

surfaces

Urea Transporter

Organic Cation Transporter

Na/K+ Pump most important primary active transporter;
utilizes ATP to extrude Na and uptake K

Active Transport = process that can transfer a solute

uphill across a membrane against electrochemical
gradient
o
Primary Active Transport driving force needed
to cause net transfer of solute against
electrochemical gradient comes from ATP (eg.
carrying heaving material to air; PUMPS
energized by ATPases)
o
Secondary Active Transport driving force is
provided by coupling uphill and downhill
movement of one or more solute which is a
favorable electrochemical gradient exists (eg
seesaw)
Na/K Pump

Occurs between a and b subunits

o
-subunit

has 10 transmembrane segments

catalytic subunit
o
-subunit

one transmembrane segment

essential for proper assembly and

targeting of Na-K pump

Exclusive to the basolateral membrane

Responsible for low Na and high K relative to the ECF

Member of large superfamily of pumps known as E1-E2

ATPses or P-type ATPase

Two Conformational States

o
E1 - binding sites for ions face inside of cell
o
E2 binding sites face outside the cell

STEPS:

o
o
o

o
o
o
o

#1 ATP-bound E1 ATP state. After pump has

released two K to the ICF, Na-binding sites face
the ICF and have high affinities to Na
#2 Na-bound E1 ATP 3Na State
#3 Occluded E1 P (3Na+ State) ATP
phosphorelates leaving ADP trigerring minor
conformational change in E1 form the pump now
occludes 3 Na ions within the permeation
pathway (Na inaccessible to ICF/ECF)
#4 Deocccluded E2-P 3Na State. E1 to E2 having
two effects: (1) Pump becomes deoccluded that
Na-binding site is now open to the ECF (2) Na
affinities of these binding sites decrease
#5 Empty E2-P state. 3 Na dissociate into ECF
and protein undergoes minor conformational
change to empty E2-P form which has high
affinity to K
#6 K-Bound E2-P 2K State. 2 K bind to pump
#7 Occluded E2-K State. Release of inorganic
phosphate into ICF
#8 Deocludded E1 ATP 2K State. E2 to E1 by ATP.
Effects: (1) pump becomes deocludded (2)K
affinities decreased
BACK TO STEP 1

the turbine-down the H

electrochemical gradient into the
mitochondrion
Stick is an axle (y and E subunits of
F1) that rotates with the turbine
Candy (a and b subunits of F1) is a
stationary chemical factory, energized
by the rotating axle that synthesizes
one ATP molecule for each 120 tun
of turbine

Blockers
o
Has high affinity to ECF of E2-P Cellular K (Step
5)
o
Cardiac Glycosides

Ouabain

Digoxin widely used for caridiac

conditions

Other P-type ATPases

H-K Pump
o
In parietal cells of gastric glands
o
Excretes H+ to the apical membrane to the
lumen and uptakes K+
2+

Ca Pumps (Plasma Membrane Ca ATPase-PMCA)

o
Extrudes Ca from cell; Influx of H in
sarcoplasmic reticulum in muscle cells

Other Pumps
o
Copper Pump ATP7B mutated in Wilsons dse
F-Type and V-Type ATPases Transport H+

F-type or FoF1 ATPases

o
~500kDa
o
Fx

The hand acts as a turbine that

roteates in the plane of membrane,
driven by the H ions that flow through

V-type H+ Pump
o
Has lysosomes, endosomes and secretory
vesicles, storage vesicles and the Golgi
Apparatus contain so-called vacuolar-type (vtype) H-ATPase that pumps H from cytoplasm to
interior organelles
o
Has 6 subunits
ATP-binding Casettes (ABC) Transporters = can act as
Channel, Pumps or Regulators

ABC1 Subfamily
o
Important in mediating efflux of phospholipids
and cholesterol from macrophages and other
cells

MDR Subfamily
o
Multidrug Resistance Transporters
o
ATPases and primary active transporters

MDR1/P Glycoprotein = extrudes

cationic metabolites and drugs across
cell membrane; important and
clinically antagonistic in cancer px
pumping anticancer drugs

MRP/CFTR Subfamily
o
Fx as low-conductance Cl- channel as well as
regulator of other ion channels
o
Has two membrane spanning domains (MSD1
and MSD2) with 6 membrane-spanning
segments
o
Has 2 nucleotide-binding domains (NBD1 and
NBD2)
o
Has Protein Kinase A and C as regulatory
domains
o
Two Mechanisms of ATP regulating CFTR Cl
Channel

Protein phosphorylation

Interaction with the nucleotide-binding

domains
Cotransporters, One Class of Secondary Active
Transporters are Generally Driven by the Energy of the
Inwardly Directed Na+ Gradient
Secondary Active Transporters = driving force is provided by
coupling uphill and downhill movement of one or more solute
which is a favorable electrochemical gradient exists (eg seesaw)

Cotransporters (Symporters)

Intrinsic membrane proteins that move the

driving solute (gradient provides the energy)
and driven solutes (which move uphill) in the
same direction
Exchangers (Antiporters)
o

o
o
o

Examples of Cotransporters:

Na/Glucose Cotransporter (SGLT)

o
Located at apical membrane of cells that line
the PCT and Small Intestine
o
Belongs to the SLC5 family consisting of a
single subunit

SGLT1 = moves 2 Na with each

glucose molecule

SGLT2 and 3 = move 1 Na with each

gluose

Na-Driven Cotransporters for Organic Solutes

o
Na-Driven AA Transporters = belong to SLC6
and SLC38

Na-coupled cotransporter for

Monocarboxylates, Di and Tri
o

Na/HCO3 Cotransporters
o
Belongs to SLC4; keygroup of acid-base
transporters (Regulation of intracellular pH)
o
In the basolateral membranes

Electrogenic = Efflux of HCO3 1:3

(Na:HCO3)

Electroneutral = Influx of HCO3 1:1

Na-Driven Cotransporters for Inorganic Anions
o
NaPi = members of SLC 17, 20 and 34 and
Sulfate cotransporter (SLC13)
Na/K/Cl Cotransporter
o
Belong to SLC12
o
Harness energy of the inwardly directed Na+
electrochemical gradient to drive the
accumulation of Cl and K

NKCC1 = present in the basolateral

membrane

NKCC2 = present in the apical

membrane
o
Mediate the uphill Cl transport to cell
o
Inhibited by furosemide and bumetanide which
are called Loop Diuretics bec they increase
urine flow by inhibiting transport at loop of
Henle

Exchangers, Another Class of Secondary Active

Transporters, Exchange of Ions for One Another
Other major class of secondary active transporters is the
exchangers, or antiporters

Exchangers intrinsic membrane proteins that move

one or more driving solutes in one direction and one or
more driven solutes in the opposite direction; -exchanges anion for anion and cation for cation

Na/Cl Cotransporter
o
In the apical membrane of DCT
o
K+ independent; blocked by thiazide diuretics
K/Cl Cotransporter
o
Na-independent;
o
Electrochemical gradient is outwardly directed
bec it is means to accumulate in the cell
o
K Electrochemical gradient is outwardly
directed bec it is means to accumulate in the
cell moving K and Cl inward
o
Inside gradient membrane potential causes
efflux of Cl

NKKC

Cl-HCO3

Bring Cl into the cell

Cl Electrochemical gradient is
outwardly directed bec it is
means to accumulate in the
cell
H-Driven Cotransporters

H/oligopeptide cotransporter PepT1 and related

proteins are members of SLC15
PepT1 electrogenic and responsible for uptake
of small peptides from the lumen into the cells
of renal PCT and small intestine
Mediate the electroneutral, H-couple flux of
lactate, pyruvate or other monocarboxylates
across the cell membranes of most tissues of
the body

H-driven Amino Acid Cotransporters

(ex PAT1) members of SLC36

Monocarboxylate Cotransporter (ex

MCT1)

Can operate in either the net

inward or outward direction,
depending on the lactate and
H gradients across the cell
membrane

Moves lactate out of cells that

produce lactate by glycolysis
but into cells that consume
lactate
Divalent Metal Ion Cotransporter (DMT1)

Member of SLC1

Couples the influx of H to the influx of

Ferrous Iron (Fe2+) as well as to a
variety of other divalent meals, some
of which (Cd2+, Pb2+) are toxic to cells

Expressed at high levels in kidney and

proximal part of the Small Intestine

Na-Ca Exchanger

Nearly ubiquitous Na-Ca Exchanger

(NCX) belong to SLC8

Mediate the exchange of 3:1 (Na:Ca)

Electrogenic and moves net powsitive

charge in the same direction as Na

Na (influx; as primary AT) = Ca (efflux;

as secondary AT)
Na-H Exchanger (NHE)

SLC9 (1:1)

Na (influx) H (efflux) = pH of Cell

NHE1
o
present in
nonepithelial cells
and basolateral
membranes of
epithelia
o
plays major role in
pHi regulation and
cell volume

NHE3
o
Present at apical
membranes of
several epithelia
o
Plays major role in
acid secretion or Na
absorption

Organic Cation H Exchanger

Secretes cationic metabolites

and drugs across the apical

membrane of renal proximal

tubule cells and hepatocytes
Na Driven Cl-HCO3 Exchanger (NDCBE)

Important for pHi regulation

SLC4 member (1:1:2)

Uses inwardly directed Na

electrochemical gradient to drive
uphill entry of HCO3 into the cell

Cl-HCO3 Exchanger

Involved in acid-base transport that

fxs independently of Na

Members of either SLC4 or SLC26 (1:1)

(SLC4) Anion Exchangers (AE1-AE3)

AE1 = Important for transport

of HCO3 into the RBC in the
lung and out of the RBC in
peripheral tissues

AE2 and 3
o
Inwardly directed Cl
gradient almost
always drives out of
the cell; plays a role
in cell volume
regulation

Other

(SLC26)

Play important roles in

epithelial Cl and HCO3
transport

multifunctional
Anion Exchangers
Cl-Formate Exchange (CFEX) and ClOxalate Exchange

Present in the apical

membranes of renal PCT

Important for the secondary

active uptake of Cl
Pendrin = mediates Cl-HCO3 exchange
and transports Iwhich is important in
the thyroid gland
Organic Anion Transporting
Polypeptides (OATP)

Members of SLC21

Mediates the uptake of bile

acids, bilirubin and the test
substrate bromosulphthalein
in the liver
Prostaglandin Transporter (PGT)

Mediates the uptake of

prostanoids

(eg. prostaglandins E2 and F2a

and thromboxane B2)
Organic Anion Transporters (OAT)

SLC22

Mediate the uptake of

endogenous organic anions
by exchange or facilitated
diffusion

Regulation of Intracellular Ion Concentrations

The Na-K Pump Keeps Na inside the Cell Low and K High

Inhibited by OUABAIN ~ Nai rises and Ki falls

Plays important role in generating the inside-negative

membrane voltage ~ (60mV) in a typical cell

Accomplishes in two ways

o
3:2 (Na:K) ~ Electrogenic = causing outward
current of positive charge across the plasma
membrane
o
Active K accumulation creates a conc gradient
that favors exit of K from cell through K
channels
The tendency of K to exit through these channels, with
unmatched negative charges left behind, is the main cause of the
insidenegative membrane voltage. For this reason Nas
inwardly directed Na-electrochemical gradient allows passive
entry of Na
o

Sodium (Na)

Predominant in ECF at 145mM in Conc

Gradient is maintained by active

extrusion of Na from cell by Na-K
Pump
Potassium (K)

4.5mM in ECF; predominant in ICF

about 25-30 fold

Its channel is inhibited such as Ba2+

and Vm becomes less negative (cell
depolarizes)

Cell Harness the Na Energy for Three Major Purposes:

1. In certain epithelial cells, amiloride-sensitive Na
channels (ENaC) are largely restricted to the apical or
luminal surface of cell. Na-K pumps are restricted to the
basolateral surface of the cell. In this way,
transepithelial Na transport takes place than a futile
recycling of Na back and forth across a single plasma
membrane
2. In excitable cells, passive entry of Na entry occurs
through voltage-dependent Na channels and plays
critical role in generation of action potential. Na is cycled
at high energy cost across the plasma membrane for
important physiological purpose of information transfer
3. Virtually every cell in the body tissues uses Na gradient
across plasma membrane to drive the second active
transport of nutrients and ions
Ca2+ Pump and the Na-Ca Exchanger Keep Intracellular
Ca Four Orders of Magnitude Lower than EC Ca

EC Ca ~ 1mM, IC 100nM = Conc Gradient 10-fold

Brought by Voltage-Gated Ion Channel

o
Ca2+ Pumps (SERCA) in Organelle Membranes

Ca pumps (ATPases) are present on

membranes that surrounds various IC
organelles such as sarcoplasmic
reticulum and ER

Actively sequester cytosolic Ca in IC

stores then released into the
cytoplasm in bursts as part of signal
transduction process in response to
membrane depolarization or humoral
agents
Ca Pump (PMCA) on the Plasma Membrane

Major route of Ca extrusion

Pump itself is incapable of this type of

feedback control because it has a high
Km for Cai.

As Cai rises, the Ca binds to a protein

called calmodulin which has a high
affinity to Ca.

Newly formed Ca-CaM binds to Ca

Pump, lowers the pumps Km for Cai
into the physiological range and thus
stimulates Ca extrusion

Cai falls = Ca-CaM levels IC falls so CaCaM dissociates from Ca pump,

returning pump to its inactive state
resulting 100nM at resting state

Na-Ca Exchanger (NCX) on Plasma Membrane

For Ca efflux only when Cai rises

above normal levels

Important in restoring low Cai when

large influxes of Ca occur

Contributes to the excitable cells

Via Voltage-Gated Ca Channels

In Most Cells, Cl is Mosdestly Above Equilibrium Because

Cl Uptake by Cl-HCO3 Exchanger and Na/K/Cl
Cotransporter Balances Passive Cl Efflux Through
Channels

Chloride
o
ICF; ECF
o
Can passively pass through membrane because
of the cells anion-selective channels
o
60mV inside-negative membrane voltage

Cl-HCO3 Exchanger = most common pathway for Cl

uptake
Na/K/Cl Cotransporter
o
mediate uphill Cl transport
o
Passive extrusion of Cl through anion-selective
channels in PM opposes Cl uptake machanisms
K/Cl Cotransporter

Na-H Exchanger and Na-Driven HCO3 Transporters Keep

the Intracellular pH and HCO3 Above Their Equilibrium

Acid Extruders = secondary active transporters that are

energized by the electrochemical Na gradient across the
cell membrane
o
Sensitive to pHi changes
o
Stimulized when acidic; Inhibited if alkalinized
ICF
o
Important Extruders:

Na-Driven Cl-HCO3 Exchangers (1:2)

Na/HCO3 Cotransporters (1:1)

o
Important AcidSecretors

V-type H

H-K Pumps

Acid Loaders = balance acid extrusion

Water Transport and the Regulation of Cell Volume
Water Transport is Driven by Osmotic and Hydrostatic
Pressure Difference Across Membranes

Always passive

LOW FLUIDITY = LOWER H2O PERMEABILITY due to

presence of phospholipids and long chains of fatty acids
with few double bonds

Single water molecules can dissolve in lipid bilayers &

move across cell membranes at a low but finite rate by
simple diffusion

Ease of H2O diffuses through lipid bilayer depends on

lipid composition of bilayer
AQUAPORINS (AQP1)

Specialize water channels that serve as a passive

conduits of water transport

Its presence increases membrane water permeability

Collecting duct cells of kidney regulate the H2O

permeability of their apical membranes by inserting
AQP2 water channels into their apical membrances
under control of ARGININE and VASOPRESSIN

Erythrocytes or renal proximal tubule, AQP1 is always

present in membrane

Net Driving Force

2 Driving Forces:
o
Chemical Potential Energy Difference = water
conc dependent to the two sides of the
membrane
o
Energy difference, per mole of water, that
results from the difference in HYDROSTATIC
PRESSUURE (H2O, pressure) across the membrane

P = hydrostatic pressure

Vw = partial morlar volume of H2O

OSMOLALITY
o
concentration of osmotically active solutes
o
UNIT: osmoles/kg of H2O
Colloid Osmotic Pressure/Oncotic Pressure
o
Difference in osmotic pressure that tends
pullback of fluid
Ultrafiltration
o
The resulting movement of water out of the
capillary when hydrostatic pressure difference
exceeds the colloid osmotic pressure difference

Because of the Presence of Impermeant Negatively

Charged Proteins Within the Cell, DONAN FORCES Will
Lead to Cell Swelling

Na-K Pump Absence = SWELLING

Model: NEGATIVELY CHARGED, IMPERMEANT

MACROMOLECULES