Membrane Transport

Systems in Living Cells

Prof. Ramkrishna Sen
rksen@iitkgp.ernet.in
Department of Biotechnology
Passive transport, facilitated passive transport (neutral molecules)
Osmosis (water transport)
Passive Ion channels for transporting Na
+
, K
+
, Cl
-
, Ca
+2
ions
Active transport ( primary active and secondary active) transport for ions,
charged molecules
Nernst equation for the equilibrium of ion transport (electrical gradient =
chemical gradient) for individual ion
Donnan equilibrium (ion transport in presence impermeable ion in cellular
compartment)
Goldman equation ( modified form of Nernst equation) for membrane
potential considering all the transporting ions
Electrical properties of Ion transporters and electrical equivalent (R-C)
circuit [ resistor-capacitor]
Bulk transport
Membrane Transport System
The cell
membrane
contains
many proteins
including ion
channels
How the cells exchange nurtrients and excretory products between inside and
outside of the cell ?
Ultrastructure of a typical animal cell:
Particles forced through a filter or a membrane by hydrostatic
pressure.
Hydrostatic pressure( mechanical potential energy)
Fluid pressure of the blood generated by the left ventricle
Opposed by osmotically (chemical potential energy) active particles
in the blood (plasma proteins).
Examples
Blood pressure generated by the heart and blood vessels forces tissue
fluid out of tiny openings in the capillary wall and leaving larger particles
of blood cells and protein molecules inside the capillary.
Coffee filters work by the pressure from weight of the water above the
coffee grounds forcing the flavored water through the filter and leaving
the large particles of coffee grounds on the filter paper.
Filtration and osmosis are the major processes in the capillaries
of tissues and the kidney.
These are passive transport process in capillaries of tissue to provide the
nutrients in tissue level
Hydrostatic and osmotic pressure (tissue level):
Interstitial fluid (or tissue fluid) is a solution that bathes and surrounds the cells of multicellular
animals. It is the main component of the extracellular fluid, which also includes plasma and
trnascellular fluid. The interstitial fluid is found in the interstitial spaces, also known as the tissue
spaces.

Blood flows from the heart to arteries, which branch and narrow into arterioles, and then narrow
further still into capillaries. After the tissue has been perfused, capillaries branch and widen to
become venules and then widen more and connect to become veins, which return blood to the heart.
The forces across a semi permeable
membrane and allows calculation of the net
flux:

P
c
: Capillary hydrostatic pressure
P
i
:Interstitial hydrostatic pressure

c
:Capillary oncotic pressure( colloid pressure )

i
: Interstitial oncotic pressure
K
f
Filtration coefficient
:Reflection coefficient
K
f
is the proportionality constant, and
J
v
is the net fluid movement between
compartments
([P
c
P
i
] [
c

i
]) Net driving force
How tissue fluid (interstatial fluid) movement occurs
All cells acquire the molecules and ions they need from their surrounding extracellular fluid
(ECF). There is an unceasing traffic of molecules and ions
In and out of the cell through its plasma membrane
Examples: glucose, Na
+
,K
+
, Ca
2+

In eukarytoic cells, there is also transport in and out of membrane-bounded
intracellular compartments such as the nucleus, endoplasmic reticulum,
and mitochondria.
Examples: proteins,mRNA, Ca
2+
, ATP
How cells acquire molecules, ions from tissue fluid (interstatial fluid)

r
of surrounding ~ 80
electrical permittivity

r
( dielectric
constant) about 2
Lipid Bilayer -- primary barrier selective based upon size and polarity of molecules
a) Nonpolar molecules (O
2
, hydrocarbons, fatty acids)-- pass freely
b) Small uncharged polar molecules (H
2
O, CO
2
) pass freely but more slowly than nonpolar
molecules
c) Large polar molecules and ions -- don't pass freely
d) Macromolecules (proteins, nucleic acids, polysaccharides) cannot pass unless a special
mechanism exists (signal hypothesis).
Cells solve the problem of transporting ions and small molecules across their membranes:
Facilitaed diffusion :Transmembrane proteins create a water-filled pore through which ions and
some small hydrophilic molecules can pass by diffusion. The channels can be opened (or closed)
according to the needs of the cell.
Active transport : Transmembrane proteins, called transporters, use the energy of ATP to force
ions or small molecules through the membrane against their concentration gradient.
Protein mediated diffusion differs from non-mediated diffusion of O
2
and CO
2
in
four distinct ways:
1. the speed and specificity of the transport
2. the transport capacity is finite, i.e., exhibits saturation kinetics
3. transport can be competitively inhibited (antagonists)
4. transport can be chemically inactivated (heat, high salt concentration)
Solving the problems of transporting polar and ionic molecules
Comparison of properties of carriers, pores and channels
Membrane Carriers: How do they function?
Chemical driving force
Difference in energy due to a concentration gradient that causes a molecule to
move from high to low concentration
Electrical driving force
Difference in energy due to a separation of charge that acts to move ions from
high energy to low energy
Electrochemical driving force
Sum of the chemical and electrical driving forces
Both hydrostatic force (P, mechanical potential energy) and osmotic force (Osm, chemical
potential energy) regulate fluid flow through membrane pores.
These force are interconvertible and net driving force for water between cell and extra cellular
solution = RT (Osm
cell
- Osm
ext
) + (P
cell
P
ext
)
Experimental measurements made in cells that show no net transmembrane hydrostatic or
osmotic pressure difference, but a net difference in transmembrane electrical potential?
Driving force responsible for membrane transport
Plasma Membrane : Environmental Boundary (Barrier)
Ionic imbalance (particularly, Na
+
and K
+
) between inside and outside created by membrane ionic
pumps, ion exchangers and channels, establishes resting menbrane potential. This is used to
drive other process (such as molecule import), as well as for information processing (e.g. nerve
cells).
Activities of plasma membrane ionic pumps are energized by hydrolysis of ATP.
N.B: All the live cells establish and maintain the membrane potential.
Types of Transport Processes
There are 4 basic mechanisms

1. DIFFUSION : Movement of molecules through the membrane in which
-no energy is required
-molecules move in response to a concentration gradient, Passive transport

2. OSMOSIS: Movement of water from an area of high to low concentration of water
-movement of water toward an area of high solute concentration

3. FACILITATED DIFFUSION: Movement of a molecule from high to low concentration with the
help of a carrier protein.
-is specific
-is passive
-saturates when all carriers are occupied

4. ACTIVE TRANSPORT: Requires energy for transport molecules against concentration
gradient , energy expanded in the form of ATP

5. BULK TRANSPORT: Bulk transport of substances through endocytosis and exocytosis

Net movement of particles from area of high concentration to area of low concentration
Due to their constant, random motion
Difference between the high and low concentrations is a concentration gradient
Diffusion tends to eliminate the gradient
Also known as movement down the concentration gradient
Simple diffusion -Nonpolar and lipid-soluble substances
- Diffuse directly through the lipid bilayer
-Diffuse through channel proteins
Factors influence diffusion process:
Distance
The shorter the distance, the more quickly gradients are eliminated
Few cells are situated greater than 125 m from a blood vessel
Molecular Size
Ions and small molecules diffuse more rapidly
Temperature
| temp., | motion of particles
Steepness of concentrated gradient
The larger the gradient, the faster diffusion proceeds
Membrane surface area
The larger the area, the faster diffusion proceed

Diffusion Transport Process
Diffusion Across Membranes
Simple Diffusion
Lipophilic substances can enter cells easily because they diffuse through the lipid
portion of the membrane
Examples are fatty acids, steroids, alcohol, oxygen, carbon dioxide, and urea,
Channel-Mediated Diffusion
Membrane channels are transmembrane proteins
Only 0.8 nm (8) in diameter
Used by ions, very small water-soluble compounds
Much more complex than simple diffusion
Are there enough channels available?
Size and charge of the ion affects which channels it can pass through

But, A is the same molecule whether its inside or
outside, so..
Passive Transport: Permeability of a membrane for a nonelectrolyte
solute (glucose)
Steps of membrane diffusion (any can be
rate limiting):

enter the membrane (potential barrier) (1)
diffusion through the bilayer core (2)
exit the membrane (potential barrier) (3)
Flux= mol solute/(Sec.cm
2
)
J = P ( C
1
C
2
) P = permeability coefficient( cm/sec) =

d
K
p
D
m

K
p
- partition coefficient ( C
membrane
/ C
aqueous
); D
m
Diffusion coefficient of molecule in the
membrane; d thickness of membrane
n - number of molecules inside cell
(mol)
t - time (seconds)
P - permeability constant for a
particular molecule (cm/sec)

Diffusion coefficient
(D,cm
2
/sec)/thickness of
membrane (x,cm)
A - surface area of the cell
membrane (cm
2
)
C - concentration of diffusing
molecule (mol/cm
3
)
X - width of cell membrane (cm)
Variables in Simple Diffusion
The concentration gradient, dC/dx,
equivalent to (C
out
- C
in
)/dx where C
out
and
C
in
are the substrate concentrations inside
and outside the cell, and dx is the width of
the cell membrane.
When the concentration outside the cell
(C
out
) is larger than inside the cell (C
in
), the
concentration gradient (dC/dx) will be
positive, and net movement will be into the
cell (positive value of dn/dt).
D
P ( c )
n - number of molecules inside cell
t - time (seconds)
V
max
- saturation constant
(mol/cm
3
/sec)
K - constant determining speed of
saturation (mol/cm
3
)
C - concentration of diffusing
molecule (mol/cm
3
)
X - width of cell membrane (cm)
Facilitated diffusion variables
Facilitated diffusion involves a limited number of
carrier proteins.
At low concentrations, molecules pass through
the carrier proteins in a way similar to that of
simple diffusion.
At high solute concentrations, all the proteins
are occupied with the diffusing molecules.
Increasing the solute concentration further will
not change the rate of diffusion.
There is some maximum rate of diffusion (Vmax)
when all the carrier proteins are saturated.
The rate of diffusion will increase with increasing
solute concentration, but must asymptotically
approach the saturation rate, V
max
.
How quickly the carrier proteins become
saturated can be described by the variable K, the
concentration gradient at which the rate of
diffusion is 1/2 V
max
.
K and Vmax depend on properties of the
diffusing molecule, such as its permeability (P),
as well as the surface area (A) of the cell, but for
simplification we give the above equation.
Large polar molecules such as glucose
and amino acids, cannot diffuse
across the phospholipid bilayer. Also
ions such as Na
+
or Cl
-
cannot pass.

These molecules pass through protein
channels instead. Diffusion through
these channels is called FACILITATED
DIFFUSION.

Movement of molecules is still
PASSIVE just like ordinary diffusion,
the only difference is, the molecules
go through a protein channel instead
of passing between the phospholipids.
Carrier Mediated Transport: Facilitated Diffusion
The sugar is bound by the protein, a flip-flop mechanism reverses the membrane direction of the sugar-protein
complex, the sugar is released and the protein flips around once more to initiate a new cycle. Transport activity is
dependent upon the sugar concentrations and the number of transport proteins in the outer cell membrane. In
principle the GLUT family can transport glucose both into and out of cells. In most tissues the internal glucose
concentration is quite low; transport can only proceed from the extracellular area into the cell.
Turnover rate (s
-1
) of glucose transporter: 0.1-1.3x10
4

Glucose and amino acids are insoluble in lipids and too large to fit through membrane channels
Passive process, i.e. no ATP used
Solute binds to receptor on carrier protein
Latter changes shape then releases solute on other side of membrane
Substance moved down its concentration gradient
A well studied example is the glucose transport across red blood cell membranes (erythrocyte
membrane).
Comparing the diffusion coefficient D for glucose across synthetic phospholipid membranes (vesicle
membrane; bilayer) with that of erythrocyte cell membranes shows a 10
+6
fold increase of glucose
diffusion across the cell membranes.
[ D(bilayer) 2.4x10
-10
cm
2
/s ; D(erythrocyte)=2x10
-4
cm
2
/s ]
Carrier Mediated Transport: Facilitated Diffusion
1. You graphed dn/dt as a function of dC/dx. What is the slope of this line? What do
increases or decreases in the slope mean biologically?
2. Now assume the concentration gradient is a constant. How does the rate of
diffusion (dn/dt) change with the surface area (A) of the cell and the permeability (P)
of the diffusing molecule? Graph dn/dt as a function of A or P and describe the
function.
3. Look at the equation for facilitated diffusion and find the horizontal asymptote.
What happens to dn/dt as dC/dx approaches infinity?
4. Try graphing this equation with different values for K. How does this change the
concentration at which the carrier proteins are saturated?
5. Compare simple and facilitated diffusion of glucose into erythrocytes by graphing
rate of diffusion (micromoles per hour) as a function of external glucose
concentration (mmol/cm
3
). For facilitated diffusion, V
max
= 500 micromoles per hour
and K=1.5 mmol/cm
3
. For simple diffusion, A x P is 3 cm
3
/hour.
6. How do rates of simple and facilitated diffusion differ in response to a
concentration gradient?
7. How is the permeability of a molecule across the lipoprotein membrane related to
the molecule's solubility in lipids and size?
Questions on diffusion and facilated diffusion
It is passive transport of water through a semi-permeable membrane
Why does water move in that particular direction?
The flow of water across a selectively
permeable membrane
Always from an area of high water
concentration to an area of low water
concentration.
A special case of diffusion of water across a
selectively permeable membrane, such as
the plasma membrane.
A semi-permeable membrane is freely
permeable to water but not to solutes
Osmosis moves water through
aquaporins toward the hypertonic
solution.
It is a very important process because water
is found throughout cells and extra-cellular
areas of the body.
Osmosis ( a special case of diffusion)
t = MRT
M-molarity of solute( moles/litre)
t - Osmotic pressure of solution ( atm or torr)
Osmolarity = the total solute concentration in an aqueous solution
Osmolarity = molarity (mol/L) of particles in solutions
A 1 M Glucose solution = 1 Osmolar (Osm)
But a 1 M NaCl soln = 2 Osmolar because NaCl dissociates into 2 particles (Na and Cl)
whereas Glucose does not
Physiological solutions are expressed in milliosmoles per liter (mOsm/L)
blood plasma = 300 mOsm/L or 0.3 Osm/L
Tonicity - property of a solution to affect fluid volume and pressure within a particular cell (depends
on concentration and permeability of solute,)
Isotonic solution
solution with the same solute concentration as that of the cytosol; normal saline[0.15M NaCl]
Hypotonic solution
lower concentration of nonpermeating solutes than that of the cytosol (high water
concentration)
cells absorb water, swell and may burst (lyse)
Hypertonic solution
has higher concentration of nonpermeating solutes than that of the cytosol (low water
concentration)
cells lose water + shrivel (crenate)

Osmolarity and Tonicity





















t
cell
= t
out
solute
solute
solute
solute
Water is in thermodynamic equilibrium across cell membranes
Hypotonic Isotonic Hypertonic
Transport of water can be induced either by osmotic pressure differences, giving rise to the osmotic
permeability (P
f
), or by a difference in tracer concentrations, giving rise to the diffusion permeability
(P
d
).
The diffusion permeability accounts for water molecules transported through the entire channel,
Osmotic permeability accounts for water entering and leaving the channel at its exits such that P
f
/P
d

is greater than one.
The ratio P
f
/P
d
measures the number of essential steps that water molecules need to pass the channel.
For aquaporin with an osmotic permeability (P
f
) of 6 x 10
-20
m
3
s
-1
per channel subunit, an equivalent
hydraulic conductivity of 4.4 x 10
-22
m
3
s
-1
MPa
-1
per channel subunit can be calculated.
For a gradient of 0.1 MPa (1 bar) in osmotic pressure, which is physiologically realistic, 1.5 x 10
6

water molecules will flow through each channel subunit per second.

This is similar to the flux through an ion channel for a typical driving force.
Schematic
depiction
of water
movement
through
the narrow
selectivity
filter of the
aquaporin
channel
Aquaporin (the water channel): The HIGHWAY for water transport
Transport of ions through membrane
Ion Channels referred the ion it permeate
Three basic properties of ion channels:
To conduct ions rapidly ( high turn over number)
Exhibit high selectivity: only certain ion species flow while others are excluded
Conduction be regulated by processes known as gating, i.e. ion conduction is
turned on and off in response to specific environmental stimuli
The flux of ions through the ion channels : passive

Channel
Substrate Turnover (s
-1
)
Na-channel (V) 7 x 10
6

Ca-channel (V) 1.9 x 10
6

K-channel (Ca, V) 0.2-3 x 10
7

ACh receptor 2.3 x 10
7


Ion channels do not bind the ions that pass through them.
They are selective in determining which ion can pass.
Types of gated ion channels: voltage-gated, stretch-gated, phosphorylation-gated and ligand-gated
channels.
Ion channels are with oligomeric arrangement with Intrinsic symmetry and the pore Size
Correlates with the number of Subunits

Voltage-Dependent
(Na
+
, K
+
, Ca
++
)

Ligand-Gated
Mechanosensitive
Connexins
(Gap Junctions)
Ion Channels
Turnover rate (s
-1
) of Na-K ATPase pump: 5x10
2

Turnover rate (s
-1
) of Ca - ATPase pump: 2x10
2


Compartmentation of Ionic Pools and Electrochemical Driving Forces
Ion Channels: Uniporter(passive), antiporter( primary active/seconadary active) ,symporter (seconadry
active, cotransporter)
Compartmentation of Ionic Pools and Electrochemical Driving Forces
Ion Channels: Uniporter(passive), antiporter( primary active/seconadary active) ,symporter (seconadry
active, cotransporter)
Categories of Ion Permeability Pathways
Lodish et al. (2000) Molecular Cell Biology
4th Edition [W.H. Freeman & Co.]
Primary Active Transport
Solute pumping
Pump or protein carrier
An enzyme-like protein carrier that pumps or carries solutes such as ions of
sodium, potassium, and calcium, into or out of the cell against their
concentration gradients.
ATPase
The enzyme on the protein carrier or pump that catalyzes the breakdown or
phosphorylation of ATP producing energy that drives the pump.
This action may require up to 40% of a cells supply of ATP
Sodium-potassium pump
(Na+/K+ ATPase Pump)


Maintains the resting membrane potential of nerve and muscle cells
Sodium
Primary extra-cellular ion that is constantly leaking into cells.
Potassium
Primary intracellular ion that is constantly leaking out of cells.
The sodium/potassium pump constantly pumps 3 sodium ions out and 2
potassium ions into the cell, maintaining the relative negativity inside the cell.
All cells have a negative charge inside because of this mechanism.
3Na
+
ATP
3Na
+
2K
+
2K
+
Secondary Active Transport
Movement of a molecule that is coupled to the active transport of another molecule
One substance moves down its electrochemical gradient and releases energy in the
process
This energy is then used to drive the movement of another substance against its
electrochemical gradient




Cotransport (Symport)
- Movement of 2 substances in the same direction
-Example
Sodium-linked glucose transport
Couples the inward flow of sodium with the inward flow of glucose
Sodium movement with its electrochemical gradient releases energy that drives the
movement of glucose against its concentration gradient

Countertransport (Antiport exchange)
Movement of 2 substances in opposite directions
Example
Sodium proton exchange
Couples the inward flow of sodium with the outward flow
of protons (H
+
)
Energy released from the inward flow of sodium along its
electrochemical gradient is used to drive the outward flow
of protons against its electrochemical gradient

PASSIVE SOLUTE TRANSPORT - SUMMARY
Passive transport moves towards the electrochemical equilibrium.

Simple diffusion depends on the concentration gradient for an uncharged
solute. In case of a charged solute, conc. gradients and electrical effects
contribute to diffusion.

Polar organic solutes (glucose, amino acids) move across membranes with
the help of transporter proteins in the direction of the electrochemical
equilibrium (facilitated diffusion).

The permeability of a membrane for a lipid solute depends on how readily the
the solute enters into and moves across the membrane lipid bilayer simple
diffusion.

For inorganic ions, the permeability depends on the number of ion channels.
Solute transport is active if it can move solutes away from electrochemical equilibrium.

Active transport is primary if the transporter is an ATPase (energy comes
directly from ATP),
example:
Na
+
-K
+
-ATPase is a primary active transport mechanism
Other important ATPases:
Ca
2+
-ATPase in the sarcoplasmic reticulum of muscle cells.
H
+
-K
+
-ATPase is the proton-pump that acidifies stomach content.
vesicular or v-type H
+
-ATPase in gills and kidneys.

Active transport is Secondary if the energy comes from a solute electrochemical gradient
(indirectly from ATP). Organic solutes are pumped by secondary-active transport
mechanism.
The transport of ions can create voltage differences (electrogenic) or not
(electroneutral).
Summary of Active Transport
Nernst Equation: It describes the flux of the ion under combine influence of concentration gradient
and the electric gradient field
Passive transport of ions across the membrane driven by two factors:
a) Concentration gradient b) electrical gradient
The direction of diffusional and electrical fluxes is opposing
J
total
= J
conc.grad
+ J
elec.grad

Z F D
ion

RT
C
d
dX
J
elec..grad

= -
D - diffusion
coefficent of ion
C - conc. of ion
- electrical
gradient(membrane
potential)
X- thickness of
membrane
Z-valency of ion
F-Faraday constant
R-gas constant
T-temp.
At equilibrium J
total
= 0
J
conc.grad
= J
elec.grad

RT
ZF
ln
C
in

C
out

(volt) =
in
-
out
= -
Effect of the passive transport of ions across the semi permeable membrane
- D
ion
dC
dX
J
conc.grad
=
Z F D
ion

RT
C
d
dX
-
J
total

= - D
ion
dC
dX

Nernst Equation : Equilibrium ionic potential of individual ion( E
ion
), when ion
concentration gradient is equivalent to electrical gradient
E
Na =
61.54mV log [Na]o/[Na]I = 62 mV

E
K =
61.54mV log [K]o/[K]I = -80 mV

E
Ca
= 30.77mV log [Ca]o/[Ca]I = 123 mV

C
Cl
= -61.54mV log [Cl]o/[Cl]I = - 65 mV
Ion Inside Outside
(mM) (mM)
K+ 125 5
Na+ 12 120
Cl- 5 125
H2O 55,000 55,000
Anion- 108 0
E
ion
= 2.303 RT/zF log [ion]
o
/[ion]
in
E
ion
= ionic equilibrium potential
Z= charge of ion
F= Faradays constant
T= absolute temperature (0Kelvin/-273C)
R= gas constant
Goldman equation
The membrane potential experimentally measured is often different than the Nernst potential for
any given cell, due to the existence of other ions.
The Goldman equation (also called the Goldman-Hodgkin-Katz equation)which quantitatively
describes the relationship between membrane potential and permeable ions.
According to the Goldman equation, the membrane potential is a compromise between various
equilibrium potentials, each dependent on the membrane permeability and absolute ion
concentration.
For multiple ions, the ion flux through the membrane at the resting state is zero, consider the three
major ions involved in the nerve cell stimulation, Na+, K+, and Cl ions.
where P
Na
, P
K
, and P
Cl
are the relative membrane permeabilities (D/L) of Na
+
, K
+
, and Cl
-
ions,
respectively. [D-diffusion coefficient of ion, L-thickness of ion channel]
Goldman prediction of membrane potential = 84 mV (almost equivalent to equilibrium ionic
potential of K
+
)
P
Na
, P
K
, and P
Cl
= 1:0.1:1
|
|
.
|

\
|
+ +
+ +
=
+ +
+ +
o Cl i Na i K
i Cl O Na O K
m
Cl P Na P K P
Cl P Na P K P
F
RT
V
] [ ] [ ] [
] [ ] [ ] [
ln

Valid when the total membrane current equals zero;

I
m
=I
K
+I
Na
+I
Cl
=0


Goldman-Hodgkin-Katz equation.
|
|
.
|

\
|
+
+
=
+ +
+ +
i i
O O
m
Na K
Na K
F
RT
V
] [ ] [
] [ ] [
ln
o
o
Dependence of the resting
membrane potential on [K+]o
and on the PNa/Pk ratio, a.
The blue line describes an
instance in which there is no
Na+ permeability (i.e.,
PNa/Pk = 0). The three
orange curves describe the
Vm predicted by the GHK
Equation for a values greater
than zero. The deviation of
these orange curves from
linearity is greater at low
values of [K+]o, where the
[K+]o is relatively larger.
Ignoring the contribution Cl
-
for membrane potential development, the Goldman
equation becomes:
Goldman and Nernst equation for membrane potential
Osmotic swelling is an unavoidable problem for all cells
The swelling arises from the presence of negatively-charged proteins( Y
-
) trapped in the
cytoplasm
First, imagine that a water-permeable membrane separates two rigid compartments.
One compartment has a 150 mmolal concentration of NaCl.
The other one has 150 mEq/liter of Na
+
and an equal quantity of anionic charge as protein
however, the protein concentration is only 1 mmolal.
Is there an osmotic gradient?
Is there a solute gradient?
Intermediate conditions: Cl
-
diffused
down its gradient; why did Na
+
move
against its gradient? Notice that
there is now a gradient of electrical
charge this is a Donnan potential.
Now imagine water trying to move
osmotically is there a gradient of
hydrostatic pressure? The system
has come into Gibbs-Donnan
equilibrium all forces are balanced.
Initial conditions
AP=RTAOsm
RT
F
ln
C
Cl,in

C
Cl,out

RT
F
ln
C
Na,in

C
Na,out

-
=
Gibbs-Donnan equilibrium: A
semipermeable membrane
separates two compartments
that have rigid walls and equal
volumes. The membrane is
permeable to Na+, Cl, and water,
but not to the macromolecule Y,
which carries 150 negative
charges.
+ C
Na,out
= C
Na,in
C
Na,total

C
Cl,in

C
Cl,out

C
Na,out

C
Na,in

=
+ C
Cl,out
= C
Cl,in
C
Cl,total

+ C
Y-
= C
Cl,in
C
Cl,out

= C
Na,out
C
Na,in

Animal cells could never attain Gibbs-Donnan Equilibrium
Why not? The plasma membrane cannot sustain a hydrostatic pressure
gradient.
Without the evolution of some means of avoiding Gibbs-Donnan equilibrium,
there would be no protein-containing cells.
The Na
+
/K
+
Pump counteracts G-D equilibration
The Na
+
/K
+
pump undergoes
cycles in which it spends an
ATP to eject 3 Na
+
from the cell
and at the same time to take 2
K
+
into the cell.
On the average, this
counteracts leakage of Na
+
and
K
+
across the membrane down
their electrochemical
gradients.
The bottom-line effect of this is
to make the cell effectively
impermeable to NaCl.
Gibbs-Donnan equilibrium is
not approached and the cell
does not swell, in spite of the
presence of protein anion (X
-
).
When Na/K pump stop
working?
The effect of Gibbs Donnan in the living cell
Cells contain impermeable anions( e,g, proteins, nucleic acids etc.)

If these anions are at equilibrium with out side and inside salt
concentration, the ratio of all monovalent cations ( C
Na,out
/ C
an,in
) would
be the same

There will be osmotic pressure difference causing the cells to swell

Active transport maintains a homeosttaic steady state in which there
is osmotic balance regulating the cell volume
1. What is the resting for Na+ ions, if the outside concentration is 140 mM and the
inside concentration is 50 mM?


2. A cell contains 100 mM of KCl and 500 mM of protein chloride. The outside of the cell
medium has 400 mM KCl. Assuming that the cell membrane is permeable to Cl and K+
ions and impermeable to the protein, determine the equilibrium concentrations and the
membrane potential. Also assume that these compounds can completely dissociate.
Problems: Nernst, Donnan
Ohms law
An open ion channel follows Ohms law!
Electrical circuit model of ion-channel
Reduced circuit obtained by
combining the ion-specific
pathways using the
Goldman equation
Electrophysiologists model the effects of ionic concentration differences, ion channels, and membrane capacitance in
terms of an equivalent circuit, which is intended to represent the electrical properties of a small patch of membrane. The
equivalent circuit consists of a capacitor in parallel with four pathways each consisting of a battery in series with a variable
conductance. The capacitance is determined by the properties of the lipid bilayer, and is taken to be fixed. The voltage of
each ionic pathway is determined by the concentrations of the ion on each side of the membrane. The conductance of each
ionic pathway at any point in time is determined by the states of all the ion channels that are potentially permeable to that
ion, including leakage channels, ligand-gated channels, and voltage-dependent channels.
Equivalent circuit
Electrical equivalent circuit model of membrane: Sodium (Na
+
) and potassium (K
+
)
channels, which are the primary contributors to the nerve action potential, are
represented by their equivalent channel resistance (R
Na
, R
K
), Nernst potentials (E
Na
, E
K
),
and channel currents (I
Na
, I
K
). The leak current (I
L
) represents current due to chloride
ions, calcium, and magnesium to simplify the model and also because these channels
are passive and not voltage-dependent like the Na
+
and K
+
channels.
K
+

(150mM)
K
+

(4mM)
Na
+

(145mM)
Na
+

(20mM)
Na/K-ATPase
1
Ca
++

(0.1M)
Ca
++

(2.5mM)
Na
2- Na/Ca-

Exchanger
3 - Ca -
ATPase
Electrogenic pumps contribute several mv (negative) to resting membrane potential

Na/K ATP ase( 3Na/2K)

Ca- ATPase

Na/Ca-Exchanger(3Na/1Ca)

Electrogenic pump contribution to membrane potential

1. Concentration gradient of Na
+
, K
+
, Ca
+2
across the membrane

2. The relative permebility( electrical conductance ) of each of the ions
( regulated by ion channels)

3. Electrogenic pumps (Na/K - ATPase[3Na in 2K out of the cell], Ca -
ATPase, Na/Ca -exchangers[3Na exchange for 1Ca)

Therefore, to determine E
m
, the individual ion equilibrium potential
are multiplied by relative membrane permeabilities( conductance) and
summed up
What determines transmembrane potential( Em )?
Bulk Transport
Endocytosis occurs when the plasma membrane envelops food particles and
liquids.
1. phagocytosis the cell takes in particulate matter
2. pinocytosis the cell takes in only fluid
3. Receptor-mediated endocytosis specific molecules are taken in after they
bind to a receptor
Exocytosis movement of materials out of the cell

Endocytosis
Exocytosis occurs when material is discharged from the cell.
-vesicles in the cytoplasm fuse with the cell membrane and release their contents to
the exterior of the cell
-used in plants to export cell wall material
-used in animals to secrete hormones, neurotransmitters, digestive enzymes
Exocytosis
Equilibrium is the state of a system without input of energy or matter
from outside.
Passive transport moves towards, active transport can move against the
electrochemical equilibrium of a system.
Uncharged molecules equalize concentrations by simple diffusion, charged
molecules are also moved by electrical forces.
The permeability of a membrane for a solute depends how readily it
dissolves into the membrane and the number of channels (inorganic ions).
Active transport uses metabolic energy (ATP) to transport a solute against
the electochemical gradient.
Organic solutes can be moved across membranes by transporters, but only
towards the electrochemical equilibrium (facilitated diffusion).
Active transport is primary when it directly uses ATP (ATPases), secondary
if the energy for transport comes from a concentration gradient.
MEMBRANE TRANSPORT SUMMARY
Questions

a. Draw a situation where a molecule of NaCl will enter the cell. Assume
that a transport protein is needed
Is the extracellular environment hypo-, hyper-, or isotonic?
Direction of water?

b. What is membrane potential? How it is generated? How this help for
transport of ions?

c. How Carrier mediated transport help transport of Glucose. Is it active
or passive transport process.

d. Describe primary and secondary active transport mechanism with
example.
Problem: Facilitated Diffusion of Glucose
Assume: a concentration of glucose inside the cell of
0.5 millimolar and a concentration of glucose outside
the cell of 5 millimolar (mM) at body temperature of
37C, so an absolute temperature of 37 + 273 = 310K,
and the plasma membrane is permeable to glucose.
Calculate the amount of free energy released.
G = (2)(273+37) x ln (0.5/5)
= (2)(310) x ln (0.1)
= (620)(2.3) = 1426 cal/mole
= 1.4 kcal/mole
(If you prefer to work with log
10
, multiply the log
10
by
2.303, thus log
10
(0.1) = 1; 1 x 2.303 = 2.3)
Because the process proceeds with the release of free
energy, it can proceed spontaneously.

In this case, facilitated diffusion would be required
because glucose is not permeamble to membrane and
needs transport channels to allow it to pass through the
lipid bilayer of the plasma membrane.
Problem: Active Transport of Glucose
Filtration of the blood in the glomeruli of the kidneys produces a nephric filtrate with a
concentration of glucose the same as that of the blood (~ 5 mM). All of this glucose is
normally reclaimed by active transport.
Problem: What is the free energy needed to move glucose
back from the tubular fluid to the blood when the
concentration in the tubular fluid has dropped to 0.005 mM?
The problem is to pump glucose into the cell (where it is about
0.5 mM) and then across the plasma membrane at the
basolateral surface of the cell into the interstitial fluid, where
the glucose concentration is 5 mM (the same as in the blood).
So the total gradient through which the glucose must be
pumped is 0.005 mM -> 5 mM.
G = (2)(310) x ln (5/.005)
= 620 ln (1000) = (620)(6.91) = + 4284 cal/mole
= + 4.3 kcal/mole
Where is the needed energy to come from?
The Na
+
/glucose Cotransporter
The active transport of glucose is mediated by the Na
+
/glucose cotransporter.
This is a symporter; that is, both the sodium ion and the glucose molecule are passing through the membrane in
the same direction:
sodium DOWN its gradient of about
140 mM outside to
10 mM inside
while glucose is going UP its gradient (0.005 mM -> 5 mM).

A mole of sodium ions (Na
+
) moving down this concentration gradient releases 1.6 kcal of free energy.
G = (2)(310) ln (10/140)
= (620) ln (0.07) = (620)( 2.64) = 1637 cal/mole
= 1.6 kcal/mole
Is this enough to move a mole of glucose?
No, but there is another force we must consider.
Sodium ions carry a single positive charge and the interior of the cell is negatively charged .
So the attraction between opposite charges provides a second force for bringing Na
+
into the cell.
This, too, can be quantified by G = (z)(F)(V
m
)
where z = the charge on the ion (+1 in this case)
F = 23,062 = the calories released as one mole of charge moves down a voltage gradient of 1 volt (1000 mV)
V
m
= the membrane potential, about 70 mV in mammalian cells.
Still not enough to move a mole of glucose, so at least two sodium ion are needed to bring one molecule of
glucose into the cell.
So the driving force for the active transport of glucose (and other small organic molecules, e.g., amino acids) is
the force provided by the movement of sodium ions following their electrochemical gradient. the sodium gradient
across the two sides of the plasma membrane is created by the active transport of Na
+
OUT of the cell by the
Na
+
/K
+
ATPase; the single most profligate user of energy in the our body.

Solution
Problem: Active transport of amino acids
How many sodium ions are needed to provide the free energy to transport a
molecule of glutamic acid from a concentration of 0.1 mM outside the cell to 20
mM inside the cell? Again, assume a temperature of 37C (310K) at pH 7.0.

Solution
At pH of ~7, glutamic acid molecules carry a net charge of minus 1.
So, once again, we have a problem of determining the movement of a molecule
against an electrochemical gradient; that is, against both a concentration
gradient (20/0.1 = 200) and a
electrostatic gradient (moving a negative charge against a voltage of 70 mV).
G = (R)(T) x ln(20/0.1) + (z)(F)(V
m
)
= [(2)(310) x ln(200)] + [(1)(23,062)( 0.070)
= (620) x (5.3) + 1614
= 3286 + 1614
= 4900 or 4.9 kcal/mole Because sodium ions release only 3.3 kcal/mole, at least
2 Na
+
are needed to cotransport one molecule of glutamic acid.