Transcript Slide 1

Lecture 20:
Membrane Transport
Energetics of Transmembrane Transport
Active Transport
Passive Transport
Molecules Diffuse from High to Low Concentration
There is an energy difference associated with a transmembrane concentration
difference. The energy difference become zero at equilibrium, when the
two concentrations are equal.
Equilibrium
High
Concentration
Equal
Low Concentration
Free
Energy
Concentrations
Concentration
Difference
Equilibrium
Progress of Reaction
Charged Molecules Create an Electrical Energy Difference
Charged molecules repel each other, increasing the energy difference
of a transmembrane concentration gradient.
C2
High
Concentration
Concentration
Difference
Low Concentration
+
-
C1
+
+
+
+
+
Voltage
Difference
+
+
Energy is Required to Create a Transmembrane Concentration Gradient
The energy stored in a transmembrane concentration gradient
depends on the concentration difference and the electrical potential difference.
DG = RT ln ( C2 / C1 )
Contribution of
concentration
difference
R
T
C1,C2
Z
F
DV
+
Z F DV
Contribution of
electrical
potential
difference
8.314 J/ ( mol K ) (Gas Constant)
Temperature in Kelvin
Concentrations
Charge on molecule
96.5 kJ/ ( mol V ) (Faraday Constant)
Electrical potential difference in Volts
Example: Glucose transport
C1 = 1 mM, C2 = 66 mM, T = 310K , DV = -50 mV
DG = RT ln ( C2 / C1 ) + Z F DV
= (8.314 J/(molK))(310K) ln (66mM/1mM) + 0
= 8.314 x 310 x 4.190 J/mol
= 10.8 kJ/mol
(transport from low concentration to high is unfavorable)
Example: Na+ transport
C1 = 143 mM, C2 = 14 mM, T = 310K, DV = -50 mV
DG = RT ln ( C2 / C1 ) + Z F DV
= (8.314 J/(molK))(310K) ln (14 mM / 143 mM) + (1)(96.5 kJ/(mol V))(-50mV)
= 8.314 x 310 x (-2.324) J/mol + 96.5 x ( -50 ) J/mol
= -6.0 kJ/mol + -4.8 kJ/mol
= -10.8 kJ/mol
(transport from high to low concentration is favorable,
especially if aided by the electrical potential)
ABC Transporters
The ABC transporters are a large family of proteins which have the
function of transporting substances across a membrane using the energy
of ATP hydrolysis. ABC stands for ATP Binding Cassette.
An example is the multidrug resistance protein
MDR1. This protein is capable of using ATP to
expel a wide variety of small molecules from
cells. Cultured cancer cells can become
resistant to a variety of drugs through its action.
Similar proteins are implicated in the acquisition
of drug resistance in a variety of cancers and
can contribute to the failure of chemotherapy.
Drugs that specifically inhibit these proteins
are under development.
P-type ATPases as Examples of Ion Pumps
Another large family of transmembrane transporters is the Ptype ATPases. Proteins in this family transport ions and other
substances across membranes using the energy of ATP
hydrolysis.
An example is the Ca2+ ATPase found in the
membrane of the sarcoplasmic reticulum.
Muscle contraction is triggered by high
levels of calcium ions in the cytosol of
muscle cells. The Ca2+ ATPase enables the
muscle to relax by pumping the cytosolic
calcium into a specialized calcium storage
compartment, the sarcoplasmic reticulum
(SR).
The Ca2+ ATPase is able to pump calcium
against a rather steep gradient. The pump
maintains a concentration of Ca2+ in the
cytosol of ~0.1mM, and in the SR it is ~1.5
mM, a 15,000-fold increase.
Structure of the Ca2+ ATPase
The calcium ATPase is a 110 kD protein with
4 domains.
The membrane-spanning domain contains 10
a-helices.
The three cytosolic domains (N,P,A domains)
have different functions.
The N domain binds ATP. (N for Nucleotide-binding)
The P domain contains a phosphorylation site (Asp
351) which accepts a phosphate from ATP during the
catalytic cycle. (P for Phosphorylation)
The A domain regulates Ca2+ binding and release.
(A for Actuator)
Mechanism of the Ca2+ ATPase
The catalytic cycle of the pump involves
conformational changes between two states E1
and E2 in which the calcium-binding sites are
accessible to different sides of the membrane.
ATP hydrolysis provides the energy to power
these changes in conformation. Two Ca2+ ions
are transported in each cycle of ATP hydrolysis.
1. Binding of ATP and 2
Ca2+ ions on the
cytoplasmic side to the
E1 state.
2. Phosphorylation of Asp
351 by ATP.
3. Transition to E2 state:
opening of the Ca2+ site
to the luminal side of the
membrane. (eversion)
4. Calcium release (low
affinity due to
phosphorylation.)
5. Phosphate hydrolysis
and release.
6. Return to E1 state.
(eversion)
The Na/K Pump
Another P-type ATPase is the Na/K pump.
This enzyme uses the energy of ATP hydrolysis to
simultaneously transport 3 Na+ ions out of the cell
and 2 K+ ions into the cell.
The concentration gradient established by the
Na/K pump enables nerve impulses and muscle
contraction, controls cell volume, and provides
energy for uptake of sugars and amino acids.
The importance of this protein is indicated by the
fact that fully one-third of the ATP consumed by a
resting animal is used to establish these
concentration gradients.
The Na/K pump has very similar mechanism to the
Ca2+ ATPase.
Primary and Secondary Transporters:
The ABC transporters and the P-type ATPases are two families of proteins
which use energy in the form of ATP to establish a transmembrane
concentration gradient. These are examples of primary transporters.
Another class of energy-requiring transporters are the secondary
transporters. This class uses energy in the form of a transmembrane
concentration gradient of one substance to transport another substance.
An example of a secondary transporter is the Na+-glucose symporter,
which transfers glucose from low to high concentration (“uphill”)
using the energy obtained by transferring Na+ from high to low
concentration (“downhill”).
Low glucose
High Sodium
Low sodium
High glucose
Secondary Transporters
Secondary transporters use one concentration gradient to create another.
One gradient is the “fuel” molecules, whereas the other is the “cargo”
molecules.
Symporters transport the “fuel” and “cargo” molecules in the same
direction.
Antiporters transport the “fuel” and “cargo” molecules in opposite
directions.
Energetics of Glucose Transport
Glucose uptake by the Na+/Glucose symport uses the Na+ gradient
established by the Na/K pump to power glucose uptake.
The total free energy change is the sum of the energies in the two
transport processes.
Glucose transport process:
C1 = 1 mM, C2 = 66 mM, T = 310K , DV = -50 mV
DG = +10.8 kJ/mol
Na+ transport process:
C1 = 143 mM, C2 = 14 mM, T = 310K, DV = -50 mV DG = -10.8 kJ/mol
The net free energy change is close to zero.
The Na+ gradient “pays” for the glucose transport.
Passive Transport
Primary and secondary transporters use energy to transport ions or
molecules “uphill” against a concentration gradient.
Passive transport proteins facilitate the rapid “downhill” flow of
ions or molecules, from higher concentration to lower concentration.
Flow ceases when equilibrium is reached, namely when the
DG of transport is zero.
In the absence of an electrical potential, equilibrium occurs
when the concentrations on either side of the membrane are equal.
Passive transporters only change the rate of approach to equilibrium
but not the equilibrium itself.
An example of passive transporters are ion channels.
Ion Channels
Ion channels are proteins that enable rapid flows of ions through membranes,
often at rates close to free diffusion in solution. Examples include Na+ channels,
K+ channels, and Cl- channels.
Ion passage is regulated.
Ligand-gated channels allow ion passage in response to binding of signal
molecules.
Voltage-gated channels allow ion passage in response to changes in the
transmembrane electrical potential.
Ion channels are very selective about ion type.
Ion Channels Enable Nerve Cells to Transmit Electrical Impulses
In the resting state, nerve cells have an electrically polarized membrane
(~-60 mV), with Na+ and K+ gradients established by the Na/K pump.
A nerve impulse is a propagating electrical disturbance of the membrane
potential caused by the flow of ions across the membrane.
The membrane becomes temporarily permeable to ions when ion
channels open.
Resting
High Na+
Low K+
High K+
Low Na+
-60 mV
Nerve Cell
Firing
(channels)
Na+
K+
Up to +30 mV
(propagates)
Recovering
(Na/K pump)
K+
Na+
Back to -60 mV
The Acetylcholine Receptor: A Ligand-Gated Channel
Nerve impulses are traverse synapses by causing
fusion of vesicles containing neurotransmitters
(such as acetylcholine) with the plasma
membrane, releasing them into the synaptic cleft.
Acetylcholine diffuses across the synapse to the
postsynaptic membrane, and binds to the
acetylcholine receptor. The acetylcholine receptor
is an ion channel which allows passage of Na+
and K+ across the membrane.
Binding of acetylcholine to the
acetylcholine receptor opens
the channel and allows Na+ to
flow into the cell and K+ to flow
out. This depolarization triggers
an action potential which
propagates down the nerve cell.
Model for Structural Basis of Acetylcholine Receptor Action
The acetylcholine receptor has the quaternary structure a2bgd and the
5 chains are arranged in a ring with approximate 5-fold symmetry,
forming a pore at the center. Acetylcholine binds at the a-d and
a-g interfaces and induces an allosteric shift between the closed
and opened conformations. Transmembrane helices lining the pore
are believed to rotate upon acetylcholine binding.
In the closed state, hydrophobic side-chains from these helices
occlude the pore.
In the open state, the rotation of the helices shifts the hydrophobic
side chains out of the way and causes the walls of the pore to be lined
with hydrophilic side-chains, facilitating ion transport.
Sodium and Potassium Channels: Voltage-Gated Channels
Sodium and Potassium channels in nerve cell membranes are sensitive to
the membrane potential. They open when the membrane is depolarized.
The ions that flow through open channels further depolarize the membrane,
propagating the disturbance down the nerve cell.
Na+ channels open first, initially causing the membrane potential to become
less negative. These close spontaneously as the K+ channels open- the
passage of K+ ions in the opposite direction allows the membrane potential
to return to near its starting value.
Only a miniscule fraction of the Na+ and K+ ions in the cell actually cross the
membrane. Small ion fluxes can change the transmembrane voltage
very effectively.
Relationships of Voltage-Gated Channels
Eukaryotic sodium channels consist
of 4 pseudo-repeats, each of which
probably contains 6 transmembrane
helices.
Sequence similarities between sodium channels and potassium channels
suggest a similar structure. Bacterial potassium channels are simpler,
containing only the pore-forming transmembrane helices 5 and 6. These
simpler molecules have proved suitable for structural studies.
Structure of the Bacterial Potassium Channel
The bacterial potassium channel forms a tetramer of subunits each containing
2 transmembrane helices- the pore is formed at the center of the tetramer.
A water-filled channel in the lower parts of the molecule can allow hydrated K+
to enter.
But a constriction at the top is too narrow for hydrated potassium to enter.
K+ ions are desolvated (very unfavorable) during passage through the
constriction in the channel, but polar groups in the constriction region
provide interactions which stabilize the desolvated ions.
Selectivity
The K+ channel is 100-fold more permeable to K+ ions than Na+ ions.
The selectivity can be understood as a difference in stabilization of
desolvated K+ and Na+ ions when passing through the constriction.
K+ ions are larger and the protein is capable
of forming sufficient interactions with K+ that
offset the energetic cost of breaking bonds to
water, enabling K+ to pass through.
Na+ is smaller and the protein is not capable of
forming sufficient interactions with Na+ to
offset the larger energetic cost of desolvating
Na+, making it more difficult for Na+ to pass
through.
Summary:
Transmembrane concentration differences are not at equilibrium and
therefore are a source of energy which can be used for other purposes.
Two categories of active transporters are pumps, which use the energy
of ATP hydrolysis to transport molecules across membranes, and
secondary transporters, which use the energy stored in a transmembrane
concentration gradient.
Passive transporters facilitate diffusion of molecules through membranes
and allow equilibrium to be more rapidly established. Ion channels
Are examples of passive transporters and play important roles in
nerve cell function.
Key Concepts:
Thermodynamics of transport
Active Transport
Primary transporters (pumps)
Secondary transporters (antiporters, symporters)
Passive transport
Ion channels
Ligand-gated and Voltage-gated ion channels