Lecture 20 Membrane Transport Energetics of Transmembrane Transport

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. C 2 High Concentration Difference Low Concentration + E M ry AC tte Ba Co. - C 1 + + + 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 ( C 2 / C 1 ) Contribution of concentration difference R T C 1, C 2 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 k. J/ ( mol V ) (Faraday Constant) Electrical potential difference in Volts

Example: Glucose transport C 1 = 1 m. M, C 2 = 66 m. M, T = 310 K , DV = -50 m. V DG = RT ln ( C 2 / C 1 ) + Z F DV = (8. 314 J/(mol. K))(310 K) ln (66 m. M/1 m. M) + 0 = 8. 314 x 310 x 4. 190 J/mol = 10. 8 k. J/mol (transport from low concentration to high is unfavorable) Example: Na+ transport C 1 = 143 m. M, C 2 = 14 m. M, T = 310 K, DV = -50 m. V DG = RT ln ( C 2 / C 1 ) + Z F DV = (8. 314 J/(mol. K))(310 K) ln (14 m. M / 143 m. M) + (1)(96. 5 k. J/(mol V))(-50 m. V) = 8. 314 x 310 x (-2. 324) J/mol + 96. 5 x ( -50 ) J/mol = -6. 0 k. J/mol + -4. 8 k. J/mol = -10. 8 k. J/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 MDR 1. 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 Ca 2+ 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 Ca 2+ ATPase enables the muscle to relax by pumping the cytosolic calcium into a specialized calcium storage compartment, the sarcoplasmic reticulum (SR). The Ca 2+ ATPase is able to pump calcium against a rather steep gradient. The pump maintains a concentration of Ca 2+ in the cytosol of ~0. 1 m. M, and in the SR it is ~1. 5 m. M, a 15, 000 -fold increase.

Structure of the Ca 2+ ATPase The calcium ATPase is a 110 k. D 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 Ca 2+ binding and release. (A for Actuator)

Mechanism of the Ca 2+ ATPase The catalytic cycle of the pump involves conformational changes between two states E 1 and E 2 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 Ca 2+ ions are transported in each cycle of ATP hydrolysis. 1. Binding of ATP and 2 Ca 2+ ions on the cytoplasmic side to the E 1 state. 2. Phosphorylation of Asp 351 by ATP. 3. Transition to E 2 state: opening of the Ca 2+ 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 E 1 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 Ca 2+ 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: C 1 = 1 m. M, C 2 = 66 m. M, T = 310 K , DV = -50 m. V DG = +10. 8 k. J/mol Na+ transport process: C 1 = 143 m. M, C 2 = 14 m. M, T = 310 K, DV = -50 m. V DG = -10. 8 k. J/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 m. V), 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 m. V Nerve Cell Firing (channels) Na+ K+ Up to +30 m. V (propagates) Recovering (Na/K pump) K+ Na+ Back to -60 m. V

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 a 2 bgd 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 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