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Voltage-gated Sodium Channel

The voltage-gated Na+ channel is an allosteric, multi-unit integral membrane protein that selectively allows the passive diffusion of Na+ ions through it. The direction and resulting equilibrium depend not on the application of energy but on the electrochemical driving force across the cell membrane. The channel is switched between open and closed state by changes in the state of membrane depolarization. These changes alter the conformation of the membrane, resulting in changes in ion permeability. The conductances of the individual channels can be measured using the technique of patch clamping.

Structurally, the functional unit of the voltage-gated Na+ channel is a single poly-peptide chain arranged in the membrane as 24 transmembrane segments, forming four domains, each consisting of six transmembrane segments. Within each domain is the so-called S4 region, whose amino acid sequence is highly homologous to that of the other S4 regions in the channel. The S4 region contains several positively charged amino acids, which detect membrane potential changes, and which then react to induce the conformational changes which cause activation. The S5-S6 linking units within each domain are thought to form the pathway for the ions. Near the amino-terminus is the so-called ball and chain region, which can block the open channel to inactivate it.

The channel is switched between the probability of existing in one of three main functional states by changes in the state of membrane depolarization. More positive membrane potentials (+Vm) will shift the probability towards an open activated state. If the positive potential is maintained, the channel becomes inactivated in the open state. Negative membrane potentials (-Vm) drive the gating sequence in the opposite direction, and will increase the probability that the channel will shift from the open, inactivated state to the open activated state, and then to the closed resting state.

Patch clamping techniques can be used to study the state of the ion channel. An area of the membrane is gently sucked up against a glass microelectrode with a heat-polished tip. A very tight bond is formed between the surface of the glass electrode and the membrane that does not allow any ion flow between them. The piece of membrane attached to the electrode may become detached from the rest of the membrane, hence the name patch, which refers to the piece of membrane being studied. A very high electrical resistance, measured as high as 1011 ohms, is generated between the inside of the electrode and the extracellular fluid of the membrane, which is why the seal between the two is sometimes referred to as the gi-gaohm seal. Leakage from the electrode tip is negligible, and so the voltage inside the electrode can be clamped.

If the piece of membrane sucked up includes an ion channel, the channel affords virtually the only means of current flow when it opens, because it offers the lowest resistance. Therefore, if the channel opens due to a change in the applied voltage, the current flow can be detected using a sensitive amplifier. Several trials of the current flow measurement can be made using a single channel, and the results summed and averaged.

Exchange Pump

The Na+/K+ ATPase Pump

External energy sources are required in addition to the selective opening of ion channels to maintain concentrations of ions in extracellular and intracellular compartments against their concentration gradients. Energy is supplied as ATP, which is used by ion pumps, such as the Na+/K+ ATPase pump.

The Na+/K+ ATPase pump is an integral membrane protein consisting of a and p chains. The a chains have ATPase activity on the cytoplasmic surface, and commonly have eight transmembrane segments. The pump actively translocates Na+ to the extracellular fluid, and K+ in the opposite direction. The p chains, which have sugar units on their external surface, do not appear to be necessary for ion transport. Subtle changes in conformation of the transport protein may be involved in ion translocation. In one state, the protein appears to possess three high-affinity binding sites for the Na+ ion on the cytoplasmic side, and two high-affinity binding sites for K+ on the extracellular side. Hydrolysis of ATP to ADP and inorganic phosphate (Pi) provides energy to alter the conformation of the protein, and to drive the pumping of the ions in opposite directions (the so-called 'antiport' action). ATP phosphor-ylates the ATPase on a specific aspartate residue on the enzyme. Once through the pump, Na+ ions are discharged into the extracellular fluid and K+ into the cytoplasm since the binding sites for them on these sides of the pump are of lower affinity for the ions. Sequentially, it is believed that the ATPase switches between at least two conformations, called E1 and E2. Therefore there may be at least four different conformations: E1, E1-P, E2, and E2-P.

Ion transport by the Na+/K+ ATPase pump (and other pumps) is much slower than through ion channels. Up to 106 Na+ ions can be transported through an ion channel per second, whereas about 103

Greenstein, Color Atlas of Neuroscience © 2000 Thieme

Na+ions are transported through the pump per second.

For the Na+/K+ ATPase pump to work, it requires energy in the form of ATP, which is hydrolyzed to ADP and Pj by an ATPase on the cytoplasmic surface of the a chain. As much as a third of all body utilization of ATP may be allocated to the maintenance of pump function. The ATPase is phosphor-ylated provided that Na+ and Mg2+ are present, and then dephosphorylated if K+ is present.

Pharmacological agents can inhibit the action of the pump. The ATPase activity is inhibited, for example, by the vanadate ion, which binds to the phosphorylation sites on the cytoplasmic side and thus inhibits enzyme phosphorylation. The drug was used to localize the site of phosphorylation to the cytoplasmic side of the pump. The cardiotonic steroids digoxin and digi-toxigenin are inhibitors of the pump. Digoxin binds to specific sites on the extracellular surface of the a subunits and blocks pump action, apparently by stabilizing the pump in the E2-P configuration. Inhibition of the pump leads to higher in-tracellular Na+ concentrations, which diminishes the Na+ gradient, and this in turn slows the rate of extrusion of Ca2+ions from the cell by the Na+/Ca2+ exchange pump. The raised intracellular Ca2+ increases the force of concentration of cardiac cells in conditions such as congestive heart failure.

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Cerebral Membrene

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