T J L J I J I J

Figure 22.9 • Representation of the transmission of a nerve impulse along a neuron fiber by saltatory conduction.

below - 70 mV, followed by the return of the membrane to the resting potential (Fig. 22.10). The generation of the action potential from the axon hillock to the terminal end of the nerve may result in the release of neurotransmitters that cross the synaptic cleft to deliver the "message" to the adjacent neuron or target organ.

Neuronal Membrane Ion Permeability During an Action Potential

By definition, the membrane potential is the difference in the polarity of the inside of the cell compared to the outside of the cell, with the outside of the cell conventionally set at 0 mV. During an action potential, the membrane potential changes from a -70 to a +35 mV. Exactly how does a membrane change its electrical potential? It changes its potential by the movement of ions. For an ion to move from one side of the lipophilic membrane to the other, it must go through a channel. There are many specialized protein channels that can change their three-dimensional configuration to allow ions to flow through. If the ion is moving with its concentration gradient, it can simply diffuse through an open channel, no energy would be required. For an ion to move against the electrical gradient, energy is required and the channels are therefore coupled to ATP pumps.

The axolemma is more permeable to K+ ions than to Na+ ions. These ions diffuse out of the neuron through the so-called potassium leak channels, whose opening does not appear to require a specific membrane change. The movement of K+ ions is concentration driven; K+ ions move from inside the neuron, where the concentration is high, to the extracellular fluid, where the concentration is lower. This tendency of K+ ions to leak out of the neuron (driven by the concentration gradient) is balanced to some extent by a limited movement of K+ ions back into the neuron, both by diffusion through K+ channels and by active transport mechanisms such as the sodium/potassium ATPase pump. These movements of K+ result in a potential difference across the membrane, which is a major contributor to the equilibrium potential that exists between the opposite faces

Figure 22.10 • Changes in electrical potential observed during a nerve impulse.

Firing threshold Resting potential

Millivolts (mV)

Time-

Millivolts (mV)

Time-

of a biological membrane in a normal cell at rest with a switched-on sodium pump.

The initial depolarization of the neuron (Fig. 22.10) was shown by Hodgkin and Huxley in 1953 to be a result of increased movement of Na+ into the neuron, which is followed almost immediately by increased movement of K+ ions out of the neuron. It is thought that the action potential is triggered by a stimulation that causes a momentary shift of the membrane potential of a small section of the membrane to a less negative value (depolarization of the membrane). This causes the gated Na+ channels in this section of the membrane to open, which allows Na+ to enter the cell. This process depolarizes the membrane still further, until the action potential reaches a critical value (the firing threshold), when it triggers the opening of large numbers of adjacent Na+ channels so that Na+ ions flood into the axon. This process continues until the membrane potential of this section of membrane reaches the equilibrium potential for Na+ ions when the cell is at rest. At this point, all the Na+ channels of the membrane should be permanently open. This situation is not reached, however, because each channel has an automatic closing mechanism that operates even though the cell membrane is still depolarized. Once closed, the ion channel cannot open again until the membrane potential in its vicinity returns to its original negative value, which is brought about by the leakage of K+ ions out of the neuron through K+ channels. Hodgkin and Huxley showed that a membrane becomes more permeable to K+ ions, a fraction of a millisecond after the Na+ channels have started to open. As a result, K+ ions flow out of the neuron, which reduces the electrical potential of the membrane, and so at the peak of the action potential, the membrane potential has a value of about + 40 mV. The movement of the K+ ions out of the axon, coupled with the automatic closing of the sodium channel gates and the slower action of the sodium/potassium ATPase pump that transports 3 Na+ ions out of the neuron for every 2 K+ ions into the neuron, results in a net flow of positive ions out of the neuron. This briefly hyperpolarizes the membrane and causes the membrane potential to drop below its resting potential. As the sodium channels close and K+ ions flow back into the axon, the membrane potential returns to its resting value. The entire process of depolarization and repolarization is normally accomplished within 1 millisecond.

Ligand-Gated Sodium Channel Structure and Function

Based on mutation studies and electrophysiology studies, a three-dimensional picture relating the structure of the sodium channel to its function is emerging. The sodium channel is a complicated protein with multiple polypeptide sections that are responsible for specific functions of the channel. The channel must (a) be selective for sodium ions, (b) be able to detect and then open when the membrane is slightly depolarized, (c) be able to detect and then close when the membrane becomes hyperpolarized, and (d) convert to a resting state ready to depolarize again. There is much work currently being conducted on all of these functions of the sodium channel.

The mammalian brain sodium channel is comprised of an a subunit and one or more auxiliary 3 subunits (Figs.

22.11-22.12). The a subunit is composed of four domains (DI-DIV) that fold to make the pore that the sodium ions pass through. Each of the four domains is composed of six transmembrane a-helical segments (S1-S6). The 3 sub-units are involved in the kinetics and voltage dependence of sodium channel opening and closing. The 3 subunits have large extracellular domains with many sites of glyco-sylation and only one transmembrane segment.57

The sodium channel contains specific amino acids that act as a selectivity filter, only allowing sodium ions to pass through the channel. The amino acids that make up the selectivity filter of an ion channel are referred to as the P region. Sodium channels have a P region that gives specificity to sodium ions, whereas potassium and calcium channels have their own P regions that confer selectivity for their respective ions. The selectivity filter of the sodium channel is composed of two rings made up of amino acids from the four homologous domains (DI-DIV). The first ring is composed entirely of negatively charged amino acids. Approximately two to three amino acids deeper into the pore, the second ring is found. The second ring of the P region of the sodium channel is composed of the amino acid sequence DEKA (Asp Glu Lys Ala), whereas the P region of a calcium channel is EEEE (Glu Glu Glu Glu).58 By selectively mutating the four amino acids from DEKA to EEEE, selectivity for calcium could be conferred to the sodium channels.59 Other studies also showed that when external solutions where made highly acidic the negative charges of the selectivity filter amino acids could be neutralized (COO- ^ COOH) and ion conductivity decreased.58

The sodium channel must also be able to change conformations in response to small changes in the membrane potential. How do sodium channels detect voltage changes and then change shape in response to them? The voltage sensing units of the sodium channels are the S4 segments of the a subunit. These segments contain positively charged amino acids at every third residue. It is postulated that the S4 segments move in response to the change in the local membrane potential and cause a further conformational change that opens the gate of the sodium channel, thus allowing sodium to flow in.60 The S4 voltage sensors are also responsible for causing conformational changes in the receptor that close the channel to sodium conductance. Exactly how the conformational changes occur is being studied (Fig. 22.12).

Further down the pore of the sodium channel, beyond the selectivity filter lays the putative local anesthetic binding site (Fig. 22.13). Site directed mutagenesis studies and molecular modeling studies suggest that local anesthetic binding involves multiple interactions. The positively charged nitrogen of the local anesthetic molecule may form a cation - w electron interaction with a phenylalanine residue from the DIVS6 domain.61,62 The aromatic ring of the anesthetic may also interact with a tyrosine amino acid in the DIVS6 domain. The putative local anesthetic binding site is believed to involve the S6 subunits of the a DI, DIII, and DIV domains. The exact amino acids involved depend on the source and the state of the sodium channel being studied. These studies also suggest that the positively charged nitrogen of the local anesthetic may lie in the center of the pore to create an electrostatic repulsive force that, in addition to the steric block, would prevent sodium ion passage through the pore (Fig. 22.13).57

02 a 01

02 a 01

Modulation

Figure 22.11 • Structure of voltage-gated sodium channels. Schematic representation of the sodium-channel subunits. The a subunit of the Nav1.2 channel is illustrated together with the p1 and p2 subunits; the extracellular domains of the p subunits are shown as immunoglobulin-like folds, which interact with the loops in the a subunits as shown. Roman numerals indicate the domains of the a subunit; segments 5 and 6 (dark gray) are the pore-lining segments and the S4 helices (light gray) make up the voltage sensors. Light gray circles in the intracellular loops of domains III and IV indicate the in-activation gate IFM motif and its receptor (h, inactivation gate); P, phosphorylation sites, in dark gray circles, sites for protein kinase A; in dark gray diamonds, sites for protein kinase C; f, probable W-linked glycosylation site. The circles in the re-entrant loops in each domain represent the amino acids that form the ion selectivity filter (the outer rings have the sequence EEDD and inner rings DEKA). (Reprinted from Catterall, W. A.: Ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26:13-25, 2000, with permission from Elsevier.)

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