Molecular Properties of the Voltagesensitive Sodium Channel

Ion channels are proteins, and like all proteins, the sequence of amino acids making up the protein of a particular ion channel is coded for by a particular gene. Thus, it is possible to study the properties of ion channels by applying techniques of molecular biology to isolate and analyze the corresponding gene. This has been done for an increasing variety of ion channels, including the voltage-sensitive sodium channel that underlies the action potential. The sodium channel is a large protein, containing some 2000 individual amino acids. A model of how the protein folds up into a three-dimensional structure has been developed, and this model is summarized schematically in Figure 6-11. According to the model, the protein consists of four distinct regions, called domains. Each domain consists of six separate segments that extend all the way across the plasma membrane (transmembrane segments), which are labeled S1 through S6. Within a domain, the protein threads its way through the membrane six times (Figure 6-11). The amino-acid sequences of each of the six transmembrane segments within a particular domain are similar to the corresponding segments in the other domains. Thus, the overall structure of the channel can be thought of as a series of six transmembrane segments, repeated four times.

It is thought that the four domains aggregate in a circular pattern as shown in Figure 6-11b to form the pore of the channel. The lining of the pore determines the permeation properties of the channel and gives the channel its selectivity for sodium ions. Interestingly, it seems that the lining is actually made up of the external loop connecting segments S5 and S6 within each of the four domains. In order for this external loop to form the transmembrane pore through which sodium ions cross the membrane, it must fold down into the pore in the manner shown schematically in Figure 6-11c.

One important question about the channel is what part of the protein is responsible for detecting changes in the membrane potential and thus imparts voltage sensitivity to the channel. Here, attention has focused on the fourth transmembrane segment of each domain, segment S4, which is marked with a + in Figure 6-11a. Segment S4 has an unusual accumulation of positive charge (because of positively charged arginine and lysine residues in that part of the protein), which should give S4 high sensitivity to the electric field across the membrane. Also, the positive charges in S4 are located within the membrane, which is the correct position to be acted upon by the transmembrane voltage. To test the idea that the charges in S4 are the voltage sensors, W. Stuhmer and co-workers have constructed artificial sodium channels by altering the DNA so that one or more of the arginines or lysines in S4 was replaced with a neutral or negatively charged amino acid. These artificial channels were less voltage dependent than the normal channels, suggesting that the charges in S4 are indeed the voltage sensors that detect depolarization of the membrane and activate the opening of the m gate.

Another important issue is to establish the identity of the sodium inactiva-tion gate, the h gate. Here, Stuhmer and co-workers found that the part of the protein connecting domains III and IV (marked with * in Figure 6-11) is important. If that region was deleted or altered, the inactivation process was greatly impaired, though activation seemed normal. Note that this part of the protein is on the intracellular side of the membrane, which is where we have drawn the

Plasma membrane

Domain I Domain II Domain III Domain IV

Outside

Domain I Domain II Domain III Domain IV

Outside

Plasma membrane

Pore

Plasma membrane

Pore

S4 S3 S2 SI

S3 SI

Domain II

Domain I

Figure 6-11 The molecular structure of the voltage-sensitive sodium channel. (a) The molecule consists of four domains of similar make-up, labeled with Roman numerals. Each domain has six transmembrane segments (S1-S6). The highly positively charged segment S4 is indicated in each domain by a plus sign (+). The linkage between domains III and IV, indicated by an asterisk (*), is involved in inactivation gating. (b) The domains are shown in a linear arrangement in (a), but in reality, the domains likely form a circular arrangement with the pore at the center. (c) The extracellular loop between S5 and S6 of each domain may fold in as indicated to line the entry to the pore. This region controls the ionic selectivity of the channel.

h gate in our cartoon diagrams of sodium channels in earlier figures in this chapter.

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