The screwhelical mechanism of voltagegating

It is clear from the structural studies that every voltage-gated ion channel incorporates four S4 voltage-sensors that operate in parallel, there being as shown in Fig. 5.2 an identical one in each domain of the tetrameric potassium channels. In the monomeric sodium channels there are four sensors that vary

0.20

Time (jjs)

Fig. 5.3. Superimposed family of sodium gating currents recorded from a squid axon dialysed with 350 mM TMAF (tetramethylammonium fluoride) and bathed in an artificial sea water with the sodium ions replaced by Tris and containing 1 ^M TTX. Test pulses -57 to +83 mV in steps of 10 mV. Holding potential -80 mV. Temperature 10°C. Number of sweeps averaged was 32. (b) Initial rise of /Na for a pulse to -23 mV after subtraction of gating current, in another axon bathed in an artificial sea water in which 4/5 of the sodium ions were replaced by Tris. From Keynes & Elinder (1998).

between four and eight in the number of positive charges carried in the individual domains, while the members of the related family of calcium channels, not shown in Fig. 5.2, carry four or five charges. In order for a single channel to be opened, it is agreed from measurements on Shaker K+ channels, and also on sodium channels from squid axons and from non-inactivating skeletal muscle fibres expressed in oocytes, that 12 or slightly more electronic charges (e0) have to be transferred, so that the transfer brought about by each S4 unit is 3 e0. Careful measurements of gating charge kinetics in squid axons have shown that each separate step in the opening, inactivation and late reopening of the channels involves just 1 e0 and no more. Studies of gating current noise in Shaker K+ channels and sodium channels expressed in oocytes have suggested the existence of 'single shots' of current carrying 2.3 e0 accompanied by others carrying a single charge, but these extra large pulses do not appear to be created by a single voltage-sensor, and are more likely to arise from an especially close temporal grouping of the four 1 e0 pulses responsible for the third transitions in the S4 segments.

The screw-helical theory of voltage-gating put forward in 1986 by Catterall, Guy and others suggested that the positive charges located in every third position on S4 were arranged on the a-helix as a spiral ribbon, and in the resting state were paired with fixed negative charges on neighbouring helices. When a depolarization of the membrane increased the outward force acting on the positive charges, the S4 helix was free to undergo a screw-like motion by rotating through 60° and moving 0.45nm outwards, so that each movable positive charge could proceed to pair up with the next fixed negative charge. Such a movement would bring an unpaired positive charge to the outer surface of the membrane, and at the same time would create an unpaired negative charge at the inner surface, so transferring approximately one electronic charge outwards. By twisting three times in succession through 60°, each S4 segment could therefore transfer a total of 3 e0 as is observed.

This proposition meets with difficulty if the hydrophobic central pore extends across the whole depth of the membrane, because there are more positive charges on the S4 segments than negative charges on the other five transmembrane segments to pair up with them. However, the elegant studies on the accessibility of the S4 charges to hydrophilic reagents on both sides of the membrane, carried out by Yang, George & Horn (1996) on sodium channels, and by Larsson et al. (1996) on Shaker K+ channels, have demonstrated that the hydrophobic section of the central pore must be appreciably shorter than was originally thought. Taking this into account, the revised version of the screw-helical gating mechanism illustrated in Fig. 5.4 could explain how the S4 units might move in three or more discrete steps each transferring 1 eo.

Closed °Pen

Closed intermediate depolarized

Closed °Pen

Closed intermediate depolarized

Fig. 5.4. A strictly diagrammatic representation of the screw-helical outward movement of the positive charges carried by S4 in a Shaker K+ channel. Each outward step transfers one electronic charge from the interior of the cell to the external solution. The charges are represented as close to the surface of the a-helix, but in fact project on flexible connections about 0.6 nm in length. The three negative charges shown on the left occupy fixed positions on S2 and S3, and salt bridges are formed with two of the positive charges in the closed hyperpolarized state and three in the intermediate and open states. From Keynes & Elinder (1999).

Fig. 5.4. A strictly diagrammatic representation of the screw-helical outward movement of the positive charges carried by S4 in a Shaker K+ channel. Each outward step transfers one electronic charge from the interior of the cell to the external solution. The charges are represented as close to the surface of the a-helix, but in fact project on flexible connections about 0.6 nm in length. The three negative charges shown on the left occupy fixed positions on S2 and S3, and salt bridges are formed with two of the positive charges in the closed hyperpolarized state and three in the intermediate and open states. From Keynes & Elinder (1999).

Their completion would be followed allosterically by a further conformational change that is electrically invisible, and whose precise nature remains to be defined, which finally opens the channel.

A vitally important feature of the system is that the individual steps made by the voltage-sensors are stabilized by the formation of salt bridges between three of the movable positive charges and three fixed negative charges located on segments S2 and S3. As may be seen in Fig. 5.2, two of the negative charges are located respectively nine places from the inner end of S2 and six places from the inner end of S3, while the third ones are more widely scattered near the outer ends of S2 or S3. There is evidence from several sources for the occurrence in the open state of ion pairing between the particular residues shown in Fig. 5.2, while a modelling exercise has shown that these interactions would be geometrically compatible with a structure in which S2 and S3 are parallel a-helices, while S4 is another a-helix tilted to cross them at an angle. In the absence of high resolution structural evidence, the correctness of this proposition still lacks final proof, but a powerful argument in its support is, as pointed out by Keynes & Elinder (1999), that it fits satisfactorily with the nearly perfect conservation of the location of the negative charges in the sequences of S2 and S3 in every voltage-gated ion channel, whether selective for K+, Na+ or Ca2+, across the entire animal kingdom.

It is now accepted that, unlike those in squid that inactivate only very slowly, there are other types of potassium channel that display a rapid but voltage-independent inactivation. Nevertheless, in sodium channels the process of inactivation as well as activation is voltage-dependent and is controlled primarily by voltage-sensor IVS4 acting in conjunction with the internal link between domains III and IV seen in Fig. 5.1. In order to account for the extra length of IVS4, inactivation probably involves a fourth transfer of 1 eo in this voltage-sensor accompanied by another undefined conformational change affecting the hydration pathway of the central activation gate. A fifth transfer of a single charge brings about delayed reopenings of the channel that account for the small inward flow of Na current taking place during the inactivated steady state.

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