The sodium gating current

An essential avenue towards a detailed understanding of the mode of operation of voltage-gated ion channels is to investigate the kinetics of the macroscopic ion currents in the manner adopted in the classical paper of Hodgkin & Huxley (1952). However, such studies are in one respect limited in their scope, because they throw light only on the kinetics of the open state, and reveal relatively little about the series of closed states through which the system must certainly pass during activation and inactivation.

It was pointed out by Hodgkin & Huxley that the voltage-dependence of the sodium conductance implies that the gating mechanism itself is charged, and further that whenever a change in membrane potential operates the gate, there must be a movement down the electric field of the charged side groups that it carries, giving rise to a displacement current that necessarily precedes the flow of ion current. The asymmetry current or gating current, as it has come to be called, remained undetected for some years because the corresponding transfer of charge within the membrane is so small compared with the transfer of ions across it. However, in 1973/4 Armstrong and Bezanilla working at Woods Hole, followed shortly afterwards by Keynes and Rojas at Plymouth, succeeded in recording an asymmetrical component of the sodium current in voltage-clamped squid axons. To achieve this, it was first necessary to block the passage of Na+ ions through the open sodium channels by bathing the axon in a sodium-free solution containing a high concentration of TTX, which fortunately turned out to seal up the channel at its mouth without interfering with the operation of the voltage-gate. The K+ channels were also blocked by perfusion or internal dialysis with a caesium or tetramethylammonium fluoride solution. The symmetrical capacity transient that arises when charging or discharging the passive membrane capacity was eliminated electronically.

Although gating currents have now been recorded at the node of Ranvier, in various other types of nerve and muscle, and in sodium and potassium channels expressed in Xenopus oocytes, the preparation that enables the best possible time resolution to be achieved is still the squid giant axon. Even so, some 20 years elapsed before the kinetics of the sodium gating current were well enough understood for their detailed interpretation in terms of the molecular structure of the channel. The recordings in Fig. 5.3(a) show that although for the most negative test pulses the current rises quickly and decays exponentially, for the pulse to — 17mV a small delayed rise can clearly be seen to come in just after the start of the relaxation. For larger pulses the slowly rising phase becomes increasingly prominent, reaching its peak with a roughly constant delay of about 30 |xs. This initial part of the gating current has been shown to be generated by the first two transitions in the four S4 units operating in parallel. The trace (b) shows that the rise of sodium current itself begins only around 75 |xs after the start of the test pulse, so that the two activating steps are nearly complete before the third and final step opens the channel. These findings have at last provided a satisfactory basis for relating the kinetics of the

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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).

gating current to the kinetics of the open state, and to the structure of the channel and other experimental evidence.

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