INDEPENDENT VERSUS COUPLED INACTIVATION IN SODIUM-CHANNELS - ROLE OF THE DOMAIN-2 S4 SEGMENT

The voltage sensor of the sodium channel is mainly comprised of four p ositively charged S4 segments. Depolarization causes an outward moveme nt of S4 segments, and this movement is coupled with opening of the ch annel. A mutation that substitutes a cysteine for the outermost argini ne in the S4 segment of the second domain (D2:R1C) results in a channe l with biophysical properties similar to those of wild-type channels. Chemical modification of this cysteine with methanethiosulfonate ethyl trimethylammonium (MTSET) causes a hyperpolarizing shift of both the p eak current-voltage relationship and the kinetics of activation, where as the time constant of inactivation is not changed substantially. A c onventional steady state inactivation protocol surprisingly produces a n increase of the peak current at -20 mV when the 300-ms prepulse is d epolarized from -190 to -110 mV. Further depolarization reduces the cu rrent, as expected for steady state inactivation. Recovery from inacti vation in modified channels is also nonmonotonic at voltages more hype rpolarized than -100 mV. At -180 mV, for example, the amplitude of the recovering current is briefly almost twice as large as it was before the channels inactivated. These data can be explained readily if MTSET modification not only shifts the movement of D2/S4 to more hyperpolar ized potentials, but also makes the movement sluggish. This behavior a llows inactivation to have faster kinetics than activation, as in the HERG potassium channel. Because of the unique properties of the modifi ed mutant, we were able to estimate the voltage dependence and kinetic s of the movement of this single S4 segment. The data suggest that mov ement of modified D2/S4 is a first-order process and that rate constan ts for outward and inward movement are each exponential functions of m embrane potential. Our results show that D2/S4 is intimately involved with activation but plays little role in either inactivation or recove ry from inactivation.