The voltage sensor of the sodium channel is mainly comprised of four positively charged S4 segments. Depolarization causes an outward movement of S4 segments, and this movement is coupled with opening of the channel. A mutation that substitutes a cysteine for the outermost arginine in the S4 segment of the second domain (D2:R1C) results in a channel with biophysical properties similar to those of wild-type channels. Chemical modification of this cysteine with methanethiosulfonate-ethyltrimethylammonium (MTSET) causes a hyperpolarizing shift of both the peak current–voltage relationship and the kinetics of activation, whereas the time constant of inactivation is not changed substantially. A conventional steady state inactivation protocol surprisingly produces an increase of the peak current at −20 mV when the 300-ms prepulse is depolarized from −190 to −110 mV. Further depolarization reduces the current, as expected for steady state inactivation. Recovery from inactivation in modified channels is also nonmonotonic at voltages more hyperpolarized 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 hyperpolarized potentials, but also makes the movement sluggish. This behavior allows inactivation to have faster kinetics than activation, as in the HERG potassium channel. Because of the unique properties of the modified mutant, we were able to estimate the voltage dependence and kinetics of the movement of this single S4 segment. The data suggest that movement of modified D2/S4 is a first-order process and that rate constants for outward and inward movement are each exponential functions of membrane potential. Our results show that D2/S4 is intimately involved with activation but plays little role in either inactivation or recovery from inactivation
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