Voltage-activated gating of sodium channels

This dissertation addressed the question of sodium channel gating. The study began with an investigation of a human muscle disease--paramyotonia congenita (PC). PC is an autosomal dominant disease in which muscle stiffness is triggered by exposure to cold temperature. Genetically it is caused by point mutations in the human skeletal muscle sodium channel (hSkM1) $\alpha$-subunit. Using recombinant DNA and patch clamp techniques, we compared five PC mutants expressed in tsA201 cells with wildtype hSkM1 channels. PC mutants from diverse locations in the $\alpha$-subunit (A1156T, T1313M, L1433R, R1448H, R1448C) all exhibit a similar disturbance in channel inactivation characterized by a reduced macroscopic rate, accelerated recovery, and altered voltage dependence. There is no significant abnormality in activation of PC mutants. In contrast, mutation T704M from another muscle disease--hyperkalemic periodic paralysis (HYPP)--shows a normal inactivation rate but shifts the steady-state activation and inactivation functions along the voltage axis. This study helps in understanding of the biophysical basis of the clinical manifestations of PC and HYPP. It also revealed Na channel structural parts affecting inactivation gating. Transmembrane segment 4 (S4) has long been believed to be the voltage sensor for voltage-gated ion channels. Although without direct evidence, it had been postulated that S4s move in respond to changes of membrane potential. A further study on the PC mutant R1448C gives the first evidence of voltage dependent S4 movement. Using hydrophilic cysteine reagents (methanethio-sulfonate derivatives--MTSs), we have shown that the modification of the cysteine residue at position 1448 (C1448) with MTSs requires depolarization. The kinetics of the appearance of C1448 is also consistent with the gating kinetics of the channel. We further mutated all 8 basic residues in S4 of domain 4 to cysteines and probed the accessibilities of these cysteines to MTSs from both sides of the membrane. The second and third positions--R2C and R3C are accessible from both sides of the membrane, depending on membrane potential. The external accessibility requires depolarization and internal accessibility requires hyperpolarization. The residues from R4C to R8C stayed internally accessible at all membrane potentials. These data describe a new picture of the mechanism of charge movement in the process of Na channel gating. The new model, in which S4 moves across a short barrier, solves multiple problems in understanding the Na channel gating process