Fig. 3.

Time course and voltage dependence of disulfide locking of E43C:R2C and E43C:R3C channels. (A) E43C:R2C (Upper) and E43C:R3C channels (Lower) were unlocked by a 5-s prepulse to −170 mV. Cells were then depolarized by prepulses to V_1/2 + 40 mV of the indicated durations. After 5 s at −140 mV, disulfide-locked channels were assayed with a 100-ms test pulse to V_1/2 + 40 mV (WT, 0 mV; E43C, 60 mV; R2C, −20 mV; E43C:R2C, −30 mV; E43C:R3C, 0 mV). Peak test pulse currents were normalized to the test pulse current in the absence of a prepulse. Mean values (±SEM) were plotted vs. prepulse duration (n = 6). (B) Comparison of the rate of disulfide locking and channel activation (blue line, after subtraction of the exponential effect of inactivation) for E43C:R2C and E43C:R3C in 2 mM H₂O₂. (C) The voltage dependence of channel activation (lines with error bars) and of disulfide locking (symbols) measured in the presence of 2 mM H₂O₂. To measure the voltage dependence of disulfide locking of E43C:R2C (n = 9), channels were unlocked by a 5-s pulse to −170 mV, then depolarized with 500-ms prepulses to the indicated potentials. After 5 s at −140 mV, the number of channels locked during the prepulse was assessed with a 100-ms test pulse to 0 mV. Because H₂O₂-induced disulfide locking of E43C:R3C channels cannot be reversed by hyperpolarization, disulfide locking could only be tested at one potential for each cell (n = 5–6 for each potential). (D) Rate of disulfide locking as a function of membrane potential estimated from the data in C at potentials where the fraction of channels locked was between 0.1 and 0.9 as ln[(Fraction of I_Na)]/(−0.5 s).