Figure 5. Disulfide locking the voltage sensor in resting and activated states.

NaChBac wild-type (WT), single Cys mutants D60C and R3CA, and double Cys mutant D60C:R3C were expressed in tsA-201 cells and recorded using the whole-cell voltage clamp mode. A. Reversible disulfide locking. Mean normalized peak currents elicited by a 0.1 Hz train of 500-ms depolarizations to 0 mV from a holding potential of −140 mV in tsA cells transfected with D60C, R3C, or D60C:R3C channels. After 2 min in control saline conditions, cells were exposed to 1 mM β-mercaptoethanol (DeCaen et al., 2008). B. Time course of voltage-sensor locking. D60C:R3C channels were first unlocked by a 5-s prepulse to −160 mV. Cells were then depolarized for the indicated times to approximately V_1/2 + 20 mV (WT, −20 mV; D60C & R3C, 0 mV; D60C:R3C, −30 mV), returned to −120 mV for 5 s and depolarized for 100 ms test pulse to 0 mV. Peak test pulse current at 0 mV was normalized to the control pulse current in the absence of a prepulse and mean (± SEM) was plotted versus prepulse duration (red circles with error bars). Sodium current recorded during a −30 mV prepulse (black); time course of activation in the absence of inactivation (blue trace) estimated by fitting an exponential to the current decay and adding the inactivated component back to the total current (DeCaen et al., 2008). C. Disulfide locking of R1 and Glu43 in the resting state. D. Disulfide locking of R2 and Glu43 in resting and activated states. E. Disulfide locking of R3 and Glu43 in the activated state. Disulfide locking was induced by depolarization in the presence of 1 mM H₂O₂ (DeCaen et al., 2011).