PMC

Full atom and molecular surface representation of the voltage-sensing module of NaChBac in three sequential activated states. Segments S1 through S4 colored individually and labeled. Side chains of gating-charge-carrying arginines (labeled R1 through R4 and colored blue) in S4, and D60 and E70 (colored red) in S2 shown in space filling representation. A probe radius of 1.4 Å was used to scan the molecular surface of each structural model. Figure generated using Chimera.

Disulfide locking the D60C:R4C voltage sensor. Mean normalized peak I_Na elicited by trains to 40 mV positive to the potential of half activation (V_1/2 + 40 mV). Holding potential = −120 mV. (A) Effect of 10 mM H₂O₂ (n = 7). (B and C) Reversal of H₂O₂ effect with 10 mM DTT (B) or 1 mM TCEP (C) (V = +80 mV; n = 8). (D) Test for disulfide locking with H₂O₂ during 2 min (t = 1–3 min, bar) at −120 mV without pulsing.

Disulfide locking the E70C:R4C voltage-sensor. (A) I_Na from the first and last pulses in a 2-min 0.1 Hz train of 500-ms depolarizations to 0 mV from a holding potential of −120 mV. (B) Mean normalized peak currents during trains (n = 7). After 3 min, pulsing was stopped for 5 min to test for channel recovery. (C) Effect of 10 mM H₂O₂ (bar; n = 5). (D and E) Effects of 10 mM βME (D) or 1 mM TCEP (E) (Bars; holding potential = −140 mV; n = 6.)

Time course of disulfide locking. (A) Voltage protocol. Channels were unlocked by a 5-s prepulse to −160 mV. Cells were then depolarized by prepulses to (V_1/2 + 40 mV) of the indicated duration. Following 5 s at −120 mV, disulfide-locked channels were assayed with a 100-ms test pulse to V_1/2 + 40 mV (WT, 0 mV; D60C, 20 mV; E70C, −30 mV; R4C, 60 mV; D60C:R4C, 80 mV; E70C:R4C, −40 mV). (B) Peak test pulse currents were normalized to the test pulse current in the absence of a prepulse. Mean values (±SEM) were plotted versus prepulse duration (n = 6). (C–E) Comparison of the rate of disulfide locking (red circles) and channel activation (blue line) for (C) E70C:R4C, control, (D) E70C:R4C, 2 mM H₂O₂, and (E) D60C:R4C, 2 mM H₂O₂. (F) Ratio of the time constant for disulfide locking (τ_S–S) to the time constant for activation (τ_Act) for E70C:R4C and D60C:R4C channels in 2 mM H₂O₂. For E70C:R4C, τ_S–S = 33 ± 2 ms, τ_act = 20 ± 2 ms (n = 6). For D60C:R4C, τ_S–S = 45 ms ± 2 ms (n = 7), τ_act = 4 ± 0.5 ms (n = 7).

Reversal of disulfide locking of E70C:R4C by hyperpolarization_. (A) Voltage dependence of reversal of disulfide locking. Before each trial, five 500-ms pulses to 0 mV were applied to disulfide-lock and fully inactivate all voltage sensors. Fully locked channels were hyperpolarized for 5 s to potentials from −200 to −120 mV followed by a 500-ms depolarization to 0 mV to assay the unlocked channels. Each cell was tested in control and in 10 mM βME or 10 mM H₂O₂, and currents were normalized to the largest current in control. Mean normalized peak currents were plotted against prepulse potential (n = 6; ±SEM). (B) Time-course of reversal of disulfide locking. Fully locked channels were hyperpolarized to −160 mV for the indicated times followed by a test pulse to 0 mV. Each cell was tested in control and then in the presence of 10 mM βME or 10 mM H₂O₂; peak test pulse currents were normalized to the peak current following the 32-s hyperpolarization in control. Mean normalized peak currents (±SEM) are plotted against prepulse duration and fit to a single exponential (n = 6). Error bars are smaller than data points where not visible. (C) Rate constants for disulfide locking. Assuming a bimolecular reaction, inset, forward α (black circles), and reverse β (green squares), rate constants were calculated from the rate and extent of reversal of unlocking at −160 mV from (B) as detailed in SI Methods.

Voltage dependence of disulfide locking and rates of inactivation of disulfide-locked states. The voltage dependence of channel activation (white circles) and of disulfide locking (blue circles) measured in the presence of 2 mM H₂O₂ are compared for E70C:R4C (A) and D60C:R4C (B). The voltages for half-maximal channel activation and disulfide locking were determined from fits of a Boltzmann equation to the data. To measure the voltage dependence of disulfide locking of E70C:R4C (n = 9), channels were unlocked by a 5 s pulse to −160 mV, then depolarized with 500-ms prepulses to the indicated potentials. After 5 s at −120 mV, the number of channels locked during the prepulse was assessed with a 100-ms test pulse to +80 mV. Since H₂O₂-induced disulfide locking of D60C:R4C channels cannot be reversed by hyperpolarization, disulfide locking could only be tested at one potential for each cell (n = 5–6 for each potential). (C) Rates of inactivation of disulfide-locked channels. Sodium current traces from the indicated NaChBac channels elicited by 500-ms depolarizations to V = V_1/2 + 40 mV from −120 mV (black traces). The time constant of inactivation was determined by an exponential fit to the decay of I_Na (red lines). I_Na is outward for D60C:R4C because its positive voltage dependence of activation requires test pulses to V = V_1/2 + 40 mV, that are beyond the reversal potential. (D) Mean time constants of inactivation measured at the indicated potentials (WT, n = 10; D60C:R4C, n = 8; E70C:R4C, n = 10; ± SEM).