Fig. 4: Voltage dependence of inactivation for mutations involved in the noncanonical path in hERG.

a Currents elicited using a classical three-pulse voltage protocol shown inset in a. A voltage pulse to +20 mV (P1) was applied for 1 s to fully activate/inactivate the channel, followed by a voltage pulse (P2) applied to different potentials from −180 to +60 mV for 30 ms. This pulse was used to remove inactivation without allowing sufficient time for significant deactivation to occur. A final pulse (P3) was then applied to +20 mV for the reopening and allowed the channels to reenter into the inactivated state. For better appreciation of the currents elicited by pulses 2 and 3, we expanded the time window of the current traces (dashed grey square in a). b–j are, respectively, the representative expanded time window for WT, T425L, L524R, A527L, L529H, L532H, W563L, A614G, and T618L current traces. j Voltage dependence curve for inactivation. The voltage dependence of inactivation was assessed by the peak currents elicited by P3 (red arrow in b and normalized by its maximum (I/I_MAX). For hyperpolarized potentials, <−120 mV, the channels start to deactivate which reduces the current elicited by P3, which underestimates the peak current. In order to account for this process, we corrected by extrapolating the currents back to the start of P2 from the falling phase of the currents elicited by P2¹⁸. For the right-shifted mutant T425L, for the mutant A527L P1 and P3 were set to +80 mV to account for the shift in the G-V curve and to fully inactivate the channel. Similarly, for T618L mutant, P1 and P3 were set to +100 mV to fully inactivate the channel. k Voltage dependence data for mutants. The voltage dependence data were fitted using Eq. 7 and the best-fitted values were displayed in Table 1. Data are presented as mean ± SD (WT (n = 4), T425L (n = 6), L524R (n = 4), A527L (n = 5), L529H (n = 3), L532H (n = 5), W563L (n = 3), A614G (n = 5), and T618L (n = 4), where n is the number of biologically independent cells).