Restoration of wild type–like closure with point mutations of D342. (A) The position of D342 in the activated VSD. For clarity, only two subunits are shown, and one of them is color coded: the N-terminal PAS domain is shown in cyan, the VSD in yellow, the S4–S5 linker in magenta, the PD helices in gray, the C linker in dark red, and the CNBHD in orange. The crucial residues are color coded in both subunits: D342 in red, Q476 (S6 bundle crossing) in blue, and Q477 in green. (B) Representative currents of L341 split and selected D342 mutants in L341 split. The voltage protocol is shown at the top. (C) Normalized, mean current values plotted against voltage (n = 7–18). Error bars denote SEM. Insets contain normalized current at −100 and −160 mV plotted against the residue at 342 position (mean ± SEM). The order from left to right reflects residue size, and dashed lines indicate the current level of the wild type. Asterisks indicate statistical significance (P < 0.05 in t test with Bonferroni correction) with respect to the continuous channel.