Characterization of individual cysteine mutants. (A) Elution profiles from size exclusion chromatography for WT and representative spin-labeled cysteine mutants before reconstitution. (B) Whole-cell current traces for representative Na_VSp1 cysteine mutants recorded from HEK-293 cells in response to an activation protocol consisting of 500-ms depolarizations from −80 to +100 mV in 5-mV increments from a holding potential of −120 mV with a sweep-to-sweep interval of 4 s. The bottom panel is the voltage dependence of activation for WT and cysteine mutants. The V_1/2 of activation is WT (28.9 ± 0.5 mV, n = 3); P156C (26.1 ± 0.5 mV, n = 3); D157C (25.5 ± 0.7 mV, n = 3); F170C (21.2 ± 1.1 mV, n = 4); V186C (29.0 ± 0.4 mV, n = 4); and V189C (29.0 ± 0.4 mV, n = 3). The vertical lines show SD from “n” individual measurements.