ENC electrostatic contributions are critical in DI and DII only. (A and D) Sample traces for currents recorded for WT and mutants at the S1 and the S2 ENC in DI–DIV. (B, C, E, and F) G-V (B and E) and SSI curves (C and F) for WT and mutants at the S1 and the S2 ENC; the insets in C and F show a bar graph representing the average time constants for fast inactivation (τ) for a depolarizing voltage step to −15 mV for WT and the mutants; *, statistical difference to WT values in an unpaired t test (P < 0.01). Note that Asn1389 in the S2 ENC in DIV had been mutated to both acidic and basic side chains previously with no functional consequence and was thus not studied further here (Groome and Winston, 2013). Bars: horizontal, 5 ms; vertical, 200 nA. Voltage steps were from −40 to +20 mV in 10-mV increments. Insets show energy-minimized structures and ESP maps of side chains (red, −100 kcal/mol; green, 0 kcal/mol; blue, +100 kcal/mol; see Pless et al., 2011b, for details).