K525 and K538 may interact with a conserved negative charge in S2. (A–D) Representative current traces from oocytes expressing the single D466E mutation and the double D466E/K525R, D466E/R537K, or D466E/K538R mutant constructs. Oocytes were held at −80 mV and subjected to either 2- or 10-s depolarizing pulses to +60 mV in 10-mV increments. Tail currents were recorded at −60 mV. Arrows indicate the zero current level. The exception to this was D466E/K525R, which was held at −110 mV, and tail currents were recorded at −130 mV. (E) Comparison of the G-V relationships for the D466E and D466E/K525R mutants. (F) Plot of the G-V relationships for the D466E and D466E/K525Q mutants. (G) Comparison of the G-V relationships for the D466E and D466E/R537K mutants. (H) Comparison of the G-V relationships for the D466E and D466E/K538R mutants. In E–H, lines represent fits of the data to a Boltzmann function. Data points represent mean ± SEM (error bars). n values and parameters from the Boltzmann fits are summarized in Table 1.