Mode shift is greatly diminished and is pH independent in D509A mutant channels. (A and D) Typical ionic current recordings from D509A mutant channels at pH 7.4 (A) or pH 6.5 (D) in response to the voltage protocols shown (insets) designed to measure the voltage dependence of activation. The membrane was held at −80 mV before 250-ms steps were applied up to +100 mV (in 10-mV increments), followed by a repolarizing step to −60 mV. Arrows mark where current measurements were made. (B and E) Typical ionic current recordings of D509A channel deactivation at pH 7.4 (B) or pH 6.5 (E). The membrane was held at −80 mV before a 250-ms depolarizing step to +100 mV, followed by 750-ms repolarizing steps down to −130 mV (in 10-mV increments). (C and F) Plots of the voltage dependence of activation and deactivation at pH 7.4 (C) and pH 6.5 (F). Data were fitted with a Boltzmann function, which yielded V_1/2 values for activation and deactivation of +17.1 ± 0.8 mV (n = 5) and +1.6 ± 1.1 mV (n = 5), respectively, at pH 7.4. The corresponding values at pH 6.5 were +21.6 ± 1.5 mV (n = 5) and +4.8 ± 1.2 mV (n = 5), respectively. The mean mode shift was −15.5 ± 0.1 mV at pH 7.4 and −16.8 mV ± 0.6 at pH 6.5 (P > 0.3; not significantly different, Student’s t test; see Table 1). Dashed lines represent baseline.