The pH sensitivity of mode shift behavior is similar to that of deactivation kinetics. (A and B) hERG channels were recruited into the relaxed state by applying a 15-s depolarizing step to +60 mV, following which deactivation kinetics were assessed during a repolarizing step to −110 mV. Current decay during repolarization was fitted to exponential function, which yielded values for τ_fast of 201 ± 11 ms (pH 8.5, n = 4), 192 ± 20 ms (pH 7.4, n = 5), 99 ± 16 ms (pH 6.5, n = 5), 79 ± 5 ms (pH 5.5, n = 5), and 73 ± 7 ms (pH 4.5, n = 5). (C) Plot of the pH dependence of the τ_fast of deactivation. Fitting the data with a Hill function yielded a pKa of pH 6.8 (Hill coefficient, n = 1.9). (D and E) Representative traces recorded during the voltage protocols (insets) designed to measure the steady state voltage dependence of activation and deactivation at pH 7.4 (D) and pH 6.5 (E). Arrows mark where current measurements were made. Plots of the normalized voltage dependence of activation and deactivation G-V at each pH are shown at right. Boltzmann fits of the data recorded at pH 7.4 yielded V_1/2 values for activation and deactivation of −34.5 ± 1.3 mV and −56.4 ± 1.2 mV, respectively (n = 12). Corresponding values measured at pH 6.5 were −27.3 ± 0.8 mV and −38.8 ± 1.2 mV (n = 5). The mode shift calculated from these data was reduced by 74% from −21.9 ± 1.4 mV at pH 7.4 to −11.5 ± 0.5 mV at pH 6.5. (F) Plot of the pH dependence of the mode shift measured as in D and E. Fitting the data with a Hill function yielded a pKa of pH 7.0 (Hill coefficient, n = 1.1; also see Table 2). Dashed lines represent baseline.