The gating curves and Cd²⁺ block in the W1716C/F1764C (WCFC) double-mutant and the corresponding single-mutant channels. (A) The activation curves (left) and the inactivation curves (right) were documented by the protocols described in Materials and methods. The duration of the inactivating pulse for plotting the inactivation curve is set at 5 s to facilitate comparison with the data (Table I) in the presence of drugs that have relatively slow binding kinetics. The findings remain qualitatively very similar with 100-ms inactivating pulses (not depicted). The curves in the left panel are fits with a Boltzmann function with V_1/2 values (in mV) of −16.9, −21.3, −15.9, and −17.2 and k values of 4.9, 5.9, 5.7, and 4.4 for the wild-type (WT; the gray line, n = 6), W1716C (n = 6), F1764C (n = 17), and WCFC (n = 15) mutant channels, respectively. The curves in the right panel are fits with a Boltzmann function with V_1/2 values (in mV) of −54.2, −64.6, −50.9, and −49.8 and k values of 4.6, 6.7, 5.1, and 6.1 for the WT (the gray line, n = 10), W1716C (n = 7), F1764C (n = 4), and WCFC (n = 14) mutant channels, respectively. (B) Cd²⁺ block of different mutant channels. The oocyte was held at −110 mV and stepped every 3 s to a test pulse of −20 mV for 100 ms. (Left) The elicited currents in the presence (red lines) and absence (black lines) of 100 μM Cd²⁺. (Right) The peak currents in the former are normalized to the peak current in the latter to give the relative current (n = 4–7).