Gating current and Q-V relations for the spHCN 462C466C mutant. (A) Schematic description of the locked-open mutant. The state of the channel with Cd²⁺ is shaded, whereas the control condition without Cd²⁺ is in the dashed area. (B and C) Traces of gating current records for spHCN 462C466C without (B) or with 130 nM free Cd²⁺ (C), from the same patch. Holding potential was 10 mV, and test pulses were from −30 to −170 mV in 10-mV increments. A −P/4 protocol was used to subtract leak and linear capacitive currents. (D and E) Normalized Q-V relations for spHCN 462C466C without Cd²⁺ (open circles) or with Cd²⁺ (closed circles). Both Q_ON (D) and Q_OFF (E) were obtained by integrating components. Smooth curves are single Boltzmann fits to the data with these parameters: Q_ON control: V_1/2 = −132.7 ± 2.1 mV, slope factor (e-fold) = 16.5 ± 1.2; Cd²⁺: V_1/2 = −103.9 ± 5.3 mV, slope factor (e-fold) = 18.2 ± 1.2; n = 5. Q_OFF control: V_1/2 = −126.7 ± 5.1 mV, slope factor (e-fold) = 21.1 ± 1.2; Cd²⁺: V_1/2 = −99.9 ± 5.3 mV, slope factor (e-fold) = 19.5 ± 1.1; n = 9. Error bars represent SEM.