Gating current and Q-V relations for the spHCN 462Y468C mutant. (A) Schematic description of the locked-closed 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 462Y468C without (B) or with 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 462Y468C without (open squares) or with Cd²⁺ (closed squares). 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 as follows: Q_ON control: V_1/2 = −116.4 mV ± 8.4, slope factor (e-fold) = 21.2 ± 4.7; 100 µM cAMP + 130 nM free Cd²⁺: V_1/2 = −131.8 ± 7.5 mV, slope factor(e-fold) = 18.1 ± 0.9; n = 5. Q_OFF control: V_1/2 = −96.7 ± 5.3 mV, slope factor (e-fold) = 16.0 ± 1.9; Cd²⁺: V_1/2 = −128.0 ± 2.3 mV, slope factor (e-fold) = 21.1 ± 2.0; n = 7. Error bars represent SEM.