Effect of Cd²⁺ on the 462C466C mutant or 462Y468C mutant. (A and B) Representative recordings from inside-out patches excised from Xenopus oocytes expressing either spHCN 462C466C (A) or spHCN 462Y468C (B) before and during the application of 130 nM free Cd²⁺. Channels were held at 10 mV, and currents were elicited by a step to −120 (A) or −130 mV (B), followed by a step to 60 mV. Currents were not leak subtracted. (C and D) To test whether the expressed channels still represent mutant phenotype in the condition of measuring the gating current (160 mM NMDA + 160 mM MeSO₃ for pipette and bath), a 160 mM K⁺ solution was applied rapidly to inside-out patches expressing spHCN 462C466C (C) or spHCN 462Y468C (D). The black trace in C indicates the response with NMDA on both sides and 130 nM free Cd²⁺, whereas the red trace shows the current response with the switch to intracellular K⁺ immediately before the voltage step from −120 to 60 mV; the sustained outward current indicates the locked-open effect. The gray arrow in C indicates the time when the solenoid was engaged; the solution switch at the patch occurred ∼50 ms later, and the actual switching speed was <1 ms. (D) The black trace shows a normal tail current for the locked-closed mutant in the absence of Cd²⁺ when intracellular K⁺ was applied immediately before the voltage step from −120 to 60 mV. The red trace again shows the response with fast perfusion of K⁺ solution, but in the constant presence of 130 nM free Cd²⁺, and the blue trace indicates the gating current alone (no K⁺ perfusion) with 130 nM free Cd²⁺ present.