Elevated concentration of Ca²⁺ causes a positive shift in the voltage dependence of the Q_OFF-V relationship for hERG1, but it does not induce a transient outward component in Ig_OFF. (A) Q_OFF-V relationships for hERG1 measured under control conditions ([Ca²⁺]_e = 2 mM) and after the elevation of [Ca²⁺]_e to 10 mM (n = 5). Averaged data were fitted to a Boltzmann function (smooth curves). 2 mM Ca²⁺: V_1/2 = −19.5 ± 0.5 mV and z = 1.87 ± 0.05; 10 mM Ca²⁺: V_1/2 = 6.8 ± 1.9 mV and z = 1.42 ± 0.06. (B) Ig traces recorded in the presence of 2 mM Ca²⁺ (control) and 10 mM Ca²⁺ at a test potential of +60 mV. Ig_OFF was measured at −110 mV. (C) Ig_OFF traces from B are shown at expanded time (initial 5 ms) and amplitude scales. (D) Gating currents of WT hERG1 channels measured during 300-ms pulses to +40 mV with a holding and return potential of −110 mV. Q_OFF-V relationships are shown in G. Extracellular concentrations of Ca²⁺ and Cd²⁺ are indicated to the left of the current traces. (E) Expanded view of initial Ig_OFF for the traces shown in D. (F) Currents recorded from an uninjected oocyte using the same pulse protocol as in D with the indicated concentrations of extracellular divalent cations. (G) Q_OFF-V relationship determined in a single oocyte in the presence of the indicated concentrations of extracellular Ca²⁺ and Cd²⁺. For 2 mM Ca²⁺: Q_OFF-max = −8.0 nC, V_1/2 = −19.6 mV, and z = 2.2. For 10 mM Ca²⁺: Q_OFF-max = −12.0 nC, V_1/2 = +7.3 mV, and z = 1.6. For 10 mM Ca²⁺ + 0.1 mM Cd²⁺: Q_OFF-max = −9.3 nC, V_1/2 = +32 mV, and z = 1.1.