[Ca²⁺]_o does not affect channel voltage dependence. Experiments were performed on cell-excised inside-out membrane patches symmetrically bathed in NMDG-Cl solution (pH 7.4) and under calcium-free bath solution. (A) Representative current recordings from two separate patches clamped at V_m 80 mV (top traces) or −80 mV (bottom traces) and under calcium-free (0 Ca_o, left) or 5 mM (5 Ca_o, right) external (pipette) conditions. The dashed lines indicate the closed channel current levels (C-). The inset is an excerpt of the trace at V_m 80 mV taken at the indicated location (asterisk) with the corresponding expanded time scale. (B) Mean N′P_o/V_m relationships under external calcium-free (□) or 5 mM Ca²⁺ (●) condition. For each [Ca²⁺]_o condition, N′P_o data were normalized to the respective N′P_o value at V_m 80 mV. Each point is the mean of five measurements, and SEM is shown as error bars when larger than symbols. Continuous and dashed lines are nonlinear least squares fits with the Boltzmann equation of calcium-free and 5 mM Ca²⁺ mean data, respectively.