Regulation of macroscopic Cx46 currents by external Ca²⁺ and Mg²⁺. (A) Representative current–voltage (I-V) relationships (top) at different external Ca²⁺ concentrations ranging from nominal (0 added) to 1.8 mM were obtained by applying slow (3 min) voltage ramps from +50 to −100 mV to a Cx46-expressing oocyte. The holding potential before and after the ramp was −40 mV. For each curve, external Mg²⁺ was maintained at 1 mM. The corresponding conductance–voltage (G-V) relationships are plotted below and show that conductance is maximum at or near 0 mV and decreases asymmetrically with voltages of either polarity. Conductance decreases robustly with hyperpolarization, but only modestly with depolarization. Increasing the extracellular Ca²⁺ concentration caused a significant decrease in overall conductance and led to a substantial rightward shift in voltage sensitivity at negative, but not positive potentials. (B) I-V (top) and corresponding G-V (bottom) relationships obtained at different external Mg²⁺ concentrations ranging from 1 to 20 mM. External Ca²⁺ was maintained at nominal levels. Increasing external Mg²⁺, like Ca²⁺, decreased overall conductance and caused a significant positive shift in voltage sensitivity at negative potentials. However, these changes required considerably higher concentrations of Mg²⁺. (C) Shown are representative currents obtained at different concentrations of external Ca²⁺ and Mg²⁺. Oocytes were clamped to a holding potential of −40 mV and 5-s voltage steps were applied from +60 to −100 mV followed by a 5-s step to −120. The currents were normalized to a constant prepulse (+10 mV) that preceded each episode. Both Ca²⁺ and Mg²⁺ had similar effects, suppressing the magnitude of the current, slowing activation and speeding up deactivation.