Intracellular Ca²⁺ and Mg²⁺ induce a negative slope at high positive potentials for I-V plots from BK channels that is not observed in their absence. (A) Representative single-channel current records from BK channels at the indicated voltages without and with 2.5 mM Ca²⁺ and Mg²⁺ in the intracellular solution. The solutions also contained 150 mM KCl and 10 mM HEPES, pH 7.2. The presented current recordings were filtered at 5 kHz for display, but the data were collected and analyzed with 10 kHz low-pass filtering for the I-V plots. The divalent cation blockers reduce the outward single-channel current amplitudes, with a greater fractional decrease at +160 mV than at +80 mV. (B) I-V plots of single-channel current amplitudes indicate that 2.5 mM of intracellular Ca²⁺ and Mg²⁺ induce a negative slope for potentials greater than +120 mV. A negative slope is not observed in the absence of Ca²⁺ and Mg²⁺ (open circles). The dashed lines are cubic spline fits constrained to pass through the origin. Each plotted point is the mean from five or more patches. The absence of visible error bars indicates that the SEM is less than the symbol size.