Voltage dependence of inhibition by BQ⁺ applied to the cytoplasmic side of Cx50 hemichannels. Single-channel I-V relationships in control (top), in 500 µM BQ⁺ (middle), and in 1 mM BQ⁺ (bottom) applied either to the cytoplasmic side (A) or extracellular side (B) of Cx50 hemichannels. I-V relations in A and B were obtained in response to 8-s voltage ramps from −100 to +50 mV applied to excised inside-out patches and outside patches, respectively. Inhibition produced by cytoplasmic application of BQ⁺ (BQ⁺_cyt) is promoted by depolarization, a voltage dependence that is opposite to that observed when BQ⁺ is applied to the extracellular side, indicating a binding site located in the pore. In addition, inhibition by intracellular BQ⁺, especially at a 1-mM concentration, occurs even at strong inside-negative voltages. Hyperpolarizing voltages are expected to prevent entry of BQ⁺ on the intracellular side into the pore, suggesting that the binding site is closer to the cytoplasmic end of the hemichannel. Solid lines superimposed on the current traces in BQ⁺ represent exponential fits to the open-state current in the absence of BQ⁺ to illustrate the decrease in conductance at high, but not lower, BQ⁺ concentrations (see Results).