The binding site for divalent cations is extracellular. (A) Recording of a single hemichannel in an outside-out patch configuration with 1 mM Mg²⁺ on the extracellular side and no added Mg²⁺ on the cytoplasmic side. Openings observed at −70 mV became more sporadic and brief upon hyperpolarization to −90 mV. (B) Recording of a single hemichannel in an outside-out patch configuration with no added Mg²⁺ on the extracellular side and 1 mM Mg²⁺ on the cytoplasmic side. In the presence of 1 mM Mg²⁺ on the cytoplasmic side, the hemichannel largely remained open at −70 mV. Closure increased with hyperpolarization to −90 mV and was robust at −130 mV. These large hyperpolarizing voltages would tend to prevent Mg²⁺, which is present only on the cytoplasmic side, from entering the pore inconsistent with voltage-dependent entry of Mg²⁺ leading to block. (C) Patch recording containing three active Cx46 hemichannels in an outside-out patch configuration. Membrane potential was held constant at −50 mV. Starting in symmetric conditions, with 1 mM Mg²⁺ present on both sides, perfusion of a solution containing 10 mM Mg²⁺ into the bath (as indicated), which exposed the extracellular side to a high concentration of Mg²⁺, produced rapid and robust closure. (D) The same experimental paradigm as in C applied to an inside-out patch containing a single hemichannel. Perfusing a solution containing 10 mM Mg²⁺ into the bath, which now exposed the cytoplasmic side to a high concentration of Mg²⁺, did not appreciably affect hemichannel behavior except for a small reduction in the magnitude of the unitary current. Shown below the recording are histograms taken from the indicated segments (solid bars) before and after application of 10 mM Mg²⁺. All currents were leak subtracted. Dashed lines indicate fully open (O) and fully closed (C) states. Currents were filtered at 1 kHz and data were acquired at 5 kHz.