Divalent cations selectively affect gating of Cx46 hemichannels at negative membrane potentials. (A and B) Multichannel cell-attached patch recordings from a Cx46-expressing oocyte recorded with (A) 1 mM Mg²⁺ present in the patch pipette and (B) no added Mg²⁺ in the patch pipette. In both cases, Ca²⁺ was buffered to very low levels with EGTA (Materials and methods). Shown are currents in response to 10-s voltage steps from −70 and +50 mV applied in 20-mV increments. No endogenous channel activity was present these patches. In the presence of 1 mM external Mg²⁺, Cx46 hemichannels essentially remained open at modest voltages (see traces at −10 and +10 mV) and tended to close for either polarity of membrane voltages. Hyperpolarization led to full and robust closure (see trace at −70 mV), whereas depolarization left a residual current (see current trace at +50 mV). The current records were leak subtracted as described in the Materials and methods. When there was no added external Mg²⁺, gating at positive voltages did not change, whereas gating at negative voltages changed significantly. In the example shown, gating was essentially abolished at hyperpolarizing voltages up to −70 mV. (C) Cell-attached patch recordings of single Cx46 hemichannels highlighting the two different types of gating at negative (−70 mV) and positive (+40 mV) voltages, termed loop gating and V_j gating, respectively. Recordings were obtained with standard IPS (containing 1 mM Mg²⁺). Loop gating is characterized by gating transitions between fully closed (C) and open (O) states (left), whereas V_j gating transitions occur between the open state (O) and a long-lasting subconductance state (right). Currents were leak subtracted as described in Materials and methods. All currents were filtered at 1 kHz and data were acquired at 5 kHz.