Biophysical Properties of Connexin-45 Gap Junction Hemichannels Studied in Vertebrate Cells

Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca²⁺, whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, V_m. These currents did not occur in nontransfected cells. The hemichannel currents were inhibited by raising extracellular Ca²⁺ or by acidification with CO₂. The unitary conductance exhibited V_m dependence (i.e., γ_hc,main increased/decreased with hyperpolarization/depolarization). Extrapolation to V_m = 0 mV led to a γ_hc,main of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, P_o, was V_m-dependent, declining at negative V_m (P_o < 0.11, V_m < −50 mV), and increasing at positive V_m (P_o ∼0.76, V_m > 50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca²⁺ level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in nontransfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca²⁺-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca²⁺. Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca²⁺ concentration is reduced.