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

Abstract

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, Vm. 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 Vm dependence (i.e., γhc,main increased/decreased with hyperpolarization/depolarization). Extrapolation to Vm = 0 mV led to a γhc,main of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, Po, was Vm-dependent, declining at negative Vm (Po < 0.11, Vm < −50 mV), and increasing at positive Vm (Po ∼0.76, Vm > 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.