Mechanisms of connexin gating and permeability revealed by connexin46 hemichannels

Abstract

Gap junctions are aggregates of specialized intercellular channels that allow direct transmission of electrolytes and small metabolites between coupled cells. In vertebrates, the gene family of connexins (Cxs) encodes the protein subunits that comprise gap junction channels. Presently, 16 different Cxs have been cloned, each with specific expression patterns, suggesting that Cxs have different physiological roles. Biophysical characterization of intercellular channels using dual whole cell voltage clamp of isolated pairs of Cx-expressing cells has proved quite useful in defining these roles, but this preparation possesses inherent difficulties that hamper mechanistic and structure/function studies. To overcome these limitations, we developed the Cx46 hemichannel preparation. Originally thought to exist only as nonfunctional precursors to the formation of intercellular channels, functional hemichannels were demonstrated in single Cx46 injected Xenopus oocytes that exhibited pharmacological sensitivities similar to intercellular channels. We showed that these functional hemichannels could be recorded in excised patches, allowing precise control of the solutions at either face. The voltage gating of unopposed hemichannels corresponds to that of hemichannels incorporated into intercellular channels; thus, the structure of the hemichannels in the two configurations is conserved. Furthermore, like intercellular channels, Cx46 hemichannels are sensitive to pH. Kinetic measurements with rapid acidification in excised patches demonstrated a direct pH gating of hemichannels and ruled out the action of both soluble cytoplasmic and membrane bound intermediates, two hypotheses that had not been eliminated in cell pair experiments. We also examined the mechanism of ion selectivity in Cx channels. Cx46 and Cx32 form cation and anion selective intercellular channels, respectively. Cx46 hemichannels are also cation selective and inwardly rectify in symmetric salts, properties best explained by fixed negative charges toward the extracellular end of the pore. These charges are located in the first extracellular loop domain (E1), as replacement of E1 in Cx46 with Cx32 sequence produced an outwardly rectifying, anion selective hemichannel. Thus, E1 must line the pore and contains the charges governing selectivity. These experiments in Cx46 hemichannels have offered tremendous insight into the operation of intercellular channels, demonstrating the utility of the preparation as a complement to studies of intercellular channels.