Distinct moieties underlie biphasic H+ gating of connexin43 channels, producing a pH optimum for intercellular communication

Most mammalian cells can intercommunicate via connexin-assembled, gap-junctional channels. To regulate signal transmission, connexin (Cx) channel permeability must respond dynamically to physiological and pathophysiological stimuli. One key stimulus is intracellular pH (pH(i)), which is modulated by a tissue's metabolic and perfusion status. Our understanding of the molecular mechanism of H+ gating of Cx43 channels-the major isoform in the heart and brain-is incomplete. To interrogate the effects of acidic and alkaline pH(i) on Cx43 channels, we combined voltage-clamp electrophysiology with pH(i) imaging and photolytic H+ uncaging, performed over a range of pH(i) values. We demonstrate that Cx43 channels expressed in HeLa or N2a cell pairs are gated biphasically by pHi via a process that consists of activation by H+ ions at alkaline pH(i) and inhibition at more acidic pH(i). For Cx43 channel-mediated solute/ion transmission, the ensemble of these effects produces a pH(i) optimum, near resting pH(i). By using Cx43 mutants, we demonstrate that alkaline gating involves cysteine residues of the C terminus and is independent of motifs previously implicated in acidic gating. Thus, we present a molecular mechanism by which cytoplasmic acid-base chemistry fine tunes intercellular communication and establishes conditions for the optimal transmission of solutes and signals in tissues, such as the heart and brain.