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 pH(i) 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.—Garciarena, C. D., Malik, A., Swietach, P., Moreno, A. P., Vaughan-Jones, R. D. Distinct moieties underlie biphasic H(+) gating of connexin43 channels, producing a pH optimum for intercellular communication.