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 (pHi), 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 pHi on Cx43 channels, we combined voltage-clamp electrophysiology with pHi imaging and photolytic H+ uncaging, performed over a range of pHi values. We demonstrate that Cx43 channels expressed in HeLa or N2a cell pairs are gated biphasically by pHivia a process that consists of activation by H+ ions at alkaline pHi and inhibition at more acidic pHi. For Cx43 channel–mediated solute/ion transmission, the ensemble of these effects produces a pHi optimum, near resting pHi. 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.

Cells of most human tissues—with the notable exception of blood cells and skeletal muscle cells—are electrically and metabolically coupled by means of gap junctional channels, assembled from connexin (Cx) proteins. The hexameric channels permit cell-to-cell solute and ion flow. This function plays a critical signaling role (1) that is particularly important for the spread of electric current in excitable tissues. The biological importance of gap junctional communication necessitates a means of regulating junctional permeability and conductance. Acute Cx channel regulation is typically exercised via post-translational modifications and may involve cellular metabolites and/or electrophysiologic maneuvers. Moreover, aberrant forms of Cx channel regulation have been implicated in pathologic states (2, 3), such as cardiac arrhythmias.

Among solutes that permeate Cx-assembled channels are H+ ions, the end products of metabolism. H+ ions are produced at a rate that reflects the tissue’s metabolic activity. They can feedback potently on cellular function via an array of protonation reactions with proteins. Essentially, all cell types are equipped with a molecular apparatus for maintaining favorable intracellular pH (pHi). Excess acid is commonly transferred from cells to the nearest functional blood capillary (4) via membrane transport proteins, such as H+-monocarboxylate transporters and Na+/H+ exchangers (NHEs). In addition, permeation of H+ ions through gap junctions allows pHi to equilibrate spatially among cells, such as those of the working myocardium. Channel-facilitated H+ dissipation reduces the spatial heterogeneity of pHi, thereby helping to unify tissue-level function, such as myocardial contractility. In contrast, some clinical conditions—for example, myocardial ischemia—can trigger abnormally large decreases of tissue pHi; permitting a large and localized intracellular acid load to spread into surrounding tissue would risk inflicting undue damage on cells that are co-opted to share the pHi disturbance. Instead, gap junctional channels tend to close by sensing low pHi.

A 1980s report first described an inhibitory effect of intracellular acidification on cell-to-cell coupling (5, 6). Subsequent expression studies on Cx43 channels have linked this to an inhibition by H+ ions, which relies on an interaction between the cytoplasmic C terminus of the Cx43 protein (residues 261–300 and 374–382) (7, 8) with its intracellular loop (a protonatable histidine residue) (9). Moreover, these domains are influenced by phosphorylation (10) and interactions with the cytoskeleton (11), which allows for additional fine tuning of Cx43 channel pHi sensitivity. More recently, an additional pHi control of gap junctional conductance and permeability has been described. Inhibition of electrical and solute coupling between mammalian ventricular myocytes—where Cx43 is the dominantly expressed gap junctional isoform—has been demonstrated at both low and high pHi. Ventricular coupling is thus modulated by pHi in a biphasic manner, with peak conductance attained at pHi ∼6.9, which is mildly acidic relative to normal resting pHi (12). The molecular structures that underpin gap junctional block at high pHi are currently unknown.

Here, by using heterologously expressed Cx43 channels, we confirm that alkaline—that is, high—pHi reversibly and robustly reduces gap junctional communication, probed electrophysiologically and from measurements of cell-to-cell H+ ion permeation down a photolytically evoked gradient of [H+]i. Furthermore, by using mutants of Cx43, we show that the C terminus of Cx43 is involved in alkaline gating and that this process is independent of the molecular apparatus responsible for channel closure at acidic—that is, low—pHi. We present an updated model of the mechanism of biphasic gating of Cx43 channels by H+ ions. Our model explains the phenomenon of optimal Cx43 channel permeability in terms of the ensemble of inhibitory and activatory effects of H+ ions operating over distinct pHi ranges.