In addition to forming gap-junction channels, a subset of connexins (Cxs) also form functional hemichannels. Most hemichannels are activated by depolarization, and opening depends critically on the external Ca ²⁺ concentration. Here we describe the mechanisms of action and the structural determinants underlying the Ca ²⁺ regulation of Cx32 hemichannels. At millimolar calcium concentrations, hemichannel voltage gating to the full open state of ≈90 pS is inhibited, and ion conduction at negative voltages of the partially open hemichannels (≈18 pS) is blocked. Thus, divalent cation blockage should be considered as a physiological mechanism to protect the cell from the potentially adverse effects of leaky hemichannels. A ring of 12 Asp residues within the external vestibule of the pore is responsible for the binding of Ca ²⁺ that accounts for both pore occlusion and blockage of gating. The residue Asp-169 of one subunit and the Asp-178 of an adjacent subunit must be arranged precisely to allow interactions with Ca ²⁺ to occur. Interestingly, a naturally occurring mutation (D178Y) that causes an inherited peripheral neuropathy induces a complete Ca ²⁺ deregulation of Cx32 hemichannel activity, suggesting that this dysfunction may be involved in the pathogenesis of the neuropathy.

Neurotransmission through electrical synapses plays an important role in the spike synchrony among neurons and oscillation of neuronal networks. Indeed, electrical transmission has been implicated in the hypersynchronous electrical activity of epilepsy. We have investigated the influence of intracellular pH on the strength of electrical coupling mediated by connexin36 (Cx36), the principal gap junction protein in the electrical synapses of vertebrates. In striking contrast to other connexin isoforms, the activity of Cx36 channels decreases following alkalosis rather than acidosis when it is expressed in Xenopus oocytes and N2A cells. This uncoupling of Cx36 channels upon alkalinization occurred in the vertebrate orthologues analyzed (human, mouse, chicken, perch, and skate). While intracellular acidification caused a mild or moderate increase in the junctional conductance of virtually all these channels, the coupling of the skate Cx35 channel was partially blocked by acidosis. The mutational analysis suggests that the Cx36 channels may contain two gating mechanisms operating with opposing sensitivity to pH. One gate, the dominant mechanism, closes for alkalosis and it probably involves an interaction between the C- and N-terminal domains, while a secondary acid sensing gate only causes minor, albeit saturating, changes in coupling following acidosis and alkalosis. Thus, we conclude that neuronal Cx36 channels undergo unique regulation by pH _i since their activity is inhibited by alkalosis rather than acidosis. These data provide a novel basis to define the relevance and consequences of the pH-dependent modulation of Cx36 synapses under physiological and pathological conditions.

The conductance, g _j , of many gap junctions depends on voltage between the coupled cells (transjunctional voltage, V _j ) with little effect of the absolute membrane potential ( V _m ) in the two cells; others show combined V _j and V _m dependence. We examined the molecular determinants of V _m dependence by using rat connexin 43 expressed in paired Xenopus oocytes. These junctions have, in addition to V _j dependence, V _m dependence such that equal depolarization of both cells decreases g _j . The dependence of g _j on V _m was abolished by truncation of the C-terminal domain (CT) at residue 242 but not at 257. There are two charged residues between 242 and 257. In full-length Cx43, mutations neutralizing either one of these charges, Arg243Gln and Asp245Gln, decreased and increased V _m dependence, respectively, suggesting that these residues are part of the V _m sensor. Mutating both residues together abolished V _m dependence, although there is no net change in charge. The neutralizing mutations, together or separately, had no effect on V _j dependence. Thus, the voltage sensors must differ. However, V _j gating was somewhat modulated by V _m , and V _m gating was reduced when the V _j gate was closed. These data suggest that the two forms of voltage dependence are mediated by separate but interacting domains.