Most gap junction channels are sensitive to the voltage difference between the two cellular interiors, termed the transjunctional voltage (V(j)). In several junctions, the conductance transitions induced by V(j) show more than one kinetic component. To elucidate the structural basis of the fast and slow components that characterize the V(j )dependence of connexin-32 (Cx32) and connexin-43 (Cx43) junctions, we created deletions of both connexins, where most of the carboxy-terminal (CT) domain was removed. The wild-type and "tailless" mutants were expressed in paired Xenopus oocytes, and the macroscopic gating properties were analyzed using the dual voltage clamp technique. Truncation of the CT domain of Cx32 and Cx43 abolished the fast mechanism of conductance transitions and induced novel gating properties largely attributable to the slow mechanism of gating. The formation of hybrid junctions comprising wild-type and truncated hemichannels allowed us to infer that the fast and slow components of gating reside in each hemichannel and that both gates close at a negative V(j) on the cytoplasmic side. Thus we conclude that the two kinetic components of V(j)-sensitive conductance are a result of the action of two different gating mechanisms. They constitute separate structures in the Cx32 and Cx43 molecules, the CT domain being an integral part of fast V(j) gating.

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Gap junctions composed of connexin-45 (Cx45) homologs from four species, zebrafish, chicken, mouse, and human, were expressed in pairs of Xenopus oocytes. The macroscopic conductance (gj) of all Cx45 junctions was modulated by transjunctional voltage (Vj) and by the inside-outside voltage (Vm), and the modulation was species specific. Although their gating characteristics varied in voltage sensitivity and kinetics, the four Cx45 junctions shared 1) maximum conductance at Vj = 0 and symmetrical gj reduction in response to positive and negative Vj of low amplitude, with little residual conductance; and 2) gj increases in response to simultaneous depolarization of the paired cells. The formation of hybrid channels, comprising Cx45 hemichannels from different species, allowed us to infer that two separate gates exist, one in each hemichannel, and that each Cx45 hemichannel is closed by the negativity of Vj on its cytoplasmic side. Interestingly, the Vm dependence of hybrid channels also suggests the presence of two gates in series, one Vm gate in each hemichannel. Thus the Vj and Vm dependence provides evidence that two independent voltage gates in each Cx45 hemichannel exist, reacting through specific voltage sensors and operating by different mechanisms, properties that have evolved divergently among species.

The conductance, gj, of many gap junctions depends on voltage between the coupled cells (transjunctional voltage, Vj) with little effect of the absolute membrane potential (V m) in the two cells; others show combined Vj and Vm 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, Vm dependence such that equal depolarization of both cells decreases gj. The dependence of gj on Vm 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 Vm dependence, respectively, suggesting that these residues are part of the V m sensor. Mutating both residues together abolished Vm 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 Vm, and Vm gating was reduced when the Vj gate was closed. These data suggest that the two forms of voltage dependence are mediated by separate but interacting domains. G ap junction channels are unique among ionic channels in that they span two cell membranes. They are composed of two hemichannels, one in each membrane, that are tightly docked head to head to form a pore that directly connects the cytoplasm of two cells (1). In vertebrates, gap junctions are formed by connexins (Cx), a gene family of protein subunits (2). Given their architecture, gap junction channels are subject to the influence of two types of voltage, that between the two cell interiors termed transjunctional voltage (V j ), and that between the interior and exterior or the membrane potential (V m ), which can differ between the cells and thus along the lumen of the channel connecting the cells. Many gap junctions are sensitive to V j with little effect of V m , whereas others show combined V j and V m dependence. The eponymous gap is accessible to small ions and because there is negligible leak through the channel wall, the potential in the gap is likely to be close to that in the bathing medium (3). V m dependence of junctional conductance (g j ) was initially described in invertebrate (deuterostome) gap junctions (4-7), which are formed by innexins, a family of proteins unrelated to connexins (8). More recently, g j dependence of V m has also been demonstrated in junctions formed of vertebrate connexins in exogenous expression systems (9-11) as well as in native cells (12,13). Junctions comprised of different connexin isoforms have divergent properties of V m dependence, varying in their polarity of closing, voltage sensitivity, and kinetics (14).V j dependence of vertebrate gap junctions has been extensively analyzed by site-directed mutagenesis (15-18), but the molecular basis of V m gating remains unknown. There is increasin...

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