Molecular determinants of membrane potential dependence in vertebrate gap junction channels

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.