pH-dependent modulation of voltage gating in connexin45 homotypic and connexin45/connexin43 heterotypic gap junctions

Intracellular pH (pHi) can change during physiological and pathological conditions causing significant changes of electrical and metabolic cell- cell communication through gap junction (GJ) channels. In HeLa cells expressing wild-type connexin45 (Cx45) as well as Cx45 and Cx43 tagged with EGFP, we examined how pHi affects junctional conductance (g(j)) and g(j) dependence on transjunctional voltage (V-j). To characterize V-j gating, we fit the g(j)-V-j relation using a stochastic four-state model containing one V-j-sensitive gate in each apposed hemichannel (aHC); aHC open probability was a Boltzmann function of the fraction of V-j across it. Using the model, we estimated gating parameters characterizing sensitivity to V-j and number of functional channels. In homotypic Cx45 and heterotypic Cx45/Cx43-EGFP GJs, pH(i) changes from 7.2 to similar to 8.0 shifted g(j)-V-j dependence of Cx45 aHCs along the V-j axis resulting in increased probability of GJ channels being in the fully open state without change in the slope of g(j) dependence on V-j. In contrast, acidification shifted g(j)-V-j dependence in the opposite direction, reducing open probability; acidification also reduced the number of functional channels. Correlation between the number of channels in Cx45-EGFP GJs and maximal gj achieved under alkaline conditions showed that only similar to 4% of channels were functional. The acid dissociation constant (pKa) of g(j)-pH(i) dependence of Cx45/Cx45 GJs was similar to 7. The pK(a) of heterotypic Cx45/Cx43-EGFP GJs was lower, similar to 6.7, between the pK(a)s of Cx45 and Cx43-EGFP (similar to 6.5) homotypic GJs. In summary, pHi significantly modulates junctional conductance of Cx45 by affecting both V-j gating and number of functional channels.