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

Intracellular pH (pH_i) 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 pH_i 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 ∼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 ∼4% of channels were functional. The acid dissociation constant (pK_a) of g_j -pH_i dependence of Cx45/Cx45 GJs was ∼7. The pK_a of heterotypic Cx45/Cx43-EGFP GJs was lower, ∼6.7, between the pK_as of Cx45 and Cx43-EGFP (∼6.5) homotypic GJs. In summary, pH_i significantly modulates junctional conductance of Cx45 by affecting both V_j gating and number of functional channels.