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 (gj) and gj dependence on transjunctional voltage (Vj). To characterize Vj gating, we fit the gj–Vj relation using a stochastic four-state model containing one Vj-sensitive gate in each apposed hemichannel (aHC); aHC open probability was a Boltzmann function of the fraction of Vj across it. Using the model, we estimated gating parameters characterizing sensitivity to Vj and number of functional channels. In homotypic Cx45 and heterotypic Cx45/Cx43-EGFP GJs, pHi changes from 7.2 to ~8.0 shifted gj–Vj dependence of Cx45 aHCs along the Vj axis resulting in increased probability of GJ channels being in the fully open state without change in the slope of gj dependence on Vj. In contrast, acidification shifted gj–Vj 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 (pKa) of gj–pHi dependence of Cx45/Cx45 GJs was ~7. The pKa of heterotypic Cx45/Cx43-EGFP GJs was lower, ~6.7, between the pKas of Cx45 and Cx43-EGFP (~6.5) homotypic GJs. In summary, pHi significantly modulates junctional conductance of Cx45 by affecting both Vj gating and number of functional channels.