Gating Modulation of Connexin45 Gap Junction Channels By Intracellular pH

Abstract

Intracellular pH (pHi) changes considerably during pathological conditions such as ischemia or epilepsy. Changes of pHi affect the way cells communicate through gap junction (GJ) channels, and therefore disturb normal tissue function. To study pHi-dependent modulation of GJ channels, we used HeLa cells expressing connexin45 (Cx45) or its fusion form with EGFP. The latter along with electrophysiological data allowed us to estimate the proportion of functional channels (NF) within a junctional plaque (JP). We examined how junctional conductance (gj) depends on pHi and how pHi affects voltage-gating properties of Cx45 homotypic and Cx45/Cx43-EGFP heterotypic GJs. Even at pHi= ∼8, where the probability of the channels being fully open approximates 1, gj was maximal but only 5 % of the channels were functional. Changes in pHi from ∼7.2 to ∼8 increased gj ∼1.8-fold in homotypic Cx45 GJs; gj-pHi dependence was sigmoidal with pKa = ∼7. We used a stochastic four-state model of contingent gating to fit experimental gj-Vj dependence, which allowed us to define parameters characterizing voltage-gating sensitivity (V0and A) and NF. We found that alkalization increases gj mainly by increasing V0, i.e., voltage at which open and closed states of hemichannel are at equilibrium. On the other hand, uncoupling by acidification was due to a decrease of both V0 and NF. In both cases, the constant A, characterizing the steepness of gj changes over Vj remained stable. These results agree with data obtained from heterotypic Cx45/Cx43-EGFP GJs in which pKa= ∼6.7, i.e., in between pKas of Cx43-EGFP and Cx45 homotypic GJs. In summary, pHi modulates Vj-gating that largely explains observed pH-dependent changes of cell-cell coupling.