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

Abstract

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 g j 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 a s 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.cell-cell coupling | pH-dependent gating | EGFP | hemichannel | connexon C hanges in intracellular pH (pH i ) take place under different physiological and pathological conditions, and H + ions have a broad effect on cell function including cell-cell electrical and metabolic communication mediated by gap junctions (GJs) and paracrine signaling through nonjunctional/unapposed hemichannels (1-4). Modest pH i changes have been observed under normal physiological conditions [e.g., changes of neuronal activity or the resting potential (5, 6)], and greater changes occur under pathological conditions such as hypoxia, ischemia, or epilepsy (4,7,8).During the last decade, significant progress has been made toward understanding the molecular mechanisms of pH-dependent modulation of GJs and hemichannels (2, 9-12). Several domains in the cytoplasmic loop and C terminus of connexin43 (Cx43) appear to be involved in pH-dependent gating (2, 9, 11, 12). Furthermore, pH-dependent interaction of connexins with other cytoplasmic proteins may be important in the remodeling of connexins and in protection from lesion spread after local ischemic injury (13,14).GJs provide channels with an inner diameter of ∼1.4 nm between the interiors of the coupled cells. This link allows the spread of electrical potential and small metabolites. Each GJ cha...

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