Abstract
Connexin 43 (Cx43), a gap junction protein seemingly fit to support cardiac impulse propagation and synchronic contraction, is phosphorylated in normoxia by casein kinase 1 (CK1). However, during cardiac ischemia or pressure overload hypertrophy, this phosphorylation fades, Cx43 abundance decreases at intercalated disks and increases at myocytes’ lateral borders, and the risk of arrhythmia rises. Studies in wild-type and transgenic mice indicate that enhanced CK1-phosphorylation of Cx43 protects from arrhythmia, while dephosphorylation precedes arrhythmia vulnerability. The mechanistic bases of these Cx43 (de)phosphoform-linked cardiac phenotypes are unknown. We used patch-clamp and dye injection techniques to study the channel function (gating, permeability) of Cx43 mutants wherein CK1-targeted serines were replaced by aspartate (Cx43-CK1-D) or alanine (Cx43-CK1-A) to emulate phosphorylation and dephosphorylation, respectively. Cx43-CK1-D, but not Cx43-CK1-A, displayed high Voltage-sensitivity and variable permselectivity. Both mutants showed multiple channel open states with overall increased conductivity, resistance to acidification-induced junctional uncoupling, and hemichannel openings in normal external calcium. Modest differences in the mutant channels’ function and regulation imply the involvement of dissimilar structural conformations of the interacting domains of Cx43 in electrical and chemical gating that may contribute to the divergent phenotypes of CK1-(de)phospho-mimicking Cx43 transgenic mice and that may bear significance in arrhythmogenesis.
Highlights
Gap junction channels made of Connexin (Cx) proteins support the propagation of electrical impulses from cell to cell, and are indispensable for synchronic heart contractions
The decrease in junctional current (Ij) observed in gap junctions (GJs) subjected to large transjunctional voltages (Vj) is called Vj-gating (Figure 1A,B) and is quantified by fitting the ratio of steady state and instantaneous junctional conductance through a broad Vj range with a Boltzmann function [12,13], with half-maximal response reported as V0
This suggests that Connexin 43 (Cx43) phosphorylation by casein kinase 1 (CK1) enhances junctional Vj-gating, and that highly Vj-dependent gap junction channels (GJChs) populate the intercalated disks
Summary
Gap junction channels made of Connexin (Cx) proteins support the propagation of electrical impulses from cell to cell, and are indispensable for synchronic heart contractions. Replacing or supplementing Cx43 with other Cx isotypes promotes arrhythmia vulnerability [3,4]. These data suggest that Cx43 is optimally endowed to support cardiac conduction. While such uniqueness may derive from Cx43 amenability to regulation by multiple kinases [5,6], the functional ramifications of such regulation remain poorly defined. To address this deficit, we aim to outline the operational profiles of Cx43 modified by specific phosphorylation events
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