Calcium-Calmodulin Regulation of Connexin43 Involves a Cytoplasmic Loop Domain

Abstract

Connexin43 (Cx43) is widely expressed throughout the mammalian body and is the predominant gap junction protein in the ventricular myocardium. Cx43 was recently reported to contain a calmodulin (CaM) binding site on its cytoplasmic loop (CL) domain near its third transmembrane domain (Zhou et al., JBC 282: 35005-17, 2007). Intracellular calcium (Cai)-dependent regulation of cardiac and Cx43 gap junctions has also been reported, but the function of this putative CaM-binding site has never been directly examined. In dual whole cell patch clamp experiments, murine neuro2a (N2a)-Cx43 cell gap junctional conductance (gj) declined by 95% within 10 min (n=3) during bath perfusion with 1 μM ionomycin + 1.8 mM external Ca2+ (Cao). Cx43 gj declined by only 20% in nominally zero Cao (n=5). This Ca2+-dependent uncoupling was demonstrated to be CaM-dependent by acute (10-15 min) pretreatment with 2 μM calmidazolium, wherein Cx43 gj declined by < 10% within 10 min (n=4). To directly test for the involvement of the Cx43 amino acid residue #136-158 domain in this Ca2+/CaM-dependent gap junction uncoupling process, 1 μM peptides were added to both whole cell patch pipettes and the 1 μM ionomycin/1.8 mM Cao perfusion experiments were repeated. The Cx43 #136-158 sequence mimetic peptide (Kd(CaM) = 860 nM) effectively prevented the Cx43 gj decline (< 3%, n=4) whereas a scrambled sequence peptide control failed to prevent the Ca2+-induced rundown of Cx43 gj (<90%, n=3). These data unequivocally demonstrate that influx of external Ca2+ induces closure of Cx43 gap junctions in a CaM-dependent process involving the Cx43 residue #136-158 CL domain. This process has significant implications for the modulation of cardiac gj by Cai and the “healing-over” of infarcted myocardium. Supported by NIH grants GM62999 & EY-05684 to JJY and HL-042220 to RDV.