title: Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain.
abstract: Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.
authors.affiliation: Department of Pharmacology, SUNY Upstate Medical University, 750 East Adams St., Syracuse, NY 13210, USA
authors.forename: Qin
authors.initials: Q
authors.lastname: Xu
authors.forename: Richard F
authors.initials: RF
authors.lastname: Kopp
authors.forename: Yanyi
authors.initials: Y
authors.lastname: Chen
authors.forename: Jenny J
authors.initials: JJ
authors.lastname: Yang
authors.forename: Michael W
authors.initials: MW
authors.lastname: Roe
authors.forename: Richard D
authors.initials: RD
authors.lastname: Veenstra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites / Q000502:physiology; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D045744:Cell Line, Tumor; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D000097003:Gap Junction alpha-5 Protein
chemical_list: D002147:Calmodulin; D018031:Connexin 43; D017630:Connexins; D000097003:Gap Junction alpha-5 Protein; C500216:GJA1 protein, human