title: Connexin 43 hemichannels contribute to cytoplasmic Ca2+ oscillations by providing a bimodal Ca2+-dependent Ca2+ entry pathway.
abstract: Many cellular functions are driven by changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) that are highly organized in time and space. Ca(2+) oscillations are particularly important in this respect and are based on positive and negative [Ca(2+)](i) feedback on inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). Connexin hemichannels are Ca(2+)-permeable plasma membrane channels that are also controlled by [Ca(2+)](i). We aimed to investigate how hemichannels may contribute to Ca(2+) oscillations. Madin-Darby canine kidney cells expressing connexin-32 (Cx32) and Cx43 were exposed to bradykinin (BK) or ATP to induce Ca(2+) oscillations. BK-induced oscillations were rapidly (minutes) and reversibly inhibited by the connexin-mimetic peptides (32)Gap27/(43)Gap26, whereas ATP-induced oscillations were unaffected. Furthermore, these peptides inhibited the BK-triggered release of calcein, a hemichannel-permeable dye. BK-induced oscillations, but not those induced by ATP, were dependent on extracellular Ca(2+). Alleviating the negative feedback of [Ca(2+)](i) on InsP(3)Rs using cytochrome c inhibited BK- and ATP-induced oscillations. Cx32 and Cx43 hemichannels are activated by <500 nm [Ca(2+)](i) but inhibited by higher concentrations and CT9 peptide (last 9 amino acids of the Cx43 C terminus) removes this high [Ca(2+)](i) inhibition. Unlike interfering with the bell-shaped dependence of InsP(3)Rs to [Ca(2+)](i), CT9 peptide prevented BK-induced oscillations but not those triggered by ATP. Collectively, these data indicate that connexin hemichannels contribute to BK-induced oscillations by allowing Ca(2+) entry during the rising phase of the Ca(2+) spikes and by providing an OFF mechanism during the falling phase of the spikes. Hemichannels were not sufficient to ignite oscillations by themselves; however, their contribution was crucial as hemichannel inhibition stopped the oscillations.
authors.affiliation: Department of Basic Medical Sciences, Physiology Group, Ghent University 9000 Ghent, Belgium.
authors.forename: Marijke
authors.initials: M
authors.lastname: De Bock
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.forename: Melissa
authors.initials: M
authors.lastname: Bol
authors.forename: Elke
authors.initials: E
authors.lastname: Decrock
authors.forename: Raf
authors.initials: R
authors.lastname: Ponsaerts
authors.forename: Geert
authors.initials: G
authors.lastname: Bultynck
authors.forename: Geneviève
authors.initials: G
authors.lastname: Dupont
authors.forename: Luc
authors.initials: L
authors.lastname: Leybaert
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling; D002229:Carbenoxolone / Q000494:pharmacology; D002460:Cell Line; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism*; D004285:Dogs; D005452:Fluoresceins / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D034622:RNA Interference; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D000097002:Gap Junction beta-1 Protein
chemical_list: D000255:Adenosine Triphosphate; D001920:Bradykinin; D002118:Calcium; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002229:Carbenoxolone; D018031:Connexin 43; D017630:Connexins; D045304:Cytochromes c; D005452:Fluoresceins; D015544:Inositol 1,4,5-Trisphosphate; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; D000097002:Gap Junction beta-1 Protein; C514423:Gap 26 peptide; C007740:fluorexon