title: Isoform-specific phosphorylation-dependent regulation of connexin hemichannels.
abstract: Connexins form gap junction channels made up of two connexons (hemichannels) from adjacent cells. Unopposed hemichannels may open toward the extracellular space upon stimulation by, e.g., removal of divalent cations from the extracellular solution and allow isoform-specific transmembrane flux of fluorescent dyes and physiologically relevant molecules, such as ATP and ions. Connexin (Cx)43 and Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43 in its cell-cell gap junction configuration and may also act to keep Cx43 hemichannels closed. In contrast, the regulation of Cx30 hemichannels by PKC is unexplored. To determine phosphorylation-dependent regulation of Cx30 and Cx43 hemichannels, these were heterologously expressed in Xenopus laevis oocytes and opened with divalent cation-free solution. Inhibition of PKC activity did not affect hemichannel opening of either connexin. PKC activation had no effect on Cx43-mediated hemichannel activity, whereas both dye uptake and current through Cx30 hemichannels were reduced. We detected no PKC-induced connexin internalization from the plasma membrane, indicating that PKC reduced Cx30 hemichannel activity by channel closure. In an attempt to resolve the PKC phosphorylation site(s) on Cx30, alanine mutations of putative cytoplasmic PKC consensus sites were created to prevent phosphorylation (T5A, T8A, T102A, S222A, S225A, S239A, and S258A). These Cx30 mutants responded to PKC activation, suggesting that Cx30 hemichannels are not regulated by phosphorylation of a single site. In conclusion, Cx30, but not Cx43, hemichannels close upon PKC activation, illustrating that connexin hemichannels display not only isoform-specific permeability profiles but also isoform-specific regulation by PKC.
authors.affiliation: Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; and.
authors.forename: Jette Skov
authors.initials: JS
authors.lastname: Alstrøm
authors.affiliation: Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; and.
authors.forename: Daniel Bloch
authors.initials: DB
authors.lastname: Hansen
authors.affiliation: Danish National Research Foundation Centre for Cardiac Arrhythmia and Department of Biomedicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Morten Schak
authors.initials: MS
authors.lastname: Nielsen
authors.affiliation: Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; and macaulay@sund.ku.dk.
authors.forename: Nanna
authors.initials: N
authors.lastname: MacAulay
mesh_terms: D000818:Animals; D053119:Benzophenanthridines / Q000494:pharmacology; D000075302:Connexin 30; D018031:Connexin 43 / Q000378:metabolism*; D017630:Connexins / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008564:Membrane Potentials; D051379:Mice; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D014982:Xenopus laevis
chemical_list: D053119:Benzophenanthridines; D000075302:Connexin 30; D018031:Connexin 43; D017630:Connexins; C580592:Gja1 protein, rat; C100629:Gjb6 protein, mouse; D007211:Indoles; D008301:Maleimides; D020033:Protein Isoforms; C116174:bisindolylmaleimide II; C016299:chelerythrine; D011493:Protein Kinase C
keywords: conductance; connexin; fluorescent dye; hemichannel activity; protein kinase C