title: Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity.
abstract: Connexin-assembled gap junctions (GJs) and hemichannels coordinate intercellular signaling processes. Although the regulation of connexins in GJs has been well characterized, the molecular determinants controlling connexin-hemichannel activity are unresolved. Here we investigated the regulation of Cx43-hemichannel activity by actomyosin contractility and intracellular [Ca(2+)] ([Ca(2+)](i)) using plasma membrane-permeable TAT peptides (100 μM) designed to interfere with interactions between the cytoplasmic loop (CL) and carboxy-terminal (CT) in primary bovine corneal endothelial cells and HeLa, C6 glioma, and Xenopus oocytes ectopically expressing Cx43. Peptides corresponding to the last 10 CT aa (TAT-Cx43CT) prevented the inhibition of Cx43-hemichannel activity by contractility/high [Ca(2+)](i), whereas a reverse peptide (TAT-Cx43CTrev) did not. These effects were independent of zonula occludens-1, a cytoskeletal-associated Cx43-binding protein. In contrast, peptides corresponding to CL (TAT-L2) inhibited Cx43-hemichannel responses, whereas a mutant peptide (TAT-L2(H126K/I130N)) did not inhibit. In these assays, TAT-Cx43CT acted as a scaffold for TAT-L2 and vice versa, a finding supported by surface plasmon resonance measurements. Loop/tail interactions appeared essential for Cx43-hemichannel activity, because TAT-Cx43CT restored the activity of nonfunctional hemichannels, consisting of either Cx43 lacking the C-terminal tail (Cx43(M239)) or intact Cx43 ectopically expressed in Xenopus oocytes. We conclude that intramolecular loop/tail interactions control Cx43-hemichannel activity, laying the basis for developing hemichannel-specific blockers.
authors.affiliation: Department Molecular Cell Biology, KU Leuven, Leuven, Belgium.
authors.forename: Raf
authors.initials: R
authors.lastname: Ponsaerts
authors.forename: Elke
authors.initials: E
authors.lastname: De Vuyst
authors.forename: Mauricio
authors.initials: M
authors.lastname: Retamal
authors.forename: Catheleyne
authors.initials: C
authors.lastname: D'hondt
authors.forename: Dieter
authors.initials: D
authors.lastname: Vermeire
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.forename: Humbert
authors.initials: H
authors.lastname: De Smedt
authors.forename: Pascale
authors.initials: P
authors.lastname: Zimmermann
authors.forename: Bernard
authors.initials: B
authors.lastname: Himpens
authors.forename: Johan
authors.initials: J
authors.lastname: Vereecke
authors.forename: Luc
authors.initials: L
authors.lastname: Leybaert
authors.forename: Geert
authors.initials: G
authors.lastname: Bultynck
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D018031:Connexin 43 / Q000378:metabolism*; D003315:Cornea / Q000166:cytology / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism; D006367:HeLa Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013917:Thrombin / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D018031:Connexin 43; D015696:Gene Products, tat; D006576:Heterocyclic Compounds, 4 or More Rings; D007473:Ion Channels; C472645:blebbistatin; D000255:Adenosine Triphosphate; D013917:Thrombin; D002118:Calcium