Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity

• Published in: The FASEB journal Vol. 24; no. 11; pp. 4378 - 4395
• Main Authors: Ponsaerts, Raf, De Vuyst, Elke, Retamal, Mauricio, D'hondt, Catheleyne, Vermeire, Dieter, Wang, Nan, De Smedt, Humbert, Zimmermann, Pascale, Himpens, Bernard, Vereecke, Johan, Leybaert, Luc, Bultynck, Geert
• Format: Journal Article
• Language: English
• Published: United States The Federation of American Societies for Experimental Biology 01.11.2010
• Subjects: Adenosine Triphosphate - metabolism; Animals; Calcium - metabolism; Cattle; Cell Line; Cell Line, Tumor; Connexin 43 - metabolism; Cornea - cytology; Cornea - metabolism; Endothelial Cells - metabolism; Gene Products, tat - metabolism; HeLa Cells; Heterocyclic Compounds, 4 or More Rings - pharmacology; Humans; Intracellular Space - metabolism; Ion Channels - drug effects; Ion Channels - metabolism; Oocytes - metabolism; Protein Binding; Rats; Thrombin - metabolism; Xenopus laevis - metabolism
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.09-153007

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 [Ca²⁺] ([Ca²⁺]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 [Ca²⁺]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-L2H¹²⁶K/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 (Cx43M²³⁹) 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.--Ponsaerts, R., De Vuyst, E., Retamal, M., D'hondt, C., Vermeire, D., Wang, N., De Smedt, H., Zimmermann, P., Himpens, B., Vereecke, J., Leybaert, L., Bultynck, G. Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity.