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Intramolecular loop/tail interactions are essential for connexin 43‐hemichannel activity
Author(s) -
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
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.09-153007
Subject(s) - connexin , chemistry , loop (graph theory) , intramolecular force , biophysics , microbiology and biotechnology , stereochemistry , biology , biochemistry , gap junction , mathematics , combinatorics , intracellular
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.—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. FASEB J . 24, 4378–4395 (2010). www.fasebj.org