Browsing by Author "Himpens, Bernard"
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Item Intramolecular loop/tail interactions are essential forconnexin 43-hemichannel activity(2010) 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, GeertConnexin-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 M) 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 zónula occludens-1, a cytoskeletal-associated Cx43- inding 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. FASEB J. 24, 4378–4395 (2010). www.fasebj.orgItem Peptides and peptide-derived molecules targeting the intracellular domains of Cx43: gap junctions versus hemichannels(Elsevier, 2013) Iyyathuraia, Jegan; D'hondt, Catheleyne; Wang, Nan; De Bock, Marijke; Himpens, Bernard; Retamal, Mauricio; Stehberg, Jimmy; Leybaert, Luc; Bultynck, GeertAbout a decade ago, the molecular determinants controlling the opening and closing of Cx43 gap junction channels have been identified. Advanced biophysical approaches revealed a critical role for structural rearrangements in the cytoplasmic loop and dimerization of the C-terminal tail, resulting in binding of the C-terminal tail to the cytoplasmic loop and Cx43 gap junction channel closure during cellular acidosis. This has spurred the development of Cx43-mimetic peptides and peptidomimetics that interfere with these loop/tail interactions, thereby preventing the closure of Cx43 gap junctions, e.g. in the heart upon ischemia. Recently, we found that loop/tail interactions control Cx43-hemichannel activity but with an opposite effect. Binding of the C-terminal tail to the cytoplasmic loop is a requisite for the opening of Cx43 hemichannels in response to different stimuli, like decreased extracellular [Ca2+], increased intracellular [Ca2+], positive membrane potentials or ischemia. Strikingly, peptides that favor the open state of Cx43 gap junctions like the L2 peptide inhibit Cx43-hemichannel opening. These tools now provide unprecedented opportunities to selectively inhibit Cx43 hemichannels while maintaining Cx43 gap junction communication, impossible to achieve with siRNA or knockdown approaches both affecting gap junctions and hemichannels. These tools not only are very helpful to unravel the role of Cx43 hemichannels in complex biological systems, but also hold therapeutic potential to counteract excessive Cx43-hemichannel activity like in ischemia/reperfusion in the brain and the heart or to prevent Cx43 hemichannel-mediated gliotransmitter release in the basal amygdala during memory consolidation in response to emotional events. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.