Browsing by Author "Ponsaerts, Raf"
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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 Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala(Federation of American Societies for Experimental Biology, 2012) Stehberg, Jimmy; Moraga-Amaro, Rodrigo; Salazar, Christian; Becerra, Alvaro; Echeverría, Cesar; Orellana, Juan; Bultynck, Geert; Ponsaerts, Raf; Leybaert, Luc; Simon, Felipe; Sáez, Juan; Retamal, MauricioRecent in vitro evidence indicates that astrocytes can modulate synaptic plasticity by releasing neuroactive substances (gliotransmitters). However, whether gliotransmitter release from astrocytes is necessary for higher brain function in vivo, particularly for memory, as well as the contribution of connexin (Cx) hemichannels to gliotransmitter release, remain elusive. Here, we microinfused into the rat basolateral amygdala (BLA) TAT-Cx43L2, a peptide that selectively inhibits Cx43-hemichannel opening while maintaining synaptic transmission or interastrocyte gap junctional communication. In vivo blockade of Cx43 hemichannels during memory consolidation induced amnesia for auditory fear conditioning, as assessed 24 h after training, without affecting short-term memory, locomotion, or shock reactivity. The amnesic effect was transitory, specific for memory consolidation, and was confirmed after microinfusion of Gap27, another Cx43-hemichannel blocker. Learning capacity was recovered after coinfusion of TAT-Cx43L2 and a mixture of putative gliotransmitters (glutamate, glutamine, lactate, d-serine, glycine, and ATP). We propose that gliotransmitter release from astrocytes through Cx43 hemichannels is necessary for fear memory consolidation at the BLA. Thus, the present study is the first to demonstrate a physiological role for astroglial Cx43 hemichannels in brain function, making these channels a novel pharmacological target for the treatment of psychiatric disorders, including post-traumatic stress disorder.