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

(2010) FASEB JOURNAL. 24(11). p.4378-4395
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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 [Ca2+] ([Ca2+](i)) using plasma membrane-permeable TAT peptides (100 mu 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 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.
Keywords
actomyosin contractility, intercellular communication, intracellular calcium, purinergic signaling, GAP-JUNCTION PROTEIN, CORNEAL ENDOTHELIAL-CELLS, INTERCELLULAR CALCIUM WAVE, C6 GLIOMA-CELLS, THROMBIN-INDUCED INHIBITION, OCULODENTODIGITAL DYSPLASIA, ZONULA OCCLUDENS-1, CARBOXYL-TERMINUS, CARDIAC MYOCYTES, ATP RELEASE

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Citation

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Chicago
Ponsaerts, Raf, Elke De Vuyst, Mauricio Retamal, Catheleyne D’hondt, Dieter Vermeire, Nan Wang, Humbert De Smedt, et al. 2010. “Intramolecular Loop/tail Interactions Are Essential for Connexin 43-hemichannel Activity.” Faseb Journal 24 (11): 4378–4395.
APA
Ponsaerts, R., De Vuyst, E., Retamal, M., D’hondt, C., Vermeire, D., Wang, N., De Smedt, H., et al. (2010). Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity. FASEB JOURNAL, 24(11), 4378–4395.
Vancouver
1.
Ponsaerts R, De Vuyst E, Retamal M, D’hondt C, Vermeire D, Wang N, et al. Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity. FASEB JOURNAL. 2010;24(11):4378–95.
MLA
Ponsaerts, Raf, Elke De Vuyst, Mauricio Retamal, et al. “Intramolecular Loop/tail Interactions Are Essential for Connexin 43-hemichannel Activity.” FASEB JOURNAL 24.11 (2010): 4378–4395. Print.
@article{1000249,
  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 [Ca2+] ([Ca2+](i)) using plasma membrane-permeable TAT peptides (100 mu 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 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.},
  author       = {Ponsaerts, Raf and De Vuyst, Elke and Retamal, Mauricio and D'hondt, Catheleyne and Vermeire, Dieter and Wang, Nan and De Smedt, Humbert and Zimmermann, Pascale  and Himpens, Bernard and Vereecke, Johan  and Leybaert, Luc and Bultynck, Geert },
  issn         = {0892-6638},
  journal      = {FASEB JOURNAL},
  keyword      = {actomyosin contractility,intercellular communication,intracellular calcium,purinergic signaling,GAP-JUNCTION PROTEIN,CORNEAL ENDOTHELIAL-CELLS,INTERCELLULAR CALCIUM WAVE,C6 GLIOMA-CELLS,THROMBIN-INDUCED INHIBITION,OCULODENTODIGITAL DYSPLASIA,ZONULA OCCLUDENS-1,CARBOXYL-TERMINUS,CARDIAC MYOCYTES,ATP RELEASE},
  language     = {eng},
  number       = {11},
  pages        = {4378--4395},
  title        = {Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity},
  url          = {http://dx.doi.org/10.1096/fj.09-153007},
  volume       = {24},
  year         = {2010},
}

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