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Gap junction protein Cx37 interacts with endothelial nitric oxide synthase in endothelial cells

Verma, Vandana
Lin, Xianming
Coombs, Wanda
Roth, Isabelle
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Published in Arteriosclerosis, thrombosis, and vascular biology. 2010, vol. 30, no. 4, p. 827-834
Abstract OBJECTIVE: The gap junction protein connexin37 (Cx37) plays an important role in cell-cell communication in the vasculature. A C1019T Cx37 gene polymorphism, encoding a P319S substitution in the regulatory C terminus of Cx37 (Cx37CT), correlates with arterial stenosis and myocardial infarction in humans. This study was designed to identify potential binding partners for Cx37CT and to determine whether the polymorphism modified this interaction. METHODS AND RESULTS: Using a high-throughput phage display, we retrieved 2 binding motifs for Cx37CT: WHK ... [K,R]XP ... and FHK ... [K,R]XXP ... , the first being more common for Cx37CT-319P and the second more common for Cx37CT-319S. One of the peptides (WHRTPRLPPPVP) showed 77.7% homology with residues 843 to 854 of endothelial nitric oxide synthase (eNOS). In vitro binding of this peptide or of the homologous eNOS sequence to both Cx37CT isoforms was confirmed by cross-linking and surface plasmon resonance. Electrophysiological analysis of Cx37 single channel activity in transfected N2a cells showed that eNOS-like and eNOS(843-854) increased the frequency of events with conductances higher than 300 pS. We demonstrated that eNOS coimmunoprecipitated with Cx37 in a mouse endothelial cell (EC) line (bEnd.3), human primary ECs, and a human EC line transfected with Cx37-319P or Cx37-319S. Cx37 and eNOS colocalized at EC membranes. Moreover, a dose-dependent increase in nitric oxide production was observed in ECs treated with Cx37 antisense. CONCLUSIONS: Overall, our data show for the first time a functional and specific interaction between eNOS and Cx37. This interaction may be relevant for the control of vascular physiology both in health and in disease.
Keywords Amino Acid MotifsAnimalsBinding SitesCells, CulturedConnexin 43/metabolismConnexins/genetics/*metabolismCross-Linking Reagents/chemistryEndothelial Cells/*enzymologyHumansImmunoprecipitationMembrane PotentialsMiceNitric Oxide/metabolismNitric Oxide Synthase Type III/genetics/*metabolismPatch-Clamp TechniquesPeptide LibraryPolymorphism, GeneticProtein BindingRecombinant Fusion Proteins/metabolismSurface Plasmon ResonanceTransfection
PMID: 20081116
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Research group L'athérosclérose (665)
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PFENNIGER, Anna et al. Gap junction protein Cx37 interacts with endothelial nitric oxide synthase in endothelial cells. In: Arteriosclerosis, thrombosis, and vascular biology, 2010, vol. 30, n° 4, p. 827-834. doi: 10.1161/ATVBAHA.109.200816 https://archive-ouverte.unige.ch/unige:20658

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Deposited on : 2012-05-22

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