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


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.
Pfenniger
Anna
A
Division of Cardiology, Department of Internal Medicine, Faculty of Medicine, University of Geneva, Switzerland. anna.pfenniger@unige.ch
Derouette
Jean-Paul
JP
Verma
Vandana
V
Lin
Xianming
X
Foglia
Bernard
B
Coombs
Wanda
W
Roth
Isabelle
I
Satta
Nathalie
N
Dunoyer-Geindre
Sylvie
S
Sorgen
Paul
P
Taffet
Steven
S
Kwak
Brenda R
BR
Delmar
Mario
M
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D042783:Endothelial Cells / Q000201:enzymology*; D006801:Humans; D047468:Immunoprecipitation; D008564:Membrane Potentials; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D019151:Peptide Library; D011110:Polymorphism, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014162:Transfection; D000097003:Gap Junction alpha-5 Protein; D000097006:Gap Junction alpha-4 Protein
D018031:Connexin 43; D017630:Connexins; D003432:Cross-Linking Reagents; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D000097003:Gap Junction alpha-5 Protein; D000097006:Gap Junction alpha-4 Protein; C496314:NOS3 protein, human; C496315:Nos3 protein, mouse