Abstract 1911: eNOS is a Binding Partner for the Gap Junction Protein Cx37 in Endothelial Cells
Connexin37 (Cx37) is a gap junction protein essential for cell-cell communication in the vascular system. A C1019T Cx37 gene polymorphism, encoding for a P319S amino acid substitution in the regulatory C-terminus of Cx37 (Cx37CT), was found to correlate with arterial stenosis and myocardial infarction in humans. The aim of this study was to identify potential binding partners for Cx37CT and to determine whether the polymorphism modified this interaction. Using a high throughput phage display, we retrieved 2 consensus binding motifs for Cx37CT: WHK. . .[K,R]XP. . . and FHK. . .[K,R]XXP. . ., the first one being more frequent for Cx37CT-319P and the second more frequent for Cx37CT-319S. One of the peptides (WHRTPRLPPPVP) showed a 77.7% homology with amino acids 843 to 854 of the endothelial nitric oxide synthase (eNOS). As eNOS plays a fundamental role in vascular biology, we further investigated this potential interaction. In vitro binding of this peptide to both forms of Cx37CT was confirmed by cross-linking and surface plasmon resonance. Electrophysiological analysis of Cx37 single channel activity in transfected N2a cells showed that eNOS-like peptides increased the frequency of occurrence of conductances higher than 300 pS. eNOS co-immunoprecipitated with Cx37 in human endothelial cells transfected with human Cx37 polymorphs and in a mouse endothelial cell line (bEnd.3). Immunofluorescence microscopy showed a co-localization of these proteins at membranes in bEnd.3 cells and a dose-dependent increase in NO production was observed when they were treated with Cx37-antisense. Overall, our data show for the first time a functional interaction between eNOS and Cx37. This interaction may be relevant for the control of vascular physiology both in health and in disease.