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

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.

Figure 1

SPR on Cx37CT and binding peptides. Representative examples (N=3) of association and dissociation curves of peptides to Cx37CT are shown for each condition. A, Cx37CT-319P and eNOS-like. B, Cx37CT-319S and eNOS-like. C, Cx37CT-319P and eNOS(843–854). D, Cx37CT-319S and eNOS(843–854). Peptides were superfused at concentrations of 1 mmol/L (red), 500 μmol/L (blue), 250 μmol/L (green), 125 μmol/L (orange), and 62.5 μmol/L (pink).

Figure 2

Cross-linking experiment on Cx37CT and peptides. The binding in solution of eNOS-like (A) and eNOS(843–854) (B) to Cx37CT-319P or Cx37CT-319S was assessed by the incubation with the cross-linker BS. Second and fifth lanes of each panel show an additional band at ≈12 kDa, which is absent in the other lanes, where either the peptide or the cross-linker is missing.

Figure 3

Single-channel data from N2a cells transfected with Cx37-319P and Cx37-319S. All events histograms of unitary conductance in cells transfected with Cx37-319P or Cx37-319S in the presence of a scrambled peptide (A, N=4, n=211; D, N=5, n=286, respectively), eNOS-like (B, N=5, n=447; E, N=4, n=205, respectively), or eNOS(843-854) (C, N=4, n=462; F, N=3, n=276, respectively) in the pipette solution. The presence of eNOS-like or eNOS(843-854) increased the number of channel openings larger than 300 pS (shaded bars) compared with the control condition. Minimum event duration was 50 ms. Bin width=6.25 pS.

Figure 4

Cx37 and eNOS interact in a mouse EC line. A, Immunoprecipitation of eNOS and Cx37 in bEnd.3. Left panel: eNOS immunoblot of Cx37 immunoprecipitate. Right panel: Cx37 immunoblot of eNOS immunoprecipitate. L indicates total lysate; IP, immunoprecipitate; and C, negative control. B, Cx37 and eNOS immunostaining in bEnd.3. eNOS was found perinuclearly and near membranes. Cx37 was detected at sites of cell-cell contact. Merged images show areas of colocalization (arrow). Scale bar represents 30 μm. C, Cx37 immunostaining (green) of bEnd.3 incubated with Cx37 sense (left) or Cx37 antisense (right). Cells were counterstained with Evans Blue (red). Scale bar represents 20 μm. D, Nitrite release by bEnd.3 incubated with Cx37 sense or 10, 25, or 50 μmol/L Cx37 antisense for 48 hours. Overnight nitrite release increased with increasing dose of antisense. N=6, error bars show SEM, and *P<0.05. E, Lysates of bEnd.3 incubated with Cx37 antisense for 48 hours were immunoblotted against eNOS, Cx40, Cx43, Cav1, and inducible nitric oxide synthase (iNOS). mΦ: stimulated mouse macrophages.

Figure 5

Cx37 and eNOS interact in primary and transfected human ECs. A, Immunoprecipitation of eNOS with Cx37 in HUVECs. Lanes are as described in Figure 4A. B, Cx37 immunostaining (green) of HUVECs in control condition (top), incubated with Cx37 sense (middle) or Cx37 antisense (bottom). Cells were counterstained with Evans Blue (red). Scale bar represents 30 μm. C, Nitrite release by HUVECs treated with Cx37 sense or 25 or 50 μmol/L Cx37 antisense for 48 hours. Overnight nitrite release increased with increasing dose of antisense. N=6 to 9, error bars show SEM, and *P<0.05. D, Immunoprecipitation of eNOS with Cx37 in EA.hy926 (human ECs) or HeLa transfected with Cx37-319P or Cx37-319S. Cx37 antibodies pulled down a protein of ≈140 kDa labeled with anti-eNOS in EA.hy926/Cx37 transfectants only.