Abstract
Aim:
TRPV4-C1 heteromeric channels contribute to store-operated Ca2+ entry in vascular endothelial cells. However, the negative regulation of these channels is not fully understood. This study was conducted to investigate the inhibitory effect of PKG1α on TRPV4-C1 heteromeric channels.
Methods:
Immuno-fluorescence resonance energy transfer (FRET) was used to explore the spatial proximity of PKG1α and TRPC1. Phosphorylation of endogenous TRPC1 was tested by phosphorylation assay. [Ca2+]i transients and cation current in MAECs were assessed with Fura-2 fluorescence and whole-cell recording, respectively. In addition, rat mesenteric arteries segments were prepared, and vascular relaxation was examined with wire myography.
Results:
In immuno-FRET experiments, after exposure of these cells to 8-Br-cGMP, more PKG1α was observed in the plasma membrane, and PKG1α and TRPC1 were observed to be in closer proximity. TAT-TRPC1S172 and TAT-TRPC1T313 peptide fragments, which contain the PKG targeted residues Ser172 and Thr313, respectively, were introduced into isolated endothelial cells to abrogate the translocation of PKG1α. Furthermore, a phosphorylation assay demonstrated that PKG directly phosphorylates TRPC1 at Ser172 and Thr313 in endothelial cells. In addition, PKG activator 8-Br-cGMP markedly reduced the magnitude of the 4αPDD-induced and 11,12-EET-induced [Ca2+]i transients, the cation current and vascular relaxation.
Conclusion:
This study uncovers a novel mechanism by which PKG negatively regulates endothelial heteromeric TRPV4-C1 channels through increasing the spatial proximity of TRPV4-C1 to PKG1α via translocation and through phosphorylating Ser172 and Thr313 of TRPC1.
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Acknowledgements
We thank Prof IC BRUCE for critical reading of the manuscript. This work was supported by National Natural Science Foundation of China (91439131 and 81572940 to Xin MA), (21305051 to Chun-lei TANG); the National High-Technology Research and Development Program (863 Program) of China (2015AA020948 to Xin MA); the Natural Science Foundation for Distinguished Young Scholars of Jiangsu Province BK20140004 (to Xin MA); the Program for New Century Excellent Talents in University of Ministry of Education of China NCET-12-0880 (to Xin MA).
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Zhang, P., Mao, Aq., Sun, Cy. et al. Translocation of PKG1α acts on TRPV4-C1 heteromeric channels to inhibit endothelial Ca2+ entry. Acta Pharmacol Sin 37, 1199–1207 (2016). https://doi.org/10.1038/aps.2016.43
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DOI: https://doi.org/10.1038/aps.2016.43
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