J Pharmacol Exp Ther. 2012 Jan;340(1):143-51. doi: 10.1124/jpet.111.187500. Epub 2011 Oct 14.

Adenylate cyclase/cAMP/protein kinase A signaling pathway inhibits endothelin type A receptor-operated Ca(2)(+) entry mediated via transient receptor potential canonical 6 channels.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Horinouchi, T., Higa, T., Aoyagi, H., Nishiya, T., Terada, K., Miwa, S.,
["Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Hokkaido, Japan."]
Receptor-operated Ca(2)(+) entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca(2)(+) concentration triggered by the activation of G(q) protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of G(q) protein-coupled endothelin type A receptor (ET(A)R)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ET(A)R and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca(2)(+) measurements with thapsigargin-induced Ca(2)(+)-depletion/Ca(2)(+)-restoration protocol to estimate ROCE showed that the stimulation of ET(A)R induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosine-cAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6(S28A)) but not Thr69 (TRPC6(T69A)) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6(S28A) and TRPC6(T69A)) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.
PMID: 22001259External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Functional consequence Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
TRP channel Interactor Method Post-translational modification Subcellular trafficking Activity Reference
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PKA In vitro PTM assay Phosphorylation (Thr-69) 22001259
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PKA In vitro PTM assay Phosphorylation (Ser-28) 22001259
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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