Abstract
Receptor-operated Ca2+ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca2+ concentration triggered by the activation of Gq 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 Gq protein-coupled endothelin type A receptor (ETAR)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ETAR 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. Ca2+ measurements with thapsigargin-induced Ca2+-depletion/Ca2+-restoration protocol to estimate ROCE showed that the stimulation of ETAR 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 (TRPC6S28A) but not Thr69 (TRPC6T69A) 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 (TRPC6S28A and TRPC6T69A) 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.
Footnotes
This study was supported in part by Grants-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science [grant 21790236] (to T. Horinouchi); Grants-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science [grant 21390068] (to S.M.); and grants from the Smoking Research Foundation of Japan (to S.M.), the Mitsubishi Pharma Research Foundation (to T. Horinouchi), the Pharmacological Research Foundation, Tokyo (to T. Horinouchi), the Shimabara Science Promotion Foundation (to T. Horinouchi), and Actelion Pharmaceuticals Japan Ltd. (to T. Horinouchi).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- TRPC
- transient receptor potential canonical
- AC
- adenylate cyclase
- BSA
- bovine serum albumin
- [Ca2+]i
- intracellular free Ca2+ concentration
- ECL
- enhanced chemiluminescence
- EPAC
- exchange protein activated by cAMP
- ER
- endoplasmic reticulum
- ET-1
- endothelin-1
- ETAR
- endothelin type A receptor
- fura-2/AM
- fura-2/acetoxymethyl ester
- GFP
- green fluorescent protein
- GqPCR
- Gq protein-coupled receptor
- HA
- hemagglutinin
- HEK293
- human embryonic kidney 293
- HRP
- horseradish peroxidase
- IB
- immunoblotting
- IP
- immunoprecipitation
- IPAH
- idiopathic pulmonary arterial hypertension
- PDE
- phosphodiesterase
- PKA
- protein kinase A
- PKG
- protein kinase G
- ROCC
- receptor-operated Ca2+ channel
- ROCE
- receptor-operated Ca2+ entry
- Rp-8-Br-cAMP
- Rp-8-bromoadenosine-cAMP sodium salt
- SA
- streptavidine
- SOCE
- store-operated Ca2+ entry
- SQ-22,536
- 9-(tetrahydro-2-furanyl)-9H-purin-6-amine
- TG
- thapsigargin
- WT
- wild type.
- Received August 31, 2011.
- Accepted October 13, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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