PT  - JOURNAL ARTICLE
AU  - Takahiro Horinouchi
AU  - Tsunaki Higa
AU  - Hiroyuki Aoyagi
AU  - Tadashi Nishiya
AU  - Koji Terada
AU  - Soichi Miwa
TI  - Adenylate Cyclase/cAMP/Protein Kinase A Signaling Pathway Inhibits Endothelin Type A Receptor-Operated Ca<sup>2+</sup> Entry Mediated via Transient Receptor Potential Canonical 6 Channels
AID  - 10.1124/jpet.111.187500
DP  - 2012 Jan 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 143--151
VI  - 340
IP  - 1
4099  - http://jpet.aspetjournals.org/content/340/1/143.short
4100  - http://jpet.aspetjournals.org/content/340/1/143.full
SO  - J Pharmacol Exp Ther2012 Jan 01; 340
AB  - 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.