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 TRPC6 channels.
AID  - 10.1124/jpet.111.187500
DP  - 2011 Jan 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - jpet.111.187500
4099  - http://jpet.aspetjournals.org/content/early/2011/10/14/jpet.111.187500.short
4100  - http://jpet.aspetjournals.org/content/early/2011/10/14/jpet.111.187500.full
AB  - Receptor-operated Ca2+ entry (ROCE) via TRPC6 (transient receptor potential canonical channel 6) is an important machinery for an increase in intracellular Ca2+ concentration triggered by 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 is to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in regulation of Gq protein-coupled endothelin type A receptor (ETAR)-mediated ROCE via TRPC6. For this purpose, HEK293 cells stably co-expressing 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 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 cAMP/PKA pathway, while it was potentiated by Rp-8-Br-cAMP, a PKA inhibitor. 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-tagTM biotin to determine 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 (about 50%) than that of wild-type. These results suggest that TRPC6 is negatively regulated by PKA-mediated phosphorylation of Ser28 but not of Thr69.