Adenylate Cyclase/cAMP/Protein Kinase A Signaling Pathway Inhibits Endothelin Type A Receptor-Operated Ca2+ Entry Mediated via Transient Receptor Potential Canonical 6 Channels

• DOI: 10.1124/jpet.111.187500
• Publication Date: 2011-10-15T02:13:09Z
• Authors: Takahiro Horinouchi; Tsunaki Higa; Hiroyuki Aoyagi; Tadashi Nishiya; Koji Terada; Soichi Miwa
• Keywords: TRPC6

Receptor-operated Ca²⁺ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular concentration triggered by the activation of G_q protein-coupled receptors. TRPC6 phosphorylated various protein kinases including kinase A (PKA). However, regulation activity PKA still controversial. The purpose this study was to elucidate role adenylate cyclase/cAMP/PKA signaling pathway endothelin type (ET_AR)-mediated ROCE TRPC6. For purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing ET_AR and (wild type) or its mutants possessing a single point mutation putative phosphorylation sites were used analyze amino acids responsible PKA-mediated measurements with thapsigargin-induced Ca²⁺-depletion/Ca²⁺-restoration protocol estimate showed that stimulation induced marked HEK293 expressing compared control cells. inhibited forskolin papaverine activate cAMP/PKA pathway, whereas it potentiated Rp-8-bromoadenosine-cAMP sodium salt, inhibitor. inhibitory effects partially cancelled replacing Ser28 (TRPC6^S28A) but not Thr69 (TRPC6^T69A) alanine. In vitro assay Phos-tag biotin determine level revealed wild-type mutant (TRPC6^S28A TRPC6^T69A) proteins PKA, these lower (approximately 50%) than wild type. These results suggest negatively regulated Thr69.