Abstract

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 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 is to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in regulation of G_q protein-coupled endothelin type A receptor (ET_AR)-mediated ROCE via TRPC6. For this purpose, HEK293 cells stably co-expressing human ET_AR 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 ET_AR 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 (TRPC6^S28A) but not Thr⁶⁹ (TRPC6^T69A) of TRPC6 with alanine. In vitro kinase assay with Phos-tag^TM biotin to determine 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 (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.