Adenylate cyclase/cAMP/protein kinase A signaling pathway inhibits endothelin type A receptor-operated Ca²⁺ entry mediated via transient receptor potential canonical 6 channels

J Pharmacol Exp Ther. 2012 Jan;340(1):143-51. doi: 10.1124/jpet.111.187500. Epub 2011 Oct 14.


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 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 was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of G(q) protein-coupled endothelin type A receptor (ET(A)R)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ET(A)R 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 ET(A)R 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 (TRPC6(S28A)) but not Thr69 (TRPC6(T69A)) 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 (TRPC6(S28A) and TRPC6(T69A)) 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclase Inhibitors
  • Adenylyl Cyclases / physiology*
  • Blotting, Western
  • Calcium / metabolism*
  • Calcium Signaling / physiology*
  • Colforsin / pharmacology
  • Cyclic AMP / physiology*
  • Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cyclic AMP-Dependent Protein Kinases / physiology*
  • Enzyme Activation / physiology
  • Enzyme Inhibitors / pharmacology
  • HEK293 Cells
  • Humans
  • Microscopy, Confocal
  • Mutation / genetics
  • Mutation / physiology
  • Papaverine / pharmacology
  • Phosphorylation
  • Protein Kinase Inhibitors / pharmacology
  • Receptor, Endothelin A / physiology*
  • Retroviridae / genetics
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*
  • Subcellular Fractions / physiology
  • TRPC Cation Channels / genetics
  • TRPC Cation Channels / physiology*
  • TRPC6 Cation Channel
  • Thapsigargin / pharmacology


  • Adenylyl Cyclase Inhibitors
  • Enzyme Inhibitors
  • Protein Kinase Inhibitors
  • Receptor, Endothelin A
  • TRPC Cation Channels
  • TRPC6 Cation Channel
  • TRPC6 protein, human
  • Colforsin
  • Thapsigargin
  • Papaverine
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Adenylyl Cyclases
  • Calcium