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J Physiol. 2008 Sep 1;586(17):4209-23. doi: 10.1113/jphysiol.2008.156083. Epub 2008 Jul 10.

Nitric oxide-cGMP-protein kinase G pathway negatively regulates vascular transient receptor potential channel TRPC6.

Author information

1
Department of Physiology, Graduate School of Medical Sciences, Fukuoka University, Fukuoka 814 0180, Japan.

Abstract

We investigated the inhibitory role of the nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway on receptor-activated TRPC6 channels in both a heterologous expression system (HEK293 cells) and A7r5 vascular myocytes. Cationic currents due to TRPC6 expression were strongly suppressed (by approximately 70%) by a NO donor SNAP (100 microm) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 microm) or after G-protein activation with intracellular perfusion of GTPgammaS (100 microm). A similar extent of suppression was also observed with a membrane-permeable analogue of cGMP, 8Br-cGMP (100 microm). The inhibitory effects of SNAP and 8Br-cGMP on TRPC6 channel currents were strongly attenuated by the presence of inhibitors for guanylyl cyclase and PKG such as ODQ, KT5823 and DT3. Alanine substitution for the PKG phosphorylation candidate site at T69 but not at other sites (T14A, S28A, T193A, S321A) of TRPC6 similarly attenuated the inhibitory effects of SNAP and 8Br-cGMP. SNAP also significantly reduced single TRPC6 channel activity recorded in the inside-out configuration in a PKG-dependent manner. SNAP-induced PKG activation stimulated the incorporation of (32)P into wild-type and S321A-mutant TRPC6 proteins immunoprecipitated by TRPC6-specific antibody, but this was greatly attenuated in the T69A mutant. SNAP or 8Br-cGMP strongly suppressed TRPC6-like cation currents and membrane depolarization evoked by Arg(8)-vasopressin in A7r5 myocytes. These results strongly suggest that TRPC6 channels can be negatively regulated by the NO-cGMP-PKG pathway, probably via T69 phosphorylation of the N-terminal. This mechanism may be physiologically important in vascular tissues where NO is constantly released from vascular endothelial cells or nitrergic nerves.

PMID:
18617565
PMCID:
PMC2652196
DOI:
10.1113/jphysiol.2008.156083
[Indexed for MEDLINE]
Free PMC Article

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