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J Physiol Volume 586, Number 17, 4209-4223, September 1, 2008 DOI: 10.1113/jphysiol.2008.156083
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CARDIOVASCULAR

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

 Shinichi Takahashi1, Hai Lin1, Naomi Geshi1,4, Yasuo Mori2, Yasuhiro Kawarabayashi1, Noboru Takami3, Masayuki X. Mori1, Akira Honda1 and Ryuji Inoue1

1 Department of Physiology
3 Radioisotope Laboratory, Graduate School of Medical Sciences, Fukuoka University, Fukuoka 814-0180, Japan
2 Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
4 Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark

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 ~70%) by a NO donor SNAP (100 µM) whether it was applied prior to muscarinic receptor stimulation with carbachol (CCh; 100 µM) or after G-protein activation with intracellular perfusion of GTP{gamma}S (100 µM). A similar extent of suppression was also observed with a membrane-permeable analogue of cGMP, 8Br-cGMP (100 µM). 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 32P 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 Arg8-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.

(Received 29 April 2008; accepted after revision 8 July 2008; first published online 10 July 2008)
Corresponding author R. Inoue: Department of Physiology, Graduate School of Medical Sciences, Fukuoka University, Fukuoka 814 0180, Japan.  Email: inouery{at}fukuoka-u.ac.jp

S. Takahashi and H. Lin contributed equally to this work.

This paper has online supplemental material.






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