Research Paper
Subject Category: Molecular and cellular mechanisms
British Journal of Pharmacology (2007) 152, 122–131; doi:10.1038/sj.bjp.0707368; published online 2 July 2007
Myosin light chain kinase-independent inhibition by ML-9 of murine TRPC6 channels expressed in HEK293 cells
J Shi1,2,3, S Takahashi2,3, X-H Jin4, Y-Q Li1, Y Ito3, Y Mori5 and R Inoue2
- 1Department of Anatomy and K.K Leung Brain Research Centre, The Fourth Military Medical University, Xi'an, China
- 2Department of Physiology, Fukuoka University School of Medicine, Fukuoka, Japan
- 3Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
- 4Department of Histology and Embryology, The Fourth Military Medical University, Xi'an, China
- 5Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan
Correspondence: Professor R Inoue, Department of Physiology, Fukuoka University School of Medicine, Nanakuma 7-45-1, Jonann-ku, Fukuoka 814-0180, Japan. E-mail: inouery@fukuoka-u.ac.jp
Received 12 April 2007; Revised 29 May 2007; Accepted 31 May 2007; Published online 2 July 2007.
Abstract
Background and purpose:
Myosin light chain kinase (MLCK) plays a pivotal role in regulation of cellular functions, the evidence often relying on the effects of extracelluarly administered drugs such as ML-9. Here we report that this compound exerts non-specific inhibitory actions on the TRPC6 channel, a transient receptor potential (TRP) protein.
Experimental approach:
Macroscopic and single channel currents were recorded from transfected HEK293 cells by patch-clamp techniques.
Key results:
Cationic currents elicited by carbachol (CCh; 100 
M) in HEK293 cells overexpressing murine TRPC6 (ITRPC6) were dose-dependently inhibited by externally applied ML-9 (IC50=7.8 M). This inhibition was voltage-dependent and occurred as fast as external Na+ removal. Another MLCK inhibitor, wortmannin (3 M), and MLCK inhibitory peptides MLCK-IP11-19 (10 M) and -IP480-501 (1 M) showed little effects on ITRPC6 density and the inhibitory efficacy of ML-9. The extent of the inhibition also unchanged with co-expression of wild-type or a dominant negative mutant of MLCK. Inhibitory effects of ML-9 on ITRPC6 remained unaffected whether TRPC6 was activated constitutively or by a diacylglycerol analogue OAG (100 M). Similar rapid inhibition was also observed with a ML-9 relative, ML-7. Intracellular perfusion of ML-9 via patch pipette, dose-dependently suppressed ITRPC6. In inside-out patch configuration, bath application of ML-9 (and ML-7) rapidly diminished 
35pS single TRPC6 channel activities. Contrarily, currents due to TRPC7 expression were rapidly enhanced by externally applied ML-9 and ML-7, which was not prevented by MLCK inhibitory peptides.
Conclusion and implications:
These results strongly suggest that ML compounds inhibit TRPC6 channels via a mechanism independent of inhibition of MLCK activity.
Keywords:
transient receptor potential, receptor-operated Ca2+-permeable cation channel, TRPC6, TRPC7, myosin light chain kinase, naphthalene sulphonamide derivative, ML-9, dominant-negative MLCK
Abbreviations:
CaM, calmodulin; CaMKII, calmodulin-dependent kinase II; ML-9, [1-(5-chloronaphthalene-1-sulphonyl)homopiperazine, HCl]; ML-7, [1-(5-iodonaphthalene-1-sulphonyl)homopiperazine, HCl]; MLCK, myosin light chain kinase; MLCK-IP, myosin light chain kinase inhibitory peptide; mut-MLCK, mutant myosin light chain kinase; NMDG, N-methyl D-glucamine; OAG, 1-oleoyl-2-acetyl-sn-glycerol; TRP, transient receptor potential protein; TRPC6, TRP canonical subfamily isoform 6; TRPC7, TRP canonical subfamily isoform 7; wt-MLCK, wild-type myosin light chain kinase
