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Myosin Light Chain Kinase-Independent Inhibition by ML-9 of Murine TRPC6 Channels Expressed in HEK293 Cells

Myosin Light Chain Kinase-Independent Inhibition by ML-9 of Murine TRPC6 Channels Expressed in HEK293 Cells

J Shi, S Takahashi, X-H Jin, Y-Q Li, Y Ito, Y Mori, R Inoue

Br J Pharmacol. 2007 Sep;152(1):122-31.

PMID: 17603544

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 microM) in HEK293 cells overexpressing murine TRPC6 (I(TRPC6)) were dose-dependently inhibited by externally applied ML-9 (IC(50)=7.8 microM). This inhibition was voltage-dependent and occurred as fast as external Na(+) removal. Another MLCK inhibitor, wortmannin (3 microM), and MLCK inhibitory peptides MLCK-IP(11-19) (10 microM) and -IP(480-501) (1 microM) showed little effects on I(TRPC6) 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 I(TRPC6) remained unaffected whether TRPC6 was activated constitutively or by a diacylglycerol analogue OAG (100 microM). Similar rapid inhibition was also observed with a ML-9 relative, ML-7. Intracellular perfusion of ML-9 via patch pipette, dose-dependently suppressed I(TRPC6). In inside-out patch configuration, bath application of ML-9 (and ML-7) rapidly diminished approximately 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP105637501-A	ML-9		105637-50-1	Price

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