Myosin light chain kinase-independent inhibition by ML-9 of murine TRPC6 channels expressed in HEK293 cells

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 nonspecific 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 mu M) in HEK293 cells overexpressing murine TRPC6 (I-TRPC6) were dose-dependently inhibited by externally applied ML-9 (IC50 = 7.8 mu M). This inhibition was voltage-dependent and occurred as fast as external Na+ removal. Another MLCK inhibitor, wortmannin ( 3 mM), and MLCK inhibitory peptides MLCK-IP11-19 (10 mu M) and -IP480-501 (1 mu M) 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 ITRPC6 remained unaffected whether TRPC6 was activated constitutively or by a diacylglycerol analogue OAG ( 100 mM). Similar rapid inhibition was also observed with a ML-9 relative, ML-7. Intracellular perfusion of ML-9 via patch pipette, dosedependently suppressed I-TRPC6. In inside-out patch configuration, bath application of ML-9 (and ML-7) rapidly diminished similar to 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.