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

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.