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 μM) in HEK293 cells overexpressing murine TRPC6 (I_TRPC6) were dose‐dependently inhibited by externally applied ML‐9 (IC₅₀=7.8 μM). This inhibition was voltage‐dependent and occurred as fast as external Na⁺ removal. Another MLCK inhibitor, wortmannin (3 μM), and MLCK inhibitory peptides MLCK‐IP_11‐19 (10 μM) and ‐IP_480‐501 (1 μM) 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 μM). 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 ∼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. British Journal of Pharmacology (2007) 152, 122–131; doi:10.1038/sj.bjp.0707368