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


METHODS
Macroscopic and single channel currents were recorded from transfected HEK293 cells by patch-clamp techniques.


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.


CONCLUSIONS
These results strongly suggest that ML compounds inhibit TRPC6 channels via a mechanism independent of inhibition of MLCK activity.
Shi
J
J
Department of Anatomy and K.K Leung Brain Research Centre, The Fourth Military Medical University, Xi'an, China.
Takahashi
S
S
Jin
X-H
XH
Li
Y-Q
YQ
Ito
Y
Y
Mori
Y
Y
Inoue
R
R
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000494:pharmacology*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000494:pharmacology; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D000074287:TRPC6 Cation Channel; D014162:Transfection; D000077191:Wortmannin
C520323:1-oleoyl-2-acetoyl-sn-glycerol; D000730:Androstadienes; D001381:Azepines; D004075:Diglycerides; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D010446:Peptide Fragments; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C492146:Trpc6 protein, mouse; C498502:Trpc7 protein, mouse; C056218:ML 9; C070571:ML 7; D002217:Carbachol; D009219:Myosin-Light-Chain Kinase; D000077191:Wortmannin