Role of calmodulin and myosin light chain kinase in the activation of carbachol-activated cationic current in murine ileal myocytes

Abstract

We investigated the effect of calmodulin (CaM) and myosin light chain kinase (MLCK) on murine ileal myocytes using the whole-cell patch-clamp technique. Under the voltage clamp, at the holding potential of -60 mV, 50 mu mol/L carbachol (CCh) induced inward currents (I-CCh), and spontaneous decay Of I-CCh occurred. The peak inward currents induced by the repetitive application of CCh (50 mu mol/L) tended to decrease in amplitude. Intracellular application of 0.2 mmol/L guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) from the patch electrode induced an inward current at a holding potential of -60mV, and the peak inward currents induced by the repetitive application of Cs tended to decrease slightly in amplitude. The amplitude of I-CCh was reduced by pretreatment either with W-7, trifluoroperazine, W-5, and melittin (CaM inhibitors) or with ML-7 and ML-9 (selective MLCK inhibitors), and the inhibitory effects were reversible. However, when we pretreated with 50 mu mol/L W-7 or 5 mu mol/L ML-7 on GTP-gamma S-induced inward currents, almost no inhibition was observed in the inward currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTP gamma S-induced currents. We conclude that CaM and MLCK modulate the activation process Of I-CCh in murine ileal myocytes and suggest that the classical type transient receptor potential (TRPC) channel 5 might be a candidate for nonselective cationic currents (NSCC) activated by muscarinic stimulation in gastrointestinal smooth muscle cells.