Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (I-CCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. I-CCh was induced by carbachol (CCh; 50 mu M) at a holding potential of -30 mV or -60 mV, ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited I-CCh concentration dependently in a reversible manner (53 +/- 8.6% at 1 mu M, mean +/- SE, n = 11). in addition, amplitudes of I-CCh were only 37 +/- 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca2+ channel current. The peak value of Ba2+ current at 0 mV was reduced to 35 +/- 7.4% (n = 9) by 3 mu M of ML-7. As I-CCh is known to have an intracellular Ca2+ dependence, we tried to exclude the possibility that ML-7 inhibited I-CCh, indirectly via suppression of Ca2+ current and the similar inhibitory effects of ML-7 on I-CCh were confirmed under the following conditions: (1) clamp of membrane potential at -60 mV; (2) clamp of intracellular [Ca2+] to 1 mu M by 10 mM BAPTA; (3) pre-inhibition of Ca2+ channel by verapamil. Different from the effects on I-CCh ML-7 barely inhibited the same cationic current induced by guanosine 5'-O-(3-thiotriphosptlate) (GTP[gamma S], 0.2 mM) in the pipette solution. These results suggest that a Ca2+/calmodulin-MLCK-dependent pathway can modulate the activation of I-CCh in guinea-pig gastric antral myocytes.