Role of calmodulin in the activation of carbachol-activated cationic current in guinea-pig gastric antral myocytes

In mammalian gastrointestinal myocytes, it is known that muscarinic stimulation activates nonselective cation channels through a G-protein and a Ca²⁺dependent pathway. We recorded inward cationic currents following application of carbachol (I_CCh) to guinea-pig gastric myocytes, which were held at –20 mV using the whole-cell patch-clamp method. I_CCh was suppressed by nicardipine or removal of Ca²⁺ from the bath solution. The peak value of inward current induced by repetitive applications of carbachol (CCh) decreased progressively (run-down phenomenon). This run-down was significantly alleviated by the addition of calmodulin to the pipette solution (0.15 mg/ml) or by using the perforated-patch whole-cell voltage-clamp technique. Moreover, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a calmodulin antagonist, was a reversible inhibitor of I_CCh. However, W-7 had only a weak inhibitory effect on the same cationic current which was induced by guanosine 5-O-(3-thiotriphosphate) (GTP[S] 0.2 mM) in the pipette solution. This GTP[S]-induced cationic current was still markedly suppressed by the Ca²⁺-free bath solution. W-7 itself had a weak inhibitory effect on voltage-operated Ca²⁺ channels as well as the effects on I_CCh. These data suggest that multiple Ca²⁺-dependent pathways are involved in the activation of CCh-gated cation channels in guinea-pig antral myocytes and a Ca²⁺/calmodulin-dependent pathway would be one of them.