Methyllycaconitine-sensitive neuronal nicotinic receptor-operated slow Ca²⁺ signal by local application or perfusion of ACh at the mouse neuromuscular junction

Local application of acetylcholine (ACh; 0.3 mM, 20 μl) elicited bi- phasic elevation of intracellular Ca²⁺ concentrations (contractile fast and non-contractile slow Ca²⁺ signal measured as aequorin luminescence) in diaphragm muscle preparation. A neuronal nicotinic antagonist methyllycaconitine (MLA; 0.01-1 μM), which did not affect the fast Ca²⁺ transients and twitch tension, concentration-dependently depressed only the slow Ca²⁺ component. Ca²⁺ channel blockers, Cd²⁺ (200 μM), nitrendipine (1 μM), verapamil (1 μM) and diltiazem (1 μM), or a Na⁺ channel blocker tetrodotoxin (TTX; 0.1 μM) failed to prevent the generation of slow Ca²⁺ response. Perfusion of ACh (1 μM) to isolated single skeletal (flexor digitorum brevis) muscle cells pretreated with TTX (0.1 μM) also elicited a slow Ca²⁺ signal measured as confocal imaging with a fluorescent dye, fluo-3, at the endplate region. MLA (1 μM) antagonized against the ACh perfusion-elicited slow Ca²⁺ signal. Perfusion of choline (1 mM), a neuronal nicotinic agonist, also elicited the MLA-sensitive slow Ca²⁺ signal. These results strongly suggest that the ACh-induced slow Ca²⁺ signal reflects Ca²⁺ entry through a postsynaptic MLA-sensitive neuronal nicotinic ACh receptor subtype at the neuromuscular junction.