Slow Ca²⁺ (RAMIC) mobilization operated by postsynaptic neuronal nicotinic receptor regulates synaptic function at the mouse neuromuscular junction

We have found that non-contractile slow Ca²⁺ mobilization (RAMIC; Receptor-Activity Modulating Intracellular Ca²⁺) is generated by motor nerve stimulation with anti-cholinesterase at the skeletal muscle, and desensitizes muscle nicotinic receptor (nAChR). To confirm this Ca²⁺mobilization without anti-cholinesterase, acetylcholine (ACh) was locally applied by N₂-gas pressure onto endplate region at the mouse phrenic nerve-diaphragm muscle preparation. ACh (0.1-3 mM, 20 |µl) elicited biphasic elevation of [Ca²⁺]_i (fast and slow Ca²⁺ mobilization measured as Ca²⁺-aequorin luminescence) in muscle cells. The peak amplitude of slow Ca²⁺ mobilization (not accompanied by contraction) was increased by ACh concentration-dependently, whereas that of fast component (accompanied by contraction) reached a maximum response at a lower concentration of ACh. The slow Ca²⁺ mobilization was blocked by lower concentrations of competitive nAChR antagonists which did not affect the fast Ca²⁺ transients. Moreover, the slow Ca²⁺ signal was selectively depressed by a neuronal nAChR antagonist methyllycaconitine. Neither Ca²⁺ channel blockers nor a Na+ channel blocker tetrodotoxin prevented the generation of the slow Ca²⁺ mobilization. These results suggest that RAMIC is mobilized through postsynaptic neuronal nAChR subtype to desensitize muscle nAChR at the neuromuscular junction.