Neuronal nicotinic receptor-operated Ca²⁺ (RAMIC) mobilization for the desensitization of muscle nicotinic receptor coexisting at the muscle endplates

Few functional roles of neuronal nicotinic receptors (N-nAChR) are clarified, although N-nAChR have been reported to be highly permeable to Ca²⁺ than the muscle nicotinic receptor (M-nAChR). We have found new regulatory Ca²⁺ mobilization, RAMIC (Receptor Activity Modulating Intracellular Ca²⁺, at the endplate region in the mouse diaphragm muscles treated with a cholinesterase inhibitor by measuring Ca²⁺ aequorin luminescences. RAMIC mobilization is enhanced via the activation of protein kinase-A by nerve derived calcitonin gene related peptide, and promotes the M-nAChR desensitization via the protein kinase C activation. The RAMIC mobilization is depressed by a N-nAChR antagonist methyllyeaconitine (0.1 1 μM) and by monoclonal antibody to β2 subunit of N-nAChR. Furthermore, β2 and α8-related subunits were identified at murine skeletal muscle endplates either by RT PCR or by immunohistochemical staining. In isolated single skeletal muscle cells loaded with a fluorescent Ca²⁺ indicator fluo-3 AM, bath applied ACh (3 μM) elicits two-phasic elevation of [Ca²⁺]i (fast and slow components) localized beneath the endplate membranes, by using a real time-confocal laser scanning analysis. Methyllycaconitine selectively inhibits the slow Ca²⁺ component at the similar concentrations (1.5 μM) used to depress RAMIC. These results demonstrate that N-nAChR coexist with M- nAChR at the muscle endplates, and operates the RAMIC mobilization to promote the M-nAChR desensitization. Thus, the neuromuscular function is regulated by dual nAChR system to avoid the overexcitation.