Abstract
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 methyllycaconitine (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.
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