Biochemical Studies on the Molecular Mechanism of Neurotransmission, Centered on Nicotinic Acetylcholine Receptor (nAChR)

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Nicotinic acetylcholine receptor (nAChR) is a membrane glycoprotein which functions as transmitter receptor at vertebrate neuromuscular synapses. nAChR from the electric organ of ray consists of four kinds of subunits assembled in a molar stoichiometry of alpha_2,beta,gamma,delta. The primary structures of all these four subunits and those from some mammalian muscle nAChR have been elucidated by cloning and sequencing cDNAs or genomic DNA encoding these polypeptides.This study was designed to elucidate the mechanism of the function of nAChR in the molecular basis using the highly purified nAChR isolated from the electric organ of Torpedo californica.Results: Major results are summarized as follows:1. nAChR was highly purified from the electric organ of Torpedo californica, by employing a cobrotoxin-Sepharose conjugate as an adsorbent for affinity chromatography.2. The role of the carbohydrate moiety of nAChR in ligand-binding was examined, using peroxidate-labelled alpha-bungarotoxin. … More The bungarotoxin-binding to nAChR was inhibited by sialic acid or ovalbumin, but the removal of sialic acid or high-mannose-type oligosaccharides from nAChR resulted in no difference from the intact receptor in its bindings. These results suggested that the carbohydrate moiety of nAChR was not necessary in the binding of cholinergic ligands.3. Protein phosphorylation is recognized as one of the major regulatory mechanism in the control of cellular metabolism. In the present study, we examined whether nAChR is phosphorylated at tyrosine residue, using anti-tyrosine antibody. The results indicated that the tyrosine residue(s) in beta- and gamma-subunits of nAChR molecule were phosphorylated in vivo. The effect of cholinergic ligandes on the extent of nAChR phosphorylation was also examined and was found to be dose dependent. These results suggest that the phosphorylation is stimulated by the conformational change of nAChR molecule by ACh-binding to the receptor and correlates with the desensitization of nAChR.4. The nonreceptor, peripheral,nu proteins (MW: 43,000 protein; 43KDa proteins) are conspicuous components of the nAChR-rich membranes and the Torpedo electrocyte. We examined on the biochemical properties of nu_1, nu_2, and nu_3 components isolated from the peripheral nu proteins and demonstrated their properties.Although the molecular natures of nAChR have been fairly demonstrated, the detailed mechanism of the neurotransmission still remains to be clarified. The elucidation of the three-dimensional structure of nAChR by X-ray or NMR-analysis will open the way to a better understanding of the problems. Less