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

烟碱型乙酰胆碱受体（nAChR）是一种膜糖蛋白，在脊椎动物神经肌肉突触中起递质受体的作用。鳐的电子器官nAChR由α_2、β、γ、δ四种亚基组成。通过对编码这些多肽的cDNA或基因组DNA的克隆和序列测定，阐明了这四个亚基以及一些哺乳动物肌肉nAChR的一级结构，本研究以从加利福尼亚电鳐（Torpedocalfornica）电器中分离的高纯度nAChR为材料，从分子水平上阐明了nAChR的功能机制。采用cobrotoxin-Sepharose偶联物作为亲和层析吸附剂，从加州电鳐（Torpedocalfornica）的电器官中高效纯化了nAChR.的碳水化合物部分的nAChR在配体结合的作用进行了检查，使用过氧化物标记的α-银环蛇毒素。 ...更多信息 银环蛇毒素结合nAChR抑制唾液酸或卵清蛋白，但从nAChR的唾液酸或高甘露糖型寡糖的去除导致在其结合的完整受体没有差异。这些结果表明，nAChR的碳水化合物部分在胆碱能配体的结合中不是必需的.蛋白质磷酸化被认为是控制细胞代谢的主要调节机制之一。在本研究中，我们检查是否nAChR是磷酸化的酪氨酸残基，使用抗酪氨酸抗体。结果表明，nAChR分子β和γ亚基的酪氨酸残基在体内均发生磷酸化。胆碱能配体对nAChR磷酸化程度的影响也进行了研究，发现是剂量依赖性的。这些结果表明，乙酰胆碱与受体结合后，nAChR分子的构象变化刺激了磷酸化，并与nAChR的脱敏作用有关.非受体、外周、nu蛋白（MW：43，000蛋白; 43 KDa蛋白）是富含nAChR的膜和电鳐电细胞的显著组分。我们从外周nu蛋白中分离出nu_1、nu_2和nu_3组分，并对其生化性质进行了研究，虽然nAChR的分子性质已经得到了较好的证实，但其神经传递的详细机制仍有待阐明。通过X射线或NMR分析阐明nAChR的三维结构将为更好地理解这些问题开辟道路。少

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