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

烟碱型乙酰胆碱受体(NAChR)是脊椎动物神经肌肉突触的递质受体，是一种膜糖蛋白。来自射线电子器官的nAChR由四种亚基组成，它们按摩尔化学计量比组装在一起，即α_2、β、γ、β。这四个亚基和一些哺乳动物肌肉nAChR的一级结构已经通过对编码这些多肽的cDNAs或基因组DNA的克隆和测序而被阐明。本研究旨在从分子水平上阐明nAChR的功能机制。结果：主要结果如下：1.利用钴溴毒素-琼脂糖偶联物作为亲和层析的吸附剂，从鱼雷的电器官中获得了高纯度的nAChR。用过氧化氢标记的α-银环蛇毒素检测了nAChR的碳水化合物部分在配体结合中的作用。…此外，唾液酸或卵清蛋白可抑制银环蛇毒素与nAChR的结合，但去除nAChR上的唾液酸或高甘露糖型低聚糖，与完整受体的结合无明显差异。这些结果表明，nAChR的碳水化合物部分不是胆碱能配体结合所必需的。蛋白质磷酸化被认为是控制细胞代谢的主要调控机制之一。在目前的研究中，我们使用抗酪氨酸抗体检测了nAChR是否在酪氨酸残基上被磷酸化。结果表明，nAChR分子的β和γ亚基中的酪氨酸残基(S)在体内被磷酸化。胆碱能配体对nAChR磷酸化程度的影响也被检测，并发现其具有剂量依赖性。这些结果表明，nAChR分子通过与受体ACh结合而发生构象变化，从而刺激其磷酸化，并与nAChR的脱敏作用有关。非受体、外周、nu蛋白(分子量：43,000蛋白；43 KDa蛋白)是富含nAChR的膜和鱼雷电子细胞的显著成分。我们研究了从外周nu蛋白中分离的nu_1、nu_2和nu_3组分的生化性质，并对它们的性质进行了论证。虽然nAChR的分子性质已经得到了较好的证明，但其神经传递的详细机制仍有待阐明。通过X射线或核磁共振分析来阐明nAChR的三维结构将为更好地理解这些问题开辟道路。较少

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