Gotti C, Moretti M, Maggi R, Longhi R, Hanke W, Klinke N, Clementi F
CNR Center of Cellular and Molecular Pharmacology, Department of Medical Pharmacology, University of Milan, Italy.
Eur J Neurosci. 1997 Jun;9(6):1201-11. doi: 10.1111/j.1460-9568.1997.tb01475.x.
Nicotinic receptors are present in the chick retina, but their structure and functional characteristics are still unclear. Using anti-alpha7 and anti-alpha8 subunit-specific antibodies, we immunopurified the alpha7 and alpha8 subtypes of chick retina neuronal nicotinic receptors. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the two purified subtypes consistently showed a similar peptide composition characterized by the presence of two major peptides of M(r) 58 +/- 1 and 54 +/- 1 kDa, and two minor peptides of 67 and 61 +/- 1 kDa. In the alpha7 subtype, the 58 kDa peptide was recognized by anti-alpha7 but not by anti-alpha8 antibodies; in the alpha8 subtype, the 58 kDa peptide was recognized only by anti-alpha8 antibodies. The alpha7 subtype had a single class of [125I]alpha-bungarotoxin binding sites with a K(D) value of 1.2 nM, whereas the purified alpha8 subtype had two classes of binding sites, one with a K(D) of 5.5 nM and the other with very high affinity (KD 52 pM), but present in only 8% of the receptors. Competition binding experiments also showed the presence on the alpha8 subtype of high- and low-affinity classes of binding sites; the affinity for cholinergic drugs of the former was greater than that of the single class present on the alpha7 subtype. When reconstituted in planar lipid bilayers, both subtypes formed ligand-gated cation channels with major conductance levels of 42 and 52 pS but with different lifetimes; the two channels were activated by agonists and blocked by d-tubocurarine and the glycinergic antagonist strychnine. In line with the binding data, the reconstituted alpha8 subtype had greater agonist sensitivity than the alpha7 subtype.
烟碱型受体存在于鸡视网膜中，但其结构和功能特性仍不清楚。我们使用抗α7和抗α8亚基特异性抗体，免疫纯化了鸡视网膜神经元烟碱型受体的α7和α8亚型。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析时，这两种纯化的亚型始终显示出相似的肽组成，其特征是存在两条主要肽段，分子量分别为58±1 kDa和54±1 kDa，以及两条次要肽段，分子量为67 kDa和61±1 kDa。在α7亚型中，58 kDa的肽段可被抗α7抗体识别，但不能被抗α8抗体识别；在α8亚型中，58 kDa的肽段仅能被抗α8抗体识别。α7亚型有一类单一的[125I]α-银环蛇毒素结合位点，解离常数（KD）值为1.2 nM，而纯化的α8亚型有两类结合位点，一类KD值为5.5 nM，另一类具有非常高的亲和力（KD 52 pM），但仅存在于8%的受体中。竞争结合实验还表明，α8亚型存在高亲和力和低亲和力两类结合位点；前者对胆碱能药物的亲和力大于α7亚型上存在的单一类结合位点。当重构于平面脂质双分子层中时，两种亚型均形成配体门控阳离子通道，主要电导水平分别为42和52 pS，但寿命不同；这两种通道均可被激动剂激活，并被d-筒箭毒碱和甘氨酸能拮抗剂士的宁阻断。与结合数据一致，重构的α8亚型比α7亚型具有更高的激动剂敏感性。
