Marritt Andrea M, Cox Brandon C, Yasuda Robert P, McIntosh J Michael, Xiao Yingxian, Wolfe Barry B, Kellar Kenneth J
Department of Pharmacology, Georgetown University School of Medicine, Washington, DC 20057, USA.
Mol Pharmacol. 2005 Dec;68(6):1656-68. doi: 10.1124/mol.105.012369. Epub 2005 Aug 29.
Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in the rat retina to determine the heteromeric subtypes. We detected seven nicotinic receptor subunit mRNA transcripts, alpha2-alpha4, alpha6, and beta2-beta4, with RNase protection assays. The density of heteromeric nAChR binding sites is approximately 3 times higher in the retina than in the cerebral cortex. Moreover, the density of the sites in the retina measured with [3H]epibatidine ([3H]EB) is approximately 30% higher than with 125I-3-(2(S)-azetidinylmethoxy)pyridine (A-85380) and more than twice that measured with [3H]cytisine or 3Hnicotine. These data suggest that the retina expresses multiple subtypes of nAChRs, including a large fraction of receptors containing the beta2 subunit and a smaller fraction containing the beta4 subunit. Consistent with this, in binding competition studies, nicotinic ligands fit a model for two affinity classes of binding sites, with the higher affinity sites representing 70 to 80% of the nAChRs in the retina. To determine the specific subtypes of nAChRs in the rat retina, we used subunit-specific antibodies in immunoprecipitation assays. Immunoprecipitation of [3H]EB-labeled nAChRs with antibodies specific to the beta2 and beta4 subunits indicated that approximately 80% of the receptors contained beta2 subunits and approximately 25% contained beta4 receptors, consistent with the binding pharmacology results. Sequential immunoprecipitation assays indicated that the rat retina contains multiple subtypes of nAChRs. The majority of the receptors measured seemed to be simple heteromeric subtypes, composed of a single type of alpha and a single type of beta subunit; but a significant fraction are mixed heteromeric subtypes, composed of two or more alpha and/or beta subunits.
为了确定异聚体亚型，我们在大鼠视网膜中测量了神经元烟碱型乙酰胆碱受体（nAChRs）。我们通过核糖核酸酶保护试验检测到了七种烟碱型受体亚基mRNA转录本，即α2-α4、α6以及β2-β4。异聚体nAChR结合位点的密度在视网膜中比在大脑皮层中约高3倍。此外，用[3H]埃博霉素（[3H]EB）测量的视网膜中这些位点的密度比用125I-3-(2(S)-氮杂环丁烷甲氧基)吡啶（A-85380）测量的约高30%，且比用[3H]金雀花碱或[3H]（-）尼古丁测量的密度高两倍多。这些数据表明，视网膜表达多种nAChRs亚型，包括很大一部分含有β2亚基的受体和一小部分含有β4亚基的受体。与此一致的是，在结合竞争研究中，烟碱型配体符合两种亲和力类型结合位点的模型，其中高亲和力位点占视网膜中nAChRs的70%至80%。为了确定大鼠视网膜中nAChRs的具体亚型，我们在免疫沉淀试验中使用了亚基特异性抗体。用β2和β4亚基特异性抗体对[3H]EB标记的nAChRs进行免疫沉淀表明，约80%的受体含有β2亚基，约25%含有β4受体，这与结合药理学结果一致。连续免疫沉淀试验表明，大鼠视网膜含有多种nAChRs亚型。所测量的大多数受体似乎是简单的异聚体亚型，由单一类型的α亚基和单一类型的β亚基组成；但相当一部分是混合异聚体亚型，由两种或更多种α和/或β亚基组成。
