Partial deletion of the nicotinic cholinergic receptor α4 or β2 subunit genes changes the acetylcholine sensitivity of receptor-mediated 86Rb+ efflux in cortex and thalamus and alters relative expression of α4 and β2 subunits
Partial deletion of the nicotinic cholinergic receptor α4 or β2 subunit genes changes the acetylcholine sensitivity of receptor-mediated 86Rb+ efflux in cortex and thalamus and alters relative expression of α4 and β2 subunits
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DOI：
10.1124/mol.108.045203
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发表时间：
2008-06-01
影响因子：
3.6
通讯作者：
Marks, Michael J.
中科院分区：
文献类型：
--
作者：
Gotti, Cecilia;Moretti, Milena;Marks, Michael J.
α4和β2烟碱能受体(NAChR)亚基可以在异源表达系统中以五聚体受体的形式组装，具有不同的亚基化学计量比，对乙酰胆碱的激活表现出不同的敏感性，产生双相浓度-效应曲线。NAChR介导的Rb-86(+)在小鼠脑突触体内的外流也呈现双时相乙酰胆碱(ACh)浓度-反应曲线。这两个阶段主要是由α4β2*-nAChR介导的，因为α4或β2亚基的缺失将使应答减少至少90%。ACh敏感性较低的Rb-86(+)外排部分缺失α4(+/-)，而部分缺失β2(+/-)则导致ACh敏感性较高的成分减少较多。选择性抗体的免疫沉淀显示，在这两个区域中，超过70%的[H-3]表巴替丁结合位点只含有α4和β2亚基。用Western blotts分析α4和β2野生型和杂合子大脑皮质和丘脑中α4和β2亚单位的含量。α4的部分缺失减少，β2的部分缺失增加了组装受体中α4亚单位的相对比例。虽然这些方法不能准确地鉴定野生型皮层和丘脑中存在的亚型的化学计量学，但它们确实证明了α4(+/-)和β2(+/-)基因型的皮质和丘脑的nAChRs在α4和β2亚基的相对表达上不同，这一结果对应于部分基因缺失后观察到的相对功能变化。这些结果有力地表明，不同化学计量比的α4β2-nAChR在自然组织中表达。
alpha 4 and beta 2 nicotinic cholinergic receptor (nAChR) subunits can assemble in heterologous expression systems as pentameric receptors with different subunit stoichiometries that exhibit differential sensitivity to activation by acetylcholine, yielding biphasic concentration-effect curves. nAChR-mediated Rb-86(+) efflux in mouse brain synaptosomes also displays biphasic acetylcholine (ACh) concentration-response curves. Both phases are mediated primarily by alpha 4 beta 2*-nAChR, because deletion of either the alpha 4 or beta 2 subunit reduces response at least 90%. A relatively larger decrease in the component of Rb-86(+) efflux with lower ACh sensitivity occurred with partial deletion of alpha 4 (alpha 4(+/-)), whereas a larger decrease in the component with higher ACh sensitivity was elicited by partial deletion of beta 2 (beta 2(+/-)). Immunoprecipitation with selective antibodies demonstrated that more than 70% of [H-3] epibatidine binding sites in both regions contained only alpha 4 and beta 2 subunits. Subsequently, alpha 4 and beta 2 subunit content in the cortex and thalamus of alpha 4 and beta 2 wild types and heterozygotes was analyzed with Western blots. Partial deletion of alpha 4 decreased and partial deletion of beta 2 increased the relative proportion of the alpha 4 subunit in assembled receptors. Although these methods do not allow exact identification of stoichiometry of the subtypes present in wild-type cortex and thalamus, they do demonstrate that cortical and thalamic nAChRs of the alpha 4(+/-) and beta 2(+/-) genotypes differ in relative expression of alpha 4 and beta 2 subunits a result that corresponds to the relative functional changes observed after partial gene deletion. These results strongly suggest that alpha 4 beta 2-nAChR with different stoichiometry are expressed in native tissue.