Structure/Function Analysis of the Neuronal alpha7 nAChR

描述(由申请人提供)：烟碱型乙酰胆碱受体(NAChRs)第二跨膜(TM2)区域的氨基酸残基已被证明在使用定点突变和异源表达系统的研究中具有重要的调节功能。肌肉和高亲和力神经元nAChRs的β亚单位TM2结构域中的位点以及同型五角体α7受体都是如此。这项提案概述了一个实验研究过程，该过程将试图确定Alpha7 TM2结构域中的同源位点的可能作用，这些同源位点先前已在β亚基中被确定为受体功能和药理学的关键决定因素。这涉及到用肌肉和神经元β亚单位TM2结构域中的氨基酸取代α7 TM2结构域中的相同残基。药理学特征将包括对激动剂激活的敏感性，对非竞争性抑制剂(NCIS)的敏感性，以及残留抑制或脱敏的程度。功能鉴定将包括受体动力学和二价离子渗透性的分析。到目前为止，初步的发现表明，α7TM2 6‘位突变包括肌肉β1亚单位序列或神经元β2/β4亚单位序列，极大地改变了突变受体的反应动力学，使得宏观电流的衰减率更接近含有受体的野生型β亚基，而不是野生型α7。这里概述的研究计划将有助于进一步对申请者进行分子生物学和电生理学方面的培训，并将有助于确定Alpha7 TM2结构域中特定位点对其功能和药理学重要方面的潜在贡献。

DESCRIPTION (provided by applicant): Amino acid residues in the second transmembrane (TM2) domain of nicotinic acetylcholine receptors (nAChRs) have been shown to have important regulatory functions in studies using site-directed mutagenesis and heterologous expression systems. This is true for sites in the beta subunit TM2 domain of both muscle and high-affinity neuronal nAChRs, as well as homopentameric alpha7 receptors. This proposal outlines a course of experimental study that will attempt to identify the possible roles of homologous sites in the alpha7 TM2 domain that have previously been established in the beta subunit as critical determinants of receptor function and pharmacology. This involves the substitution of amino acids from the muscle and neuronal beta subunit TM2 domains for identical residues in the alpha7 TM2 domain. Pharmacological characterization will include sensitivity to activation by agonist, sensitivity to non-competitive inhibitors (NCIs), and the degree of residual inhibition or desensitization. Functional characterization will involve analysis of receptor kinetics and divalent ion permeability. Thus far, the preliminary findings indicate that mutation of the alpha7 TM2 6' position to include either the muscle Beta1 subunit sequence or the neuronal Beta2/Beta4 subunit sequence, dramatically changes the response kinetics of the mutant receptors so that the decay rate of macroscopic currents is more like that of the respective wild-type beta subunit containing receptor than wild-type alpha7. The research plan outlined here will serve to further the training of the applicant in molecular biology and electrophysiology, and will help to identify the potential contribution of specific sites within the alpha7 TM2 domain to important aspects of its function and pharmacology.