Structure/Function Analysis of the Neuronal alpha7 nAChR

描述（由申请人提供）：在使用定点诱变和异源表达系统的研究中，已显示烟碱乙酰胆碱受体（nAChR）的第二跨膜（TM 2）结构域中的氨基酸残基具有重要的调节功能。这对于肌肉和高亲和力神经元nAChR以及同型五聚体α 7受体的β亚基TM 2结构域中的位点是真实的。该提案概述了一个实验研究过程，将试图确定先前已在β亚基中建立的α 7 TM 2结构域中的同源位点作为受体功能和药理学的关键决定因素的可能作用。这涉及来自肌肉和神经元β亚基TM 2结构域的氨基酸取代α 7 TM 2结构域中的相同残基。药理学表征将包括对激动剂激活的敏感性、对非竞争性抑制剂（NCI）的敏感性以及残留抑制或脱敏的程度。功能表征将涉及受体动力学和二价离子渗透性的分析。到目前为止，初步发现表明α 7 TM 2 6'位置突变为包括肌肉β 1亚基序列或神经元β 2/β 4亚基序列，显著改变了突变受体的响应动力学，使得宏观电流的衰减速率与野生型α 7相比更类似于相应的野生型含β亚基的受体。本文概述的研究计划将有助于进一步培训申请人的分子生物学和电生理学，并将有助于确定α 7 TM 2结构域内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.