Potentiation of Neuronal Nicotinic Receptors by Zinc

描述（由申请人提供）：本项目的目标是：（1）阐明锌结合并增强受体功能的神经元烟碱乙酰胆碱受体（nAChR）位点的结构，以及（2）阐明锌增强受体功能的机制。 烟碱配体可用作抗焦虑药和镇痛药，用于治疗神经系统疾病，如精神分裂症、帕金森病和阿尔茨海默病，并且也是烟碱发挥其精神活性和成瘾作用的位点。神经元nAChRs的有效药物干预需要亚型选择性配体的开发。追求这一目标，传统上是针对开发的配体，在乙酰胆碱（ACh）的结合位点的行为。我们建议追求一类新的网站上的神经元乙酰胆碱受体，锌增强这些受体的网站。神经元nAChR上的ACh结合位点位于亚基胞外结构域之间的界面处。在许多神经元nAChRs中，两个激动剂结合位点在两对不同亚基之间的界面处形成，留下三个不参与ACh结合的交替界面。我们假设一个或多个交替接口结合锌，神经元nAChR功能的调制器。在目标1中，我们将使用定点诱变和功能分析，以确定氨基酸残基的神经元乙酰胆碱受体，协调锌的增效位点。在目标2中，我们将使用取代的半胱氨酸可及性突变来探索增强位点的更大结构。在目标1和2中获得的信息将用于改进神经元nAChR的胞外结构域的同源性模型。在目标3中，我们将确定锌是否通过改变单通道开放概率、单通道电流和/或通道数量来增强神经元nAChR。开放概率的变化将根据潜在的门控机制进行表征。

DESCRIPTION (provided by applicant): The goals of this project are: (1) to elucidate the structure of the site(s) on neuronal nicotinic acetylcholine receptors (nAChRs) where zinc binds and potentiates receptor function, and (2) to elucidate the mechanism through which zinc potentiates receptor function. Nicotinic ligands are potentially useful as anxiolytics and analgesics, in the treatment of neurological disorders such as schizophrenia, Parkinson's disease, and Alzheimer's disease, and are also the sites at which nicotine exerts its psychoactive and addictive effects. Effective pharmacological intervention at neuronal nAChRs requires development of subtype selective ligands. Pursuit of this goal has traditionally been directed toward development of ligands that act at the acetylcholine (ACh) binding sites. We propose to pursue a new class of site on neuronal nAChRs, the site at which zinc potentiates these receptors. The ACh binding sites on neuronal nAChRs are at the interfaces between the extracellular domains of subunits. In many neuronal nAChRs, two agonist-binding sites are formed at interfaces between two pairs of dissimilar subunits, leaving three alternate interfaces with no involvement in ACh binding. We hypothesize that one or more of the alternate interfaces bind zinc, a modulator of neuronal nAChR function. In aim 1, we will use site-directed mutagenesis and functional analysis to identify amino acid residues on neuronal nAChRs that coordinate zinc at the potentiation site. In aim 2, we will explore the larger structure of the potentiation site using Substituted Cysteine Accessibility Mutagenesis. Information obtained in aims 1 and 2 will be used to refine a homology model of the extracellular domains of neuronal nAChRs. In aim 3, we will determine whether zinc potentiates neuronal nAChRs through changes in the single channel open probability, the single channel current, and/or the number of channels. Changes in open probability will be characterized in terms of the underlying gating mechanism.