Potentiation of Neuronal Nicotinic Receptors by Zinc

描述(申请人提供)：本项目的目标是：(1)阐明神经元烟碱型乙酰胆碱受体(NAChRs)上锌结合并增强受体功能的位点(S)的结构；(2)阐明锌增强受体功能的机制。 尼古丁配体在治疗精神分裂症、帕金森氏病和阿尔茨海默病等神经疾病方面具有潜在的抗焦虑和止痛药作用，也是尼古丁发挥其精神活性和成瘾作用的部位。对神经元nAChRs进行有效的药物干预需要开发亚型选择性配体。传统上，追求这一目标的方向是开发作用于乙酰胆碱(ACh)结合位点的配体。我们建议在神经元nAChRs上寻找一类新的位点，即锌增强这些受体的部位。神经元nAChRs上的ACh结合部位位于亚基的胞外区之间的界面。在许多神经元nAChRs中，在两对不同亚基之间的界面上形成了两个激动剂结合位点，留下了三个不参与ACh结合的交替界面。我们假设一个或多个交替的界面结合了锌，锌是神经元nAChR功能的调节器。在目标1中，我们将使用定点突变和功能分析来鉴定神经元nAChRs上的氨基酸残基，这些氨基酸残基与锌在增强位点上配位。在目标2中，我们将使用取代半胱氨酸可及性突变来探索增强位点的更大结构。在AIMS 1和AIMS 2中获得的信息将用于改进神经元nAChRs胞外区域的同源模型。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nAChRs。打开概率的变化将根据基本的门控机制来表征。

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.