Neuronal nicotinic acetylcholine receptors (nAChRs)

项目总结 神经型烟碱型乙酰胆碱受体(NAChRs)与几种 病理生理状况包括阿尔茨海默氏症和帕金森氏症，癫痫， 精神分裂症，以及尼古丁成瘾的发展。因此，药物靶向性 神经元nAChR的研究在开发治疗这些疾病的药物策略方面有希望。 多种神经元nAChR亚型(如α4β2和α7 nAChR)的存在 亚基组成和独特的生理和病理特征阻碍了发育 大多数nAChR选择性治疗药物和少数神经性nAChR药物可用于临床。 由于神经元nAChRs共享保守的ACh结合位点，已证明很难开发 具有高nAChR亚型选择性的激动剂。此外，神经元nAChRs的直接激活 由激动剂引起的胆碱能传递的改变与长时间的激活有关 和nAChRs的脱敏。因此，nAChRs的正变构调节剂(PAM) 已经成为一种新的和生理上更相关的策略来增强大脑胆碱能 变速箱。PAM不能结合ACh结合部位，也不能激活nAChRs 哎呀。相反，它们通过与ACh不同的位点(S)结合来增强ACh诱导的反应 结合部位。然而，关于nAChR PAM结合位点的位置(S)和 缺乏负责PAM行动的机制。我们的长期目标是确定 NAChR PAM结合位点的数量和位置以及nAChR亚型选择性PAM的开发 用于实验和临床用途。我们这项研究的目标是确定两个结合位点 结构无关的α4β2nAChR PAM(dFBR和CMPI)。我们的中心假设是α4β2 NAChRs有多个PAM结合位点，nAChR PAM与这些结合位点的作用方式不同 网站。我们将使用两种互补的方法：1-用光反应进行光亲和标记 NAChR PAM，用于直接识别有助于PAM结合位点的氨基酸；以及2- 突变分析与体外电生理记录相结合以确定其贡献 α-4-β-2nAChR变构调控的亚基界面。这项工作的完成将 提供对理解变构多样性很重要的结构信息 并将促进更具选择性的α4β2 nAChR的发展 帕姆斯。

Project summary Neuronal nicotinic acetylcholine receptors (nAChRs) are implicated in several pathophysiological conditions including Alzheimer’s and Parkinson’s diseases, epilepsy, schizophrenia, and in the development of nicotine addiction. Therefore, pharmacological targeting of neuronal nAChR holds promise in the development of drug strategies to treat these conditions. The presence of multiple neuronal nAChR subtypes (e.g. α4β2 and α7 nAChR) with different subunit composition and unique physiological and pathological profiles hinders the development of nAChR selective therapeutics and only few neuronal nAChR drugs are clinically available. Since neuronal nAChRs share conserved ACh binding sites, it has proven difficult to develop agonists with high nAChR subtype selectivity. Moreover, direct activation of neuronal nAChRs by agonists is associated with alteration in cholinergic transmission due to prolonged activation and desensitization of nAChRs. As a result, positive allosteric modulators (PAMs) of nAChRs have emerged as a novel and more physiologically relevant strategy to enhance brain cholinergic transmission. PAMs do not bind to the ACh binding site or activate nAChRs in the absence of ACh. Rather, they potentiate ACh-induced responses by binding at site(s) distinct from the ACh binding sites. However, information regarding the location of nAChR PAM binding site(s) and the mechanisms responsible for PAM action are lacking. Our long-term goal is to determine the number and location of nAChR PAM binding sites and to develop nAChR subtype-selective PAMs for experimental and clinical uses. Our objective for this study is to identify binding sites for two structurally-unrelated α4β2 nAChR PAMs (dFBr and CMPI). Our central hypothesis is that α4β2 nAChRs have multiple PAM binding sites and that nAChR PAMs interact differently with these sites. We will us two complementary approaches: 1- Photoaffinity labeling with photoreactive nAChR PAMs to directly identify amino acids contributing to PAM binding sites; and 2- Mutational analyses coupled with in vitro electrophysiological recording to define the contribution of subunit interfaces in allosteric modulation of α4β2 nAChR. The completion of this work will provide structural information that are important for understanding the diversity of allosteric binding sites in nAChRs and would facilitate the development of more selective α4β2 nAChR PAMs.