Neuronal nicotinic acetylcholine receptors (nAChRs)

项目摘要 神经元烟碱乙酰胆碱受体（nAChR）参与了几种神经细胞的凋亡。 病理生理状况，包括阿尔茨海默病和帕金森病，癫痫， 精神分裂症和尼古丁成瘾的发展。因此，药理学靶向 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（例如α4β2和α7 nAChR）的存在， 亚基组成和独特的生理和病理概况阻碍了发展 的nAChR选择性治疗和只有少数神经nAChR药物是临床上可用的。 由于神经元nAChRs共享保守的ACh结合位点，因此已经证明难以开发 具有高nAChR亚型选择性的激动剂。此外，神经元nAChRs的直接激活 与胆碱能传递的改变有关， 和nAChRs的脱敏。因此，nAChR的正变构调节剂（PAM） 已经成为一种新的和更生理相关的策略，以提高脑胆碱能 传输PAM不与ACh结合位点结合，也不激活nAChR。 啊。相反，它们通过与ACh不同的位点结合而增强ACh诱导的反应 结合位点。然而，关于nAChR PAM结合位点的位置和nAChR PAM结合位点的位置的信息是未知的。 缺乏负责地中海议会行动的机制。我们的长期目标是确定 nAChR PAM结合位点的数量和位置，并开发nAChR亚型选择性PAM 用于实验和临床用途。我们这项研究的目的是确定两个 结构无关的α4β2 nAChR PAM（dFBr和CMPI）。我们的中心假设是α4β2 nAChR具有多个PAM结合位点，并且nAChR PAM与这些结合位点的相互作用不同。 网站.我们将使用两种互补的方法：1-光亲和标记与光反应 nAChR PAM，以直接鉴定有助于PAM结合位点的氨基酸;和 结合体外电生理记录的突变分析，以确定 α4β2 nAChR变构调节中亚基界面的变化。这项工作的完成将 提供了结构信息，对于理解变构的多样性是重要的， nAChR中的结合位点，并将促进更具选择性的α4β2 nAChR的开发 PAM。

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.