Chemical Synapses - Biophysical Studies

描述(申请人提供)：本项目研究Cys-loop受体超家族中的分子的结构和功能：肌肉烟碱型乙酰胆碱受体(NAChR)、神经元α4beta2nAChR和5-羟色胺5-HT3受体。假设1指出，在激动剂结合部位发生了三个事件，(A)激动剂的带电的胺/铵基团被侧链上固定负电荷的单极-单极相互作用所吸引，(B)对于带有氨基(而不是季铵盐基)的激动剂，这种相互作用是通过149-150肽键的主键上的氢键稳定的，(C)最早的构象变化使激动剂在色氨酸α149处处于阳离子-pi相互作用中。假设2指出，YE M2-M3连接子在选通过程中经历了骨架构象的变化。假设3指出，在通道激活过程中，所有五个亚基的上部M2螺旋相对于相邻螺旋重新定位。假设4指出，α4beta2和P2X2受体之间动态的、依赖历史的功能相互作用是通过Beta2-M3-M4环和P2X2 C末端尾巴发生的。假设1和2将通过宏观和单通道电生理评估来检验带有非天然氨基酸侧链和非天然主干连接的受体。假设1、3和4将在基于系留探针的直接荧光、荧光共振能量转移(FRET)和基于稀土的共振能量转移(LRET)的测量中得到验证。由此产生的关于乙酰胆碱受体和5-HT3受体的知识可能为健康挑战提供病理生理学见解和更好的药物治疗，包括戒烟、帕金森氏病、阿尔茨海默病、疼痛、克罗恩病、婴儿猝死综合征、注意力缺陷障碍、常染色体显性遗传性夜间额叶癫痫和精神分裂症。

DESCRIPTION (provided by applicant): This project studies the structure and function of molecules in the Cys-loop receptor superfamily: the muscle nicotinic acetylcholine receptor (nAChR), the neuronal alpha4beta2 nAChR, and the serotonin 5-HT3 receptor. Hypothesis 1 states that three events occur in the following sequence at the agonist binding site, (a) The charged amine / ammonium group of the agonist is attracted to the site by a monopole-monopole interaction with fixed negative .charges on side chains, (b) For agonists with an amino group (not a quaternary ammonium group), this interaction is stabilized by an H-bond to the backbone carbonyl of the 149-150 peptide bond, (c) The earliest conformational change places the agonist in a cation-pi interaction at tryptophan alpha149. Hypothesis 2 states that ye M2-M3 linker undergoes a change in backbone conformation during gating. Hypothesis 3 states that during channel activation, the upper M2 helix of all five subunits re-orients with respect to neighboring helices. Hypothesis 4 states that the dynamic, history- dependent functional interaction between alpha4beta2 and P2X2 receptors occurs via the beta2-M3-M4 loop and the P2X2 C-terminal tail. Hypotheses 1 and 2 will be tested with macroscopic and single-channel electrophysiological assessments of receptors bearing unnatural amino-acid side chains and unnatural backbone linkages. Hypotheses 1, 3, and 4 will be tested in measurements based on direct fluorescence of tethered probes, fluorescence resonance energy transfer (FRET), and lanthanide-based resonance energy transfer (LRET). The resulting knowledge about acetylcholine receptors and 5-HT3 receptors may provide both pathophysiological insights and better drug therapies for health challenges including smoking cessation, Parkinson's disease, Alzheimer's disease, pain, Crohn's disease, sudden infant death syndrome, attention deficit disorder, autosomal dominant nocturnal frontal lobe epilepsy, and schizophrenia.