Chemical Synapses - Biophysical Studies

项目描述（申请人提供）：本项目研究Cys-loop受体超家族分子的结构和功能：肌肉尼古丁乙酰胆碱受体（nAChR）、神经元α 4beta2 nAChR和5-羟色胺5-HT3受体。假设1认为在激动剂结合位点发生以下三个事件：(a)激动剂的带电荷胺/铵基团通过具有固定负极的单极-单极相互作用被吸引到该位点。(b)对于带有氨基（不是季铵基）的激动剂，这种相互作用通过与149-150肽键的主羰基的氢键来稳定，(c)最早的构象变化使激动剂在色氨酸α 149处处于阳离子- π相互作用。假设2认为在门控过程中，M2-M3连接体的主干构象发生了变化。假设3表明，在通道激活过程中，所有五个亚基的M2上部螺旋相对于邻近的螺旋重新定向。假设4认为，alpha4beta2和P2X2受体之间的动态、历史依赖的功能相互作用是通过beta2-M3-M4环和P2X2 c端尾部发生的。假设1和2将通过具有非天然氨基酸侧链和非天然主链的受体的宏观和单通道电生理评估进行测试。假设1、3和4将在基于系绳探针的直接荧光、荧光共振能量转移（FRET）和基于镧系元素的共振能量转移（LRET）的测量中进行测试。由此产生的关于乙酰胆碱受体和5-HT3受体的知识可能为戒烟、帕金森病、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、常染色体显性夜间额叶癫痫和精神分裂症等健康挑战提供病理生理学见解和更好的药物治疗。

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.