Mechanisms of Neurotransmitter-gated Ion Channels

描述(申请人提供)：本提案的重点是烟碱受体超家族离子通道。我们的长期目标是阐明这些受体通道的生物物理特性如何在正常和病理条件下塑造它们的功能。为此，我们将应用多方面的方法，包括单通道和整体电生理学、蛋白质工程、X射线结晶学和理论计算。大多数实验将在肌肉烟碱型乙酰胆碱受体(AChR)及其细菌对应的ELIC和GLIC上进行。由于其大的单通道电导，强大的化学计量学，易控制的动力学和在异源表达系统中的高表达水平，肌肉AChR仍然是整个超家族的无与伦比的模型。此外，多年来，对这种特殊受体的研究已经为离子通道的物理、化学和生物学的基本问题提供了答案，这些问题可能是任何其他通道无法解决的。在这个应用中，我们建议解决离子通道功能的三个支柱，即离子传导、门控和门控调制。我们的三个具体目标是：1)根据总负电荷的数量和酸碱行为来表征乙酰胆碱受体电荷选择性过滤器中的谷氨酸环；2)确定门的性质(S)及其在通道开放、关闭和脱敏时的重排；以及3)表征假设参与代谢和膜内功能调节的乙酰胆碱受体区域。由于其作为神经递质门控离子通道的作用，从拟议的肌肉AChR实验中获得的知识将对我们理解突触后受体和一般的快速突触传递产生广泛的影响。还应该强调的是，AChR介导的神经传递不仅支持运动，而且还支持自主和认知功能。因此，除了在肌无力综合征中受损外，胆碱能神经传递在心血管疾病和神经退行性疾病(如阿尔茨海默病和精神分裂症)中也受到损害。更广泛地说，AChRs也在呼吸道、角质形成细胞、淋巴细胞和内皮细胞中表达，在这些细胞中，它们是细胞分化、增殖和抗凋亡的媒介，因此也是癌症的媒介。

DESCRIPTION (provided by applicant): The focus of this proposal is the nicotinic-receptor superfamily of ion channels. Our long-term goal is to elucidate how the biophysical properties of these receptor-channels shape their function under both normal and pathological conditions. To this end, we will apply a multi-faceted approach that includes single-channel and ensemble electrophysiology, protein engineering, X-ray crystallography and theoretical calculations. Most of the experiments will be conducted on the muscle nicotinic acetylcholine receptor (AChR) and its bacterial counterparts, ELIC and GLIC. Because of its large single-channel conductance, robust stoichiometry, tractable kinetics and high expression levels in heterologous expression systems, the muscle AChR remains an unparalleled model for the entire superfamily. Furthermore, throughout the years, research on this particular receptor has provided answers to fundamental questions on the physics, chemistry and biology of ion channels that cannot be addressed with (probably) any other channel. In this application, we propose to address the three pillars of ion-channel function, namely, ion conduction, gating, and modulation of gating. Our three Specific Aims are: 1) To characterize the ring of glutamates in the charge selectivity filter of the AChR in terms of number of total negative charges and acid-base behavior; 2) To identify the nature of the gate(s) and its rearrangement upon channel opening, closing and desensitization; and 3) To characterize the regions of the AChR hypothesized to be involved in the metabolic and intramembrane modulation of function. Because of its role as a paradigmatic neurotransmitter-gated ion channel, the knowledge derived from the proposed experiments on the muscle AChR is poised to have a broad impact on our understanding of postsynaptic receptors and fast synaptic transmission in general. It should also be emphasized that AChR-mediated neurotransmission supports not only motor, but also, autonomic and cognitive function. As a result, in addition to being compromised in myasthenic syndromes, cholinergic neurotransmission is also impaired in cardiovascular diseases and neurodegenerative disorders such as Alzheimer's disease and schizophrenia. Even more broadly, AChRs are also expressed in the airways, keratinocytes, lymphocytes and endothelial cells where they are mediators of cell differentiation, proliferation and resistance to apoptosis, and hence, of cancer.