Mechanisms of Neurotransmitter-gated Ion Channels

描述（由申请人提供）：本提案的重点是离子通道的尼古丁受体超家族。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其功能。为此，我们将采用多方面的方法，包括单通道和集合电生理学，蛋白质工程，x射线晶体学和理论计算。大部分实验将在肌肉烟碱乙酰胆碱受体（AChR）及其细菌对应物ELIC和GLIC上进行。由于其大的单通道电导，强大的化学计量学，易于处理的动力学和在异源表达系统中的高表达水平，肌肉AChR仍然是整个超家族中无与伦比的模型。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、化学和生物学方面的基本问题提供了答案，这些问题无法用（可能）任何其他通道来解决。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)从总负电荷数和酸碱行为方面表征AChR电荷选择性过滤器中的谷氨酸环；2)识别闸门的性质及其在通道开启、关闭和脱敏时的重新排列；3)表征被推测参与代谢和膜内功能调节的AChR区域。由于其作为典型的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对突触后受体和快速突触传递的理解产生广泛的影响。还应强调的是，achr介导的神经传递不仅支持运动功能，还支持自主神经和认知功能。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）中也受损。更广泛地说，achr也在气道、角化细胞、淋巴细胞和内皮细胞中表达，它们是细胞分化、增殖和抗凋亡的介质，因此也是癌症的介质。

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. PUBLIC HEALTH RELEVANCE: The main subject of this research proposal is the biophysical characterization of the nicotinic acetylcholine receptor. In the central and autonomic nervous system, nicotinic receptors play important roles in learning, memory and the modulation of cardiac function. It follows then a full understanding of not only motor, but also, cardiovascular and cognitive pathological conditions (such as Alzheimer's disease and schizophrenia) requires a thorough knowledge of the molecular underpinnings of these receptors. Furthermore, there is growing evidence that nicotinic receptors are also expressed in the lung, skin, platelets, immune cells and the cells that line the blood vessels, where they mediate cell growth and, eventually, the development of malignant tumors.