Structural biology of the alpha4beta2 nicotinic acetylcholine receptor

描述(申请人提供)：尼古丁成瘾是全球可预防死亡的主要来源。尼古丁成瘾新疗法合理设计的瓶颈是烟碱型乙酰胆碱受体的高分辨率结构信息。具体地说，尼古丁受体的alpha4beta2亚型在尼古丁中起着至关重要的作用。 上瘾。在从克隆细胞系到吸烟者大脑的各种系统中，尼古丁选择性地上调这种神经递质受体，而缺乏alpha4beta2受体的小鼠表现出显著的尼古丁自我给药行为减弱。戒烟的唯一不同于尼古丁替代的特定疗法是对α4β2尼古丁受体的部分激动剂。不幸的是，这些疗法在帮助人们戒烟方面只能起到一定的效果。这项建议的目标是通过X射线结晶学确定α4β2尼古丁受体的结构来解决这一紧迫的生物医学问题。α4beta2烟碱受体是结晶学研究中特别令人望而生畏的靶标，因为它是：(1)真核膜蛋白；(2)专性异五聚体蛋白；(3)其亚基化学计量可变，由两个α和三个β亚基或三个α和两个β亚基组成。这项提议分为两个阶段。在第一个探索阶段(R21)，目标是(1)鉴定结合在一起高水平表达并形成稳定的五聚体受体的α4和β2亚单位结构，(2)纯化单一化学计量比的受体物种，(3)获得初步晶体。在第二阶段(R33)，目标是(1)获得衍射性良好的α4β2受体晶体和(2)确定其与不同类型配体的络合物的结构。

DESCRIPTION (provided by applicant): Nicotine addiction is the leading source of preventable death worldwide. The bottleneck in rational design of novel therapeutics for nicotine addiction is high-resolution structural information for nicotinic acetylcholine receptors. Specifically, the alpha4beta2 subtype of the nicotinic receptor plays an essential role in nicotine addiction. Nicotine selectively upregulates this neurotransmitter receptor in systems ranging from clonal cell lines to smokers' brains, and mice lacking the alpha4beta2 receptor exhibit dramatically attenuated nicotine self-administration behavior. The only specific therapy for smoking cessation distinct from nicotine replacement is a partial agonist for the alpha4beta2 nicotinic receptor. Unfortunately, these therapies are only modestly effective in helping people quit smoking. The goal of this proposal is to resolve this pressing biomedical problem by determining the structure of the alpha4beta2 nicotinic receptor by X-ray crystallography. The alpha4beta2 nicotinic receptor is a particularly daunting target for crystallographic studies because it is: (1) a eukaryotic membrane protein; (2) an obligate heteropentameric protein; and (3) its subunit stoichiometry is variable, being formed from either two alpha and three beta subunits, or three alpha and two beta subunits. This proposal is organized into two phases. In the first, exploratory (R21) phase, the goals are to (1) identify alpha4 and beta2 subunit constructs that combine to express at high levels and form stable pentameric receptors, (2) purify a single stoichiometric species of receptor and (3) obtain preliminary crystals. In the second (R33) phase, the goals are to (1) obtain well-diffracting crystals of the alpha4beta2 receptor and (2) determine its structure in complex with different classes of ligands.