Structural Biology of the Sodium-Calcium Exchanger

描述(由申请人提供)：钠/钙交换器(NCX)在脊椎动物心脏心肌细胞的肌肉收缩中起着关键作用。人NCX1在心肌细胞横管(T)管质膜上高水平表达，在肌肉收缩的松弛阶段，NCX1将一种钙离子的电外流与三种钠离子的输入相偶联。NCX1基因敲除小鼠的胚胎致死性以及与钙离子异常运输相关的许多病理条件，包括心律失常和心力衰竭，突显了NCX1在调节心脏细胞内钙水平方面的重要性。虽然有大量关于NCX1及其同源物的文献，包括电生理学、免疫化学和药理学研究，但到目前为止还没有关于这些蛋白质的详细结构信息。因此，高分辨率的结构数据对于了解NCX蛋白的分子机制是必不可少的。由于细菌和古菌中存在NCX1同源物，这些蛋白质可能为获得目前研究中无法获得的高分辨率结构信息提供了一条途径。本申请提出的研究目的是：1)表达和纯化足够数量的原核生物NCX1同源物，用于结构研究，并评价其在蛋白脂质体中的单分散性和生化活性；目的：通过X射线结晶学获得原核生物NCX蛋白的高分辨结构信息。这些研究可以提供当前方法无法提供的宝贵细节，包括离子结合部位的立体化学和与运输相关的构象变化的性质。此外，了解NCX蛋白的功能结构有助于同源建模，从而有助于基于结构的药物设计人类NCX家族成员的潜在抑制剂。 与公共卫生相关。钙离子水平的上升和下降与心脏跳动的节律性肌肉收缩有关，而这些水平直接由钠/钙交换蛋白(NCX)的活性控制。由于异常的钙水平与包括心律失常和心力衰竭在内的各种心脏病理有关，对NCX机制的研究可能有助于揭示这些疾病的详细原因。

DESCRIPTION (provided by applicant): Sodium/Calcium exchangers (NCX) play a key role in muscle contraction of cardiac myocytes in the vertebrate heart. Human NCX1 is expressed at high levels in the plasma membrane of the transverse (t)- tubules of cardiac myocytes and couples the electrogenic efflux of one Ca2+ to the import of 3 Na+ ions during the relaxation phase of muscle contraction. The importance of NCX1 in regulating intracellular Ca2+ levels in the heart is underscored by the embryonic lethality of NCX1 knockout mice as well as the numerous pathological conditions associated with aberrant Ca2+ transport, including cardiac arrythmias and heart failure. Although there is a large body of literature on NCX1 and its homologs, including electrophysiological, immunochemical, and pharmacological studies, there is no detailed structural information on these proteins to date. As a result, high resolution structural data is essential to understand the molecular mechanism of NCX proteins. As there are NCX1 homologs present in bacteria and archaea, these proteins may provide an avenue to the high-resolution structural information currently unavailable in present studies. The aims of the research proposed in this application are: 1.) To express and purify adequate amounts of prokaryotic NCX1 homologs for structural studies and assess their monodispersity and biochemical activity in proteoliposomes; and 2.) To obtain high resolution structural information of a prokaryotic NCX protein by X-ray crystallography. These studies could provide invaluable details unavailable by current methods, including the stereochemistry of the ion binding sites and the nature of the conformational changes associated with transport. Furthermore, knowledge of the functional architecture of an NCX protein could aid homology modeling and hence structure-based pharmaceutical design of potential inhibitors of the human NCX family members. PUBLIC HEALTH RELEVANCE. The rise and fall of Ca2+ levels are coupled to the rhythmic muscle contraction of the beating heart, and these levels are directly controlled by the activity of the Na+/Ca2+ exchanger protein (NCX). As abnormal Ca2+ levels have been linked to various cardiac pathologies, including arrythimias and heart failure, studies of the mechanism of NCX could shed light on the detailed causes of these diseases.