Structural Biology of the Sodium-Calcium Exchanger

钠钙交换器的结构生物学

• 批准号: 7405717
• 负责人: Nathan Kyle Karpowich
• 金额: $ 4.96万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-27 至 2011-06-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7405717
• 关键词: ATP-Binding Cassette Transporters; Amino Acids; Archaea; Architecture; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Ca(2+)-Transporting ATPase; Cardiac; Cardiac Muscle Contraction; Cardiac Myocytes; Cardiology; Cell membrane; Cloning; Complex; Condition; Coupled; Couples; Crystallization; Cytochromes; Data; Detergents; Development; Disease; Drug Design; Embryo; Family member; Functional disorder; Heart; Heart failure; Homologous Gene; Homologous Protein; Homology Modeling; Human; In Vitro; Integral Membrane Protein; Investigation; Ion Channel; Ions; Knockout Mice; Knowledge; Lead; Light; Link; Lipids; Literature; Medical; Membrane Proteins; Methods; Molecular; Muscle Contraction; Names; Nature; Numbers; Pathology; Pathway interactions; Personal Satisfaction; Pharmacologic Substance; Pharmacology; Phase; Photosynthetic Reaction Centers; Play; Positioning Attribute; Protein Family; Proteins; Public Health; Publications; Regulation; Relaxation; Reporting; Research; Research Personnel; Research Proposals; Resolution; Roentgen Rays; Role; Sarcoplasmic Reticulum; Sodium-Calcium Exchanger; Solutions; Source; Structure; System; Techniques; Training; Vesicle; Work; X-Ray Crystallography; bacterial genetics; base; career; design; experience; falls; inhibitor/antagonist; insight; interest; novel; post-doctoral training; protein expression; proteoliposomes; research study; stereochemistry; structural biology

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.

描述（由申请人提供）：钠/钙交换器（NCX）在脊椎动物心脏中心肌细胞的肌肉收缩中发挥关键作用。人 NCX1 在心肌细胞横 (t) 小管的质膜中高水平表达，并在肌肉收缩的松弛阶段将 1 个 Ca2+ 的电流出与 3 个 Na+ 离子的输入耦合起来。 NCX1 敲除小鼠的胚胎致死率以及与异常 Ca2+ 转运相关的多种病理状况（包括心律失常和心力衰竭）强调了 NCX1 在调节心脏细胞内 Ca2+ 水平方面的重要性。尽管有大量关于 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，但迄今为止还没有这些蛋白质的详细结构信息。因此，高分辨率结构数据对于理解 NCX 蛋白的分子机制至关重要。由于细菌和古细菌中存在 NCX1 同源物，这些蛋白质可能为目前研究中无法获得的高分辨率结构信息提供途径。本申请提出的研究目的是： 1.)表达和纯化足够量的原核NCX1同系物用于结构研究并评估它们在脂蛋白体中的单分散性和生化活性； 2.) 通过X射线晶体学获得原核NCX蛋白的高分辨率结构信息。这些研究可以提供当前方法无法提供的宝贵细节，包括离子结合位点的立体化学以及与运输相关的构象变化的性质。此外，了解 NCX 蛋白的功能结构可以帮助同源建模，从而有助于人类 NCX 家族成员的潜在抑制剂的基于结构的药物设计。 公共卫生相关性。 Ca2+ 水平的上升和下降与跳动心脏的节律性肌肉收缩有关，并且这些水平直接受 Na+/Ca2+ 交换蛋白 (NCX) 的活性控制。由于 Ca2+ 水平异常与心律失常和心力衰竭等多种心脏病有关，因此对 NCX 机制的研究可以揭示这些疾病的详细原因。