Structure and Function of Sodium-Calcium Exchangers

钠钙交换体的结构和功能

• 批准号: 8514639
• 负责人: Dinesh A Yernool
• 金额: $ 27.78万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8514639
• 关键词: Active Biological Transport; Architecture; Binding Sites; Biochemical; Calcium; Calcium ion; Cardiac; Cardiac Muscle Contraction; Cardiac Myocytes; Cations; Cell Death; Cell division; Cell membrane; Cell physiology; Cells; Chimeric Proteins; Communities; Complex; Crystallization; Data; Development; Disease; Drug Design; Elements; Family; Fertilization; Foundations; Functional disorder; Future; Goals; Health; Heart Diseases; Homeostasis; Homologous Gene; Ii-Key; Immunoglobulin Fragments; Integral Membrane Protein; Ion Channel; Ion Transport; Kinetics; Knowledge; Laboratories; Lead; Life; Liposomes; Membrane; Membrane Proteins; Membrane Transport Proteins; Methodology; Methods; Mission; Modeling; Molecular; Molecular Genetics; Myocardial Ischemia; Neuronal Dysfunction; Neurons; Outcome; Physiological; Property; Protein Family; Proteins; Proteolysis; Public Health; Pump; Recurrence; Regulation; Research; Resolution; Signal Transduction; Sodium-Calcium Exchanger; Specificity; Structural Models; Structure; Structure-Activity Relationship; Study models; Time; Toxic effect; Transmembrane Domain; United States National Institutes of Health; X-Ray Crystallography; base; cost; design; experience; improved; inhibitor/antagonist; neuron loss; novel; novel strategies; optical imaging; overexpression; receptor; reconstitution; small molecule; structural biology; three dimensional structure; three-dimensional modeling; transport inhibitor

DESCRIPTION (provided by applicant): Flux of calcium ions, potent signals, controls many if not all cellular processes from fertilization and cell division to eventual cell death. Sodium/calcium exchangers (NCX), high-capacity integral membrane transporters, are the principal Ca2+ pumps in the plasma membranes of most cells. The unmatched ability of NCX to rapidly extrude Ca2+ using preexisting Na+-gradient potentials is critical to the normal function of excitable cells such as cardiac myocytes and neurons. These cells undergo quick, recurrent oscillations in levels of intracellular Ca2+ to achieve cardiac muscle contraction and neuronal signaling, respectively; and perturbations in NCX activity have been implicated in cardiac dysfunction and neuronal cell death. At present, an atomic-resolution structure of NCX has not been determined. Lack of 3D structure impedes the elucidation of structure-function relationships that are key to understanding NCX's mechanism of action. Our long-term goal is to describe at the atomic level the mechanism of transport and regulation of Ca2+/cation exchange in cardiac and neuronal cells. Toward this goal, the specific aims proposed here are as follows: 1. Heterologous overexpression of membrane proteins using a novel method. Optimized expression methods developed in my laboratory to increase membrane protein yields and reduce their host toxicity will enable us to produce enough protein for biochemical, biophysical, and crystallographic analyses. Initial expression targets will be prokaryotic NCX homologs selected for their significant sequence identity to eukaryotic sodium-calcium exchangers. Subsequently we will demonstrate our method's applicability to a large panel of membrane protein targets and investigate the molecular basis of overexpression toxicity. 2. Demonstration that prokaryotic NCX is an appropriate model for studies of Na+/Ca2+ exchange. This will be achieved by determining the oligomerization state and topology of the transporter, followed by characterization of structure-function and transport properties of prokaryotic NCX homologs by reconstitution into liposomes. Wild-type ion transport kinetics will be determined and inhibitors of transport identified. 3. Structure. We will crystallize a prokaryotic NCX homolog(s) alone and/or in complex with antibody fragments to determine and analyze structure(s). Combining biochemical and biophysical data with the crystal structure of an NCX homolog will lead to development of structure-based mechanistic models for Na+-Ca2+ exchange, spur new structure-based hypotheses to understand the mechanism of cardiac NCX, and may inspire structure-based drug design to find pharmacologically active agents.

描述（由申请人提供）：钙离子通量是有效的信号，控制着从受精和细胞分裂到最终细胞死亡的许多（如果不是全部）细胞过程。钠/钙交换器 (NCX) 是一种高容量的整体膜转运蛋白，是大多数细胞质膜中的主要 Ca2+ 泵。 NCX 利用预先存在的 Na+ 梯度电位快速挤出 Ca2+ 的无与伦比的能力对于心肌细胞和神经元等可兴奋细胞的正常功能至关重要。这些细胞的细胞内 Ca2+ 水平经历快速、反复的振荡，分别实现心肌收缩和神经元信号传导； NCX 活性的扰动与心脏功能障碍和神经元细胞死亡有关。目前，原子分辨率 NCX的结构尚未确定。缺乏 3D 结构阻碍了结构-功能关系的阐明，而结构-功能关系是理解 NCX 作用机制的关键。我们的长期目标是在原子水平上描述心脏和神经元细胞中 Ca2+/阳离子交换的运输和调节机制。为了实现这一目标，本文提出的具体目标如下： 1.使用新方法异源过度表达膜蛋白。我的实验室开发的优化表达方法可提高膜蛋白产量并降低其宿主毒性，这将使我们能够生产足够的蛋白质用于生化、生物物理和晶体学分析。初始表达靶标将是原核NCX同源物，因其与真核钠钙交换体具有显着的序列同一性而被选择。随后，我们将展示我们的方法对大量膜蛋白靶标的适用性，并研究过度表达毒性的分子基础。 2. 证明原核NCX是研究Na+/Ca2+交换的合适模型。这将通过确定转运蛋白的寡聚状态和拓扑结构，然后通过重构到脂质体中来表征原核 NCX 同系物的结构功能和转运特性来实现。将测定野生型离子传输动力学并鉴定传输抑制剂。 3.结构。我们将单独结晶原核NCX同源物和/或与抗体片段复合物，以确定和分析结构。将生物化学和生物物理数据与 NCX 同系物的晶体结构相结合将导致开发基于结构的 Na+-Ca2+ 交换机制模型，激发新的基于结构的假设 了解心脏 NCX 的机制，并可能启发基于结构的药物设计来寻找 药理活性剂。