Structure and Function of Sodium-Calcium Exchangers

钠钙交换体的结构和功能

• 批准号: 8656709
• 负责人: Dinesh A Yernool
• 金额: $ 28.73万
• 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/8656709
• 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; biophysical properties; 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)：钙离子的流动是强有力的信号，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所有的细胞过程。钠/钙交换器(NCX)是大多数细胞质膜上的主要钙泵，是一种高容量的整合膜转运体。NCX利用预先存在的钠离子梯度电位快速排出钙离子的无与伦比的能力，对心肌细胞和神经元等可兴奋细胞的正常功能至关重要。这些细胞分别经历细胞内钙离子水平的快速、反复的振荡，以实现心肌收缩和神经元信号传递；NCX活性的扰动与心脏功能障碍和神经细胞死亡有关。目前，一种原子分辨率 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肌和神经细胞中钙离子/阳离子交换的运输和调节机制。针对这一目标，本文提出的具体目标如下：1.利用一种新的方法实现膜蛋白的异源过表达。我的实验室开发的优化表达方法可以增加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、生物物理和结晶学分析。最初的表达目标将是原核NCX同源物，因为它们与真核细胞的钠钙交换器具有显著的序列相似性。随后，我们将演示我们的方法对一大批膜蛋白靶标的适用性，并研究过度表达毒性的分子基础。2.论证了原核生物NCX是研究Na~+/Ca~(2+)交换的合适模型。这将通过确定转运蛋白的寡聚化状态和拓扑结构来实现，然后通过重组到脂质体中来表征原核生物NCX同源物的结构功能和转运性。将测定野生型离子转运动力学，并确定转运的抑制物。3.结构。我们将对原核细胞中的NCX同源物(S)单独和/或与抗体片段形成复合体进行结晶以确定和分析结构(S)。将生化和生物物理数据与NCX同系物的晶体结构相结合，将导致基于结构的Na+-Ca~(2+)交换机制模型的发展，刺激新的基于结构的假设 了解心脏NCX的作用机制，并可能启发基于结构的药物设计找到 药理活性物质。