Physiologically relevant energetic interactions within the Na/K Pump

Na/K 泵内生理相关的能量相互作用

基本信息

批准号：
8574225
负责人：
Pablo Artigas
金额：
$ 45.26万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8574225
关键词：
ATP phosphohydrolase Active Biological Transport Address Affinity Amino Acids Animals Arrhythmia Binding Binding Sites Biochemistry Brain Cardiac Glycosides Cardiovascular Diseases Carrier Proteins Cell membrane Cells Characteristics Chemicals Complex Computer Simulation Congestive Heart Failure Cysteine Data Digitalis preparation Disease Disulfides Drug usage Electrodes Electrophysiology (science)Engineering Etiology Familial Hemiplegic Migraine Familial disease Functional disorder Gene Mutation Genes Goals Heart failure Hypertension Investigation Ions Laboratories Lead Life Ligands Link Maintenance Manuscripts Mediating Membrane Potentials Membrane Proteins Migraine Mission Modification Molecular Molecular Biology Molecular Conformation Muscle Mutagenesis Mutate Mutation Myocardium Na(+)-K(+)-Exchanging ATPase Nerve Oocytes Parkinsonian Disorders Pharmaceutical Preparations Polycystic Kidney Diseases Positioning Attribute Protein Isoforms Pump Recruitment Activity Research Resolution Rest Role Structural Models Structure Students Surface Testing Tissues Work Xenopus oocyte base clinically relevant computational chemistry conformer crosslink extracellular graduate student inhibitor/antagonist insight interest meetings molecular mechanics mutant nervous system disorder novel palytoxin patch clamp public health relevance receptor single molecule undergraduate student voltage clamp

项目摘要

DESCRIPTION (provided by applicant): All animal cells require maintenance of transmembrane gradients for Na+ and K+ ions by the Na/K pump, a plasma membrane P-type ATPase. Pathophysiologically, the Na/K pump is the target specifically inhibited by cardiotonic steroids, drugs traditionally used for the treatment of heart failure. Also, mutations in the genes encoding for two Na/K pump isoforms have been linked to migraine and Parkinsonism. The long-term goal of our laboratory is to understand the relationship between the Na/K pump structure, its function, and its multiple roles in cardiovascular and neurological diseases. All P-type ATPases alternate between two major conformers E1 and E2. The molecular forces that stabilize intermediate states in the cycle must arise as a consequence of a set of state-specific residue-residue and residue-ligand interactions. The main research goal of this AREA project is to identify and characterize energetically and mechanistically relevant interactions between pairs of residues and between residues and ions within the Na/K pump. To address this problem we use mutagenesis, heterologous expression of Na/K pumps in Xenopus oocytes, voltage clamp (two electrode voltage clamp, cut-open oocyte and patch clamp), chemical modification of engineered cysteine residues and computational chemistry to address two independent and interrelated aims, which we propose on the basis of extensive preliminary work: 1) To identify and characterize critical conformation-specific interactions that stabilize Na/K pump conformations, 2) to elucidate the mechanisms of ion-induced conformational changes. Considering the overwhelming evidence that the rate of E1 - E2 conversion underlies the molecular mechanism of this critical transporter, the work proposed here will be crucial to understanding and resolving the etiology of clinically relevant problems. Specifically, dysfunction of the Na,K-ATPase has been attributed to hypertension, congestive heart failure, familial hemiplegic migraine, and polycystic kidney disease.

描述（由申请人提供）：所有动物细胞都需要通过 Na/K 泵（质膜 P 型 ATP 酶）维持 Na+ 和 K+ 离子的跨膜梯度。从病理生理学角度来看，Na/K 泵是强心类固醇（传统上用于治疗心力衰竭的药物）特异性抑制的靶标。另外，基因突变两种 Na/K 泵亚型的编码与偏头痛和帕金森症有关。我们实验室的长期目标是了解Na/K泵结构、功能及其在心血管和神经系统疾病中的多种作用之间的关系。所有 P 型 ATP 酶在两个主要构象异构体 E1 和 E2 之间交替。稳定循环中的中间态的分子力必须是一组状态特异性残基-残基和残基-配体相互作用的结果。该 AREA 项目的主要研究目标是识别和表征 Na/K 泵内残基对之间以及残基与离子之间的能量和机械相关相互作用。为了解决这个问题，我们使用诱变、爪蟾卵母细胞中 Na/K 泵的异源表达、电压钳（两电极电压钳、切开卵母细胞和膜片钳）、工程半胱氨酸残基的化学修饰和计算化学来解决两个独立和我们在广泛的前期工作的基础上提出了相互关联的目标：1）识别和表征稳定Na/K泵构象的关键构象特异性相互作用，2）阐明离子诱导构象的机制变化。考虑到大量证据表明 E1 - E2 转化率是这一关键转运蛋白分子机制的基础，本文提出的工作对于理解和解决临床相关问题的病因至关重要。具体来说，功能障碍 Na,K-ATP酶的减少可归因于高血压、充血性心力衰竭、家族性偏瘫性偏头痛和多囊肾病。