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Molecular Physiology of the Na-K-Cl Cotransporter

Na-K-Cl 协同转运蛋白的分子生理学

基本信息

批准号：
8119129
负责人：
BLISS FORBUSH
金额：
$ 36.5万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-01-01 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8119129
关键词：
Affinity Amino Acid Transporter Behavior Biochemical Bumetanide Carrier Proteins Cations Cell Volumes Cell membrane Cells Cerebral Edema Charge Chloride Ion Chlorides Cysteine Cystic Fibrosis Defect Diagnosis Diagnostic Diarrhea Dimerization Disease Diuretics Epithelium Evaluation Family Fluorescence Resonance Energy Transfer Furosemide Goals Homeostasis Hypertension Individual Ion Transport Ions Kidney Label Lead Mechanics Mediating Membrane Transport Proteins Modeling Molecular Molecular Structure Monitor Movement Mus Mutagenesis Na(+)-K(+)-Exchanging ATPase Neurons Optics Organ Pharmaceutical Preparations Physiological Processes Physiology Play Polycystic Kidney Diseases Potassium Channel Potassium Chloride Process Protein Isoforms Proteins Regulation Reporter Reporting Research Resolution Role Salivary Glands Scanning Side Signal Transduction Site Small Interfering RNA Sodium Sodium Chloride Stroke Structure Structure of choroid plexus Structure-Activity Relationship Testing Therapeutic Agents Tissues Transgenic Mice Transgenic Organisms Transmembrane Domain Work absorption base chloride-cotransporter potassium clomeleon design dimer member mouse model nervous system disorder research study sensor tissue culture

项目摘要

DESCRIPTION (provided by applicant): The Na-K-Cl cotransporter (NKCC) is a plasma membrane transport protein that plays a central role in cellular homeostasis. In non-polarized cells including neurons the NKCC1 isoform is involved in regulation of intracellular chloride and cell volume, and in secretory epithelia, NKCC1 functions together with Cl channels, the Na pump, and K channels to bring about regulated salt movement. In the mammalian kidney another isoform, NKCC2, mediates salt absorption and is the site of action of the loop diuretic drugs furosemide and bumetanide. NKCCs are members of the cation-chloride cotransporter family, and of the APC superfamily of transporters. The long term goal of this project, which focuses on NKCC1, is to understand the molecular mechanism of the cotransporter, including the structural and functional features underlying ion translocation and its regulation, and the significance of the transporter in cell and organ function. The research is directly relevant to the understanding, diagnosis, and treatment of ion transport diseases and disease conditions including hypertension, polycystic kidney disease, secretory diarrhea, cerebral edema associated with stroke, and cystic fibrosis. The Specific Aims of the project are: 1) To examine the structure and function of the transport protein, utilizing FRET probes to look at cotransporter activation and conformational changes, testing a hypothesis of functionally interacting dimer partners, using cysteine scanning mutagenesis to investigate potential reentrant pore loops, and elucidating the mechanism of the activation switch in CCCs; 2) To further elucidate the mechanism of regulation of NKCC1 by cell volume and intracellular [Cl-] utilizing a transgenic reporter mouse encoding a fluorescent sensor of intracellular Cl- concentration and transporter activation. Diseases and disease conditions including hypertension, cerebral edema, polycystic kidney disease, secretory diarrhea, cystic fibrosis, and some diseases of the nervous system involve defects or overactivity of the cellular machinery that is responsible for salt movements across cell membranes. This research is directed to understanding one part of that cellular machinery, a protein called the Na-K-Cl cotransporter (or NKCC) that is responsible for handling coordinated sodium, potassium and chloride movements. By understanding the molecular structure of the protein, the mechanics of its action, and the mechanism of its regulation we will be better able to design diagnostic and therapeutic agents and treat these disease states.

描述（由申请人提供）：Na-K-Cl 协同转运蛋白（NKCC）是一种质膜转运蛋白，在细胞稳态中发挥核心作用。在包括神经元在内的非极化细胞中，NKCC1 亚型参与细胞内氯离子和细胞体积的调节，而在分泌性上皮细胞中，NKCC1 与 Cl 通道、Na 泵和 K 通道一起发挥作用，调节盐的运动。在哺乳动物肾脏中，另一种异构体 NKCC2 介导盐吸收，是袢利尿药物呋塞米和布美他尼的作用位点。 NKCC 是阳离子-氯化物协同转运蛋白家族和 APC 转运蛋白超家族的成员。该项目以NKCC1为重点，长期目标是了解协同转运蛋白的分子机制，包括离子易位及其调控的结构和功能特征，以及转运蛋白在细胞和器官功能中的重要性。该研究与离子转运疾病和疾病状况的理解、诊断和治疗直接相关，包括高血压、多囊肾病、分泌性腹泻、中风相关脑水肿和囊性纤维化。该项目的具体目标是：1）检查转运蛋白的结构和功能，利用FRET探针观察协同转运蛋白的激活和构象变化，测试功能上相互作用的二聚体伴侣的假设，使用半胱氨酸扫描诱变研究潜在的可重入孔环，并阐明CCC中激活开关的机制； 2) 利用编码细胞内Cl-浓度和转运蛋白激活荧光传感器的转基因报告小鼠，进一步阐明NKCC1通过细胞体积和细胞内[Cl-]调节的机制。包括高血压、脑水肿、多囊肾病、分泌性腹泻、囊性纤维化和一些神经系统疾病在内的疾病和病症涉及负责盐跨细胞膜运动的细胞机器的缺陷或过度活跃。这项研究旨在了解细胞机制的一部分，即一种称为 Na-K-Cl 协同转运蛋白（或 NKCC）的蛋白质，负责协调钠、钾和氯的运动。通过了解蛋白质的分子结构、其作用机制及其调节机制，我们将能够更好地设计诊断和治疗药物并治疗这些疾病状态。