Molecular Physiology of the Na-K-Cl Cotransporter

Na-K-Cl 协同转运蛋白的分子生理学

• 批准号: 7901772
• 负责人: BLISS FORBUSH
• 金额: $ 9.97万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-11 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7901772
• 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; polarized cell; research study; sensor; tissue culture

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，在细胞内稳态中发挥核心作用。在包括神经元在内的非极化细胞中，NKCC 1同种型参与细胞内氯离子和细胞体积的调节，在分泌性上皮中，NKCC 1与Cl通道、Na泵和K通道一起发挥作用，以实现受调节的盐运动。在哺乳动物肾脏中，另一种亚型NKCC 2介导盐吸收，并且是袢利尿药呋塞米和布美他尼的作用部位。NKCC是阳离子-氯化物协同转运蛋白家族和转运蛋白APC超家族的成员。该项目的长期目标是了解协同转运蛋白NKCC 1的分子机制，包括离子转运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意义。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，与中风相关的脑水肿和囊性纤维化。本研究的具体目的是：1）研究CCCs转运蛋白的结构和功能，利用FRET探针观察CCCs转运蛋白的激活和构象变化，验证功能相互作用的二聚体伴侣假说，利用半胱氨酸扫描突变研究CCCs潜在的凹孔环，阐明CCCs激活开关的机制; 2）利用转基因报告小鼠进一步阐明NKCC 1受细胞体积和细胞内[Cl-]调节的机制，所述转基因报告小鼠编码细胞内Cl-浓度和转运蛋白激活的荧光传感器。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的疾病和疾病状况涉及负责盐穿过细胞膜运动的细胞机制的缺陷或过度活性。这项研究旨在了解细胞机制的一部分，一种称为Na-K-Cl协同转运蛋白（或NKCC）的蛋白质，负责处理协调的钠，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。