Molecular Physiology of the Na-K-Cl Cotransporter

描述(申请人提供)：钠-钾-氯共转运蛋白(NKCC)是一种质膜转运蛋白，在细胞内稳态中起核心作用。在包括神经元在内的非极化细胞中，NKCC1亚型参与了细胞内氯离子和细胞体积的调节，而在分泌上皮细胞中，NKCC1与氯通道、钠泵和钾通道一起发挥作用，实现了盐的调节运动。在哺乳动物肾脏中，另一种异构体NKCC2介导盐的吸收，是环状利尿剂药物速尿和布美他尼的作用部位。NKCC是阳离子-氯离子共转运体家族和APC超家族转运体的成员。这个以NKCC1为中心的项目的长期目标是了解共转运蛋白的分子机制，包括潜在的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。这项研究直接关系到对离子转运疾病和疾病状况的了解、诊断和治疗，这些疾病包括高血压、多囊肾病、分泌性腹泻、与中风相关的脑水肿和囊性纤维化。该项目的具体目标是：1)检测运输蛋白的结构和功能，利用FRET探针观察共转运体的激活和构象变化，测试功能相互作用的二聚体伙伴的假说，使用半胱氨酸扫描突变来研究潜在的可重入孔环，并阐明CCCS中激活开关的机制；2)利用编码细胞内氯-浓度和转运体激活的荧光传感器的转基因小鼠，进一步阐明细胞体积和细胞内[Cl-]调节NKCC1的机制。疾病和疾病状况包括高血压、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，涉及负责跨细胞膜盐分移动的细胞机制的缺陷或过度活动。这项研究旨在了解细胞机制的一部分，一种名为钠-钾-氯协转运体(或NKCC)的蛋白质，负责处理钠、钾和氯的协调运动。通过了解蛋白质的分子结构、作用机制和调节机制，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。

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.