Molecular Physiology of the renal Na-Cl cotransporter

描述（由申请人提供）：这项为期5年的资助申请的长期目标是了解噻嗪类药物敏感Na-CI共转运体的分子生理学基本问题。吉特曼氏病是这种基因突变失活的结果这种共转运体是可能与人类高血压发展有关的基因之一。肾Na-CI共转运体是噻嗪类利尿剂的靶点，噻嗪类利尿剂是世界上最常用的处方药之一。此外，噻嗪类药物敏感的共转运蛋白的表达受到多种因素的高度调控，这些因素已知可以调节钠的肾脏排泄。因此，肾脏Na-CI共转运体在肾脏生理学、药理学和病理生理学中具有重要意义。研究人员利用一种功能表达策略，证明了哺乳动物和鱼类对噻嗪类药物敏感的Na-CI共转运体在离子易位的特异性和动力学特性、利尿结合亲和力以及对细胞体积和WNK4激酶调节的反应等方面存在重要差异。本应用程序的主要重点是确定定义这些功能差异的结构域和/或单个氨基酸残基。在本应用中要检验的具体假设是，利尿剂亲和力和结合位点的差异位于细胞外连接环，离子运输动力学的差异位于不同的跨膜结构域，调节差异位于细胞内N端和c端结构域。结合分子、生化和生理学方法，我们将a)确定肾噻嗪类敏感Na:CI共转运体的利尿结合位点；b)确定肾噻嗪类敏感Na:CI共转运体中离子亲和力的结构决定因素，c)确定细胞体积和WNK激酶对TSC/NCC调节的结构要求。

DESCRIPTION (provided by applicant): The long-term goal of this 5-year Grant Application is to understand basic issues on the molecular physiology of the thiazide-sensitive Na-CI cotransporter. The Gitleman's disease is the result of inactivating mutations of this gene and this cotransporter is one of the genes that could be implicated in the development of human hypertension. The renal Na-CI cotransporter is the target of the thiazide-type diuretics, which are among the most commonly prescribed drugs in the world. Moreover, the expression of the thiazide-sensitive cotransporter is highly regulated by multiple factors that are known to modulate the renal excretion of sodium. Thus, the renal Na-CI cotransporter is of major importance in renal physiology, pharmacology, and pathophysiology. Using a functional expression strategy in X. laevis oocytes, we have demonstrated important differences between the mammalian and fish thiazide-sensitive Na-CI cotransporter in the specificity and kinetic properties for ion translocation, the diuretic binding affinity and the response to regulation by cell volume and by WNK4 kinase. The major focus of this Application is to determine the domains and/or single amino acid residues defining these functional differences. The specific hypothesis to be examined in this application are that differences in diuretic affinity and binding site are located in the extracellular connecting loops, that differences in ion transport kinetics are located within diverge transmembrane domains and that regulatory differences are located in the intracellular N- and C-terminal domain. Using a combination of molecular, biochemical, and physiological approaches, we will a) identify the diuretic binding site of the renal thiazide-sensitive Na:CI cotransporter; b) identify the structural determinants of ion affinity in the renal thiazide-sensitive Na:CI cotransporter, and c) identify the structural requirements for TSC/NCC regulation by cell volume and by WNK kinases.