Molecular Physiology of the renal Na-Cl cotransporter

描述（由申请人提供）：本5年资助申请的长期目标是了解噻嗪敏感性NaCl协同转运蛋白分子生理学的基本问题。Gitleman病是该基因失活突变的结果，该协同转运蛋白是可能与人类高血压发展有关的基因之一。肾脏Na-Cl协同转运蛋白是噻嗪类利尿剂的靶标，噻嗪类利尿剂是世界上最常用的处方药之一。此外，噻嗪敏感性协同转运蛋白的表达受到多种已知调节钠的肾排泄的因子的高度调节。因此，肾Na-Cl协同转运蛋白在肾生理学、药理学和病理生理学中具有重要意义。在X.通过对laevis卵母细胞的研究，我们已经证明了哺乳动物和鱼类噻嗪敏感性Na-Cl协同转运蛋白在离子易位的特异性和动力学性质、利尿剂结合亲和力以及对细胞体积和WNK 4激酶调节的响应方面的重要差异。本申请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。在本申请中要检查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中，离子转运动力学的差异位于发散跨膜结构域中，并且调节差异位于细胞内N-和C-末端结构域中。使用分子、生物化学和生理学方法的组合，我们将a）鉴定肾噻嗪敏感性Na：Cl协同转运蛋白的利尿剂结合位点; B）鉴定肾噻嗪敏感性Na：Cl协同转运蛋白中离子亲和力的结构决定因素;和c）鉴定细胞体积和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.