Molecular Physiology of the renal Na-Cl cotransporter

描述(由申请者提供)：这项为期5年的资助申请的长期目标是了解有关噻嗪敏感的Na-CI共转运体的分子生理学的基本问题。吉特尔曼病是该基因失活突变的结果，而该共转运蛋白是可能与人类高血压的发展有关的基因之一。肾脏Na-CI协同转运体是世界上最常用的处方药物之一--噻嗪类利尿剂的靶标。此外，对噻嗪敏感的辅助转运蛋白的表达受到多种已知调节肾脏钠排泄的因素的高度调控。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。通过在爪哇X.laevis卵母细胞中的功能表达策略，我们已经证明了哺乳动物和鱼类对噻氮化物敏感的Na-CI共转运蛋白在离子转运的特异性和动力学特性、利尿剂结合亲和力以及对细胞体积和WNK4激酶调节的反应方面的重要差异。本申请的主要焦点是确定定义这些功能差异的结构域和/或单一氨基酸残基。在这一应用中需要检验的具体假设是利尿剂亲和力和结合位置的差异位于细胞外连接环，离子转运动力学的差异位于分歧的跨膜结构域，调控差异位于细胞内的N-末端和C-末端区域。结合分子、生物化学和生理学方法，我们将a)确定肾脏对噻嗪敏感的Na：CI共转运体的利尿剂结合部位；b)确定肾脏对噻嗪敏感的Na：CI共转运体中离子亲和力的结构决定因素；以及c)确定通过细胞体积和WNK激酶调节TSC/NCC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.