MOLECULAR MECHANISMS OF PROXIMAL TUBULAR ION TRANSPORT

该项目的总体目标是表征分子机制。 钠、氯和酸碱等效物跨细胞转运的基础 (即H，OH，HCO3)在哺乳动物近端小管中。其中一部分 提案将继续努力，详细分析 兔细胞微绒毛(腔)膜囊泡中的Na-H交换器。 这些研究将研究与内部H相互作用的动力学， Na、Li和NH4；NH4的输送；Na-Li交换； 反应机理为乒乓球或同时反应。的第二部分。 提案将检查Na-H交换器的生化特征和 将涉及使用特定于组的试剂来识别化学物质 阳离子结合部位的基团(S)并标记阳离子结合部位， 一种增溶交换剂的方法的开发 在蛋白脂质体中重组其活性，以及这一功能的使用 重组法监测增溶Na-H的纯化 蛋白质分离过程中的交换器。的第三部分 该项目将评估氯-HCO3交换器的传输性能 我们已经在狗的肾微绒毛膜中发现了它，并将涉及 分析H的可能修饰效应，即相互作用的动力学 对于内部和外部的氯和HCO3，对其他阴离子的专一性， 以及抑制剂的特异性。提案的第四部分将 氯气、氢氧化氢和碳酸氢在基侧膜中的迁移途径 从狗和兔肾中分离出的囊泡。这些实验将 K偶联的导电路径和阴离子交换机制的筛子 氯气、氢氧化氢和碳酸氢钠的迁移过程和钠偶联迁移过程。 给出了钠、氯和酸碱等效物的共同传输机制 似乎是在渗漏的上皮细胞中运作的，拟议中的研究是相关的 要了解生理学和病理生理学不仅是 近端小管，也有许多其他上皮细胞。此外，计划中的 Na-H交换器的动力学和生物化学研究很多。 与细胞生理学有关，因为Na-H的重要作用 非上皮细胞中的交换器在以下重要过程中发挥作用 细胞内pH调节、细胞体积调节与细胞调节 扩散。

The overall aim of the project is to characterize the molecular mechanisms underlying the transcellular transport of Na, Cl and acid-base equivalents (ie. H, OH, HCO3) in the mammalian proximal tubule. One portion of the proposal will continue our efforts to analyze in detail the kinetics of the Na-H exchanger in rabbit cell microvillus (luminal) membrane vesicles. These studies will investigate the kinetics of interaction with internal H, Na, Li and NH4; the transport of NH4; Na-Li exchange; and whether the reaction mechanism is ping-pong or simultaneous. A second portion of the proposal will examine the biochemical features of the Na-H exchanger and will involve the use of group-specific reagents to identify the chemical group(s) at the cation binding site and to label the cation binding site, the development of a method for solubilizing the exchanger and reconstituting its activity in proteoliposomes, and the use of this reconstitution assay to monitor purification of the solubilized Na-H exchanger during protein separation procedures. A third portion of the project will evaluate the transport properties of the Cl-HCO3 exchanger that we have identified in dog renal microvillus membranes and will involve analyzing the possible modifier effects of H, the kinetics of interaction with internal and external Cl and HCO3, the specificity for other anions, and the specificity for inhibitors. A fourth portion of the proposal will examine the transport pathways for Cl, OH and HCO3 in basolateral membrane vesicles isolated from dog and rabbit kidneys. These experiments will screen for conductive pathways, anion exchange mechanisms, K-coupled transport processes and Na-coupled transport processes for Cl, OH and HCO3. Given the common transport mechanisms for Na, Cl, and acid-base equivalents that seem to operate in leaky epithelia, the proposed studies are relevant to understanding the physiology and pathophysiology not only of the proximal tubule but of many other epithelia as well. Moreover, the planned studies on the kinetics and biochemistry of the Na-H exchanger are highly relevant to cell physiology in general, given the important roles that Na-H exchangers in nonepithelial cells play in such important processes as intracellular pH regulation, cell volume regulation and regulation of cell proliferation.