MOLECULAR MECHANISMS OF PROXIMAL TUBULAR ION TRANSPORT

该项目的总体目标是表征分子机制 作为Na、Cl和酸碱等价物跨细胞转运的基础 （即。H，OH，HCO3）。 的一部分 建议将继续我们的努力，详细分析动力学的 兔细胞微绒毛（腔）膜囊泡中的Na-H交换器。 这些研究将调查与内部H相互作用的动力学， Na、Li和NH 4; NH 4的运输; Na-Li交换;以及 反应机理为乒乓或同时进行。 的第二部分 该提案将研究Na-H交换器的生物化学特征， 将涉及到使用特定的化学试剂来识别化学物质 基团并标记阳离子结合位点， 开发了一种溶解交换剂的方法， 在蛋白脂质体中重建其活性，以及这种蛋白脂质体的用途， 用于监测溶解的Na-H的纯化的重构测定 蛋白质分离过程中的交换器。 的第三部分 该项目将评估Cl-HCO 3交换器的传输特性 我们已经在狗肾微绒毛膜中发现， 分析了H可能的改性作用， 与内部和外部Cl和HCO 3，对其他阴离子的特异性， 和对抑制剂的特异性。 提案的第四部分将 检查基底外侧膜中Cl、OH和HCO 3的转运途径 从狗和兔肾脏分离的囊泡。 这些实验将 筛选传导途径，阴离子交换机制，K-耦合 Cl、OH和HCO 3的迁移过程和Na耦合迁移过程。 考虑到Na、Cl和酸碱当量的常见转运机制， 似乎在渗漏的上皮细胞中起作用， 不仅能理解人类的生理学和病理生理学 近端小管，但也有许多其他上皮细胞。 此外，计划 对Na-H交换剂的动力学和生物化学的研究是高度 与一般细胞生理学相关，鉴于Na-H 非上皮细胞中的交换体在以下重要过程中起作用， 细胞内pH调节、细胞体积调节和细胞调节 增殖

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.