H/HCO3 transport in the collecting duct

描述（由申请人提供）：氨代谢对正常健康至关重要。肾中氨代谢不当导致代谢性酸中毒，肝中氨代谢不当导致氨性脑病。在中枢神经系统中，细胞外氨的增加改变了神经元的功能，并可导致脑病。因此，了解氨代谢的细胞和分子机制，包括氨运输，是很重要的。最近的研究已经确定了一个新的家庭铵离子（NH 4+）特异性转运。这些蛋白质首先在酵母和植物中被鉴定，并且同源物存在于整个自然界中。在模型系统中，如酵母、植物和细菌，这些是介导高亲和力、铵特异性转运的内在膜蛋白，并且其表达受到生理调节。这些蛋白质中的两种，RhBG和RhCG在肾脏的连接段和集合管中表达，并且表现出极化表达。这些观察结果使我们假设RhBG和RhCG是完整的膜，生理调节的铵离子转运蛋白，在肾氨代谢中发挥关键作用。该项目的广泛，长期目标是确定RhBG和RhCG在哺乳动物肾脏生理学中的作用。为此，本提案的具体目的是：（1）确定小鼠肾RhBG和RhCG表达和囊泡运输的调节，以响应与肾氨代谢改变相关的特定临床状况;（2）确定细胞外氨刺激的RhBG和RhCG介导的离子转运变化的机制;以及，（3）识别RhBG和RhCG的特定离子传输特征。我们将利用改变肾氨代谢、代谢性酸中毒和水肿以及低钾血症的体内动物模型来确定RhBG和RhCG表达的调节和细胞定位，培养的集合管细胞系，mIMCD-3，用于体外研究检查RhBG和RhCG介导的转运调节的细胞机制，和异源表达系统，其中定义了RhBG和RhCG的特异性离子转运特征。

DESCRIPTION (provided by applicant): Ammonia metabolism is critical for normal health. Inappropriate ammonia metabolism in the kidney leads to metabolic acidosis and in the liver leads to ammonia encephalopathy. In the central nervous system increased extracellular ammonia alters neuronal function and can lead to encephalopathy. Accordingly, understanding the cellular and molecular mechanisms of ammonia metabolism, which includes ammonia transport, is important. Recent studies have identified a novel family of ammonium ion (NH4+)-specific transporters. These proteins were first identified in yeast and in plants, and homologues are present throughout nature. In model systems, such as yeast, plants and bacteria, these are intrinsic membrane proteins that mediate high-affinity, ammonium-specific transport and whose expression is physiologically regulated. Two of these proteins, RhBG and RhCG are expressed in the connecting segment and the collecting duct of the kidney, and exhibit polarized expression. These observations lead us to postulate that RhBG and RhCG are integral membrane, physiologically-regulated ammonium-ion transporters that play critical roles in renal ammonia metabolism. The broad, long-term objectives of this project are to define the roles of RhBG and RhCG in mammalian renal physiology. To do so, the Specific Aims of the current proposal are to: (1) Define the regulation of mouse renal RhBG and RhCG expression and vesicular trafficking in response to specific clinical conditions associated with altered renal ammonia metabolism; (2) Determine the mechanism of extracellular ammonia-stimulated changes in RhBG- and RhCG-mediated ion transport; and, (3) identify the specific ion-transport characteristics of RhBG and RhCG. We will utilize in vivo animal models of altered renal ammonia metabolism, metabolic acidosis and alkalosis and hypokalemia, to define the regulation of RhBG and RhCG expression and cellular localization, a cultured collecting duct cell line, mIMCD-3, for in vitro studies examining the cellular mechanisms underlying regulation of RhBG and RhCG-mediated transport, and heterologous expression systems in which to define the specific ion transport characteristics of RhBG and RhCG.