MOLECULAR MECHANISM OF RENAL NA+-DEPENDENT COTRANSPORT

拟议研究的长期目标是确定 Na+依赖性共转运蛋白的结构影响其功能 性质，并确定Na+依赖的分子机制 共转运，通过研究克隆肾Na+依赖性共转运蛋白。 的 Na+依赖性转运或有机溶质是 肾脏在保存必需的营养物质，这些营养物质是从肾脏中过滤出来的。 血 Na+依赖性有机溶质协同转运蛋白主要是 在近曲小管细胞的顶膜上发现。 的 肾脏的许多Na+依赖性转运蛋白的性质已经被 研究，但目前还没有关于运输机制的信息。 在分子水平上。 随着重组DNA技术的发展， 技术，现在有可能分离编码肾Na+- 依赖的共转运蛋白，并研究其功能和结构 特征更详细。 现在看来，许多Na+- 依赖性共转运蛋白在结构上相关，并形成 蛋白质，包括SGLT家族，与肠道 Na+/葡萄糖协同转运蛋白（SGLT 1）。 拟议的研究将侧重于 克隆的肾转运蛋白是SGLT家族的成员： Na+/核苷协同转运蛋白（SNST 1），一种假定的转运蛋白，RK-D， 最近被测序，其中一个肾脏Na+/葡萄糖协同转运蛋白， SGLT 1. 本建议的第一个具体目标是审查 两个克隆SNST 1和RK-D在表达中的转运特性 系统包括非洲爪蟾卵母细胞、COS细胞和Sf 9细胞。 的方法 纯化含有表达的克隆的质膜， 为了能够进行更精确的动力学测定而开发。 第二个具体目的是确定SNST 1的组织分布 用北方和Western印迹法检测RK-D。 这些信息将帮助 以确定这些转运蛋白在体内的生理作用。 第三个具体的目的是检查翻译后的影响 修饰，如磷酸化和糖基化，对 SNST 1和RK-D的转运。 这些变化，特别是 磷酸化，可能是重要的调节功能，这些 运输机 最后，第四个具体目标是确定 参与赋予差异的转运蛋白区域 在底物选择性方面。 最后一节的实验将是 通过在这些结构上相关的蛋白质之间构建嵌合蛋白质， 运输机 这些研究应提供以下方面的基本信息： 这些Na+依赖性转运蛋白的功能特性，以及 这些转运蛋白在肾脏中的生理作用。

The long term goals of the proposed research are to determine how the structures of Na+-dependent cotransport proteins affect their functional properties, and to identify the molecular mechanism of Na+-dependent cotransport, by studying cloned renal Na+-dependent cotransporters. The Na+-dependent transport or organic solutes is central to the function of the kidney in conserving essential nutrients that are filtered from the blood. The Na+-dependent organic solute cotransporters are predominantly found on the apical membranes of the cells of the proximal tubule. The properties of many Na+-dependent transporters of the kidney have been studied, but there is as yet no information on the mechanism of transport at the molecular level. With the development of recombinant DNA techniques, it is now possible to isolate cDNAs that code for renal Na+- dependent cotransport proteins and to study their function and structural characteristics in greater detail. It now appears that many Na+- dependent cotransporters are structurally related, and form families of proteins, including the SGLT family, related to the intestinal Na+/glucose cotransporter (SGLT1). The proposed research will focus on cloned renal transport proteins that are members of the SGLT family: the Na+/nucleoside cotransporter (SNST1), a putative transporter, RK-D, that was recently sequenced, and one of the renal Na+/glucose cotransporters, SGLT1. The first specific aim of this proposal is to examine the transport properties of two of the clones, SNST1 and RK-D, in expression systems including Xenopus oocytes, COS cells and Sf9 cells. A method of purifying plasma membranes containing the expressed clones will be developed in order to be able to do more precise kinetic determinations. The second specific aim is to determine the tissue distribution of SNST1 and RK-D by Northern and Western blotting. This information will help to determine the physiological role of these transporters in the body. The third specific aim is to examine the effects of post-translational modifications, such as phosphorylation and glycosylation, on the transport of SNST1 and RK-D. These modifications, particularly phosphorylation, may be important in regulating the function of these transporters. Finally, the fourth specific aim is to determine the regions of the transporters that are involved in conferring differences in substrate selectivity. The experiments in this final section will be done by constructing chimeric proteins between these structurally related transporters. These studies should provide fundamental information on the functional properties of these Na+-dependent transporters, and on the physiological role of these transporters in the kidney.