MOLECULAR ARCHITECTURE OF THE NA+/GLUCOSE COTRANSPORTER

NA/葡萄糖协同转运蛋白的分子结构

• 批准号: 6177002
• 负责人: ERNEST M WRIGHT
• 金额: $ 21.15万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177002
• 关键词: Vibrio; Xenopus; Xenopus oocyte; bacterial proteins; gastrointestinal absorption /transport; glucose transport; glucose transporter; metal metabolism; microorganism culture; microorganism metabolism; molecular cloning; protein structure function; recombinant proteins; sodium; water channel

Co-transporters are an important class of membrane proteins responsible for the accumulation of sugars, amino acids, peptides, neurotransmitters and ions in cells. These proteins are now known to also co-transport water (200-500 molecules per cycle) and small polar molecules such as urea. The energy for the substrate transport comes from the Na+ (or H=) electrochemical potential gradient across the plasma membrane. Our goal is to understand how co-transporters use the free energy released from downhill Na+ transport to drive uphill substrate and water transport. Using the intestinal Na+/glucose co-transporter (SGLT1), and a bacterial homolog (Vibrio SGLT), we plan to identify and determine the architecture of the Na+ and substrate transport pathways, and to determine how Na+ changes the conformation of SGLTs to couple Na+ transport to sugar and water transport. The experimental strategy is to express truncated parts of the co-transporters in Xenopus laevis oocytes and bacteria, and to determine which parts of the protein retain partial reactions, e.g. Na+ uniport, sugar uniport, Na+/sugar co-transport, and water or urea transport. Once parts of the protein are found that exhibit the partial transport reactions, we will use a combination of molecular, biophysical and biochemical techniques to determine their structure and how they interact. Preliminary studies indicate that the C-terminal part of SGLT1 can function as a glucose uniporter, and we have identified several residues in this domain that interact with sugar during Na+/glucose co- transport. The plan is to locate the other residues in the sugar transport pathway, and carry out similar studies on the Na+ transport pathway. These studies will provide a low resolution topological map of the co- transporter and structural information about Na+/sugar/water coupling, and lay the ground work for obtaining higher resolution structures.

共转运蛋白是一类负责的膜蛋白 糖，氨基酸，肽，神经递质的积累 和细胞中的离子。现在已知这些蛋白质也可以共同运输水 （每个周期200-500个分子）和小极性分子，例如尿素。这 基板运输的能量来自Na+（或H =） 整个质膜的电化学潜在梯度。我们的目标是 了解共同转运者如何使用从 下坡NA+运输以驱动上坡基材和水运输。 使用肠道Na+/葡萄糖共转运蛋白（SGLT1）和细菌 同源物（Vibrio SGLT），我们计划识别和确定体系结构 Na+和底物传输途径，并确定Na+的方式 将sglts的构象更改为将Na+转运为糖和糖的构象和 水运。实验策略是表达截短的零件 Xenopus laevis卵母细胞和细菌中的共转运蛋白 确定蛋白质的哪些部分保留部分反应，例如Na+ Uniport，Sugar Uniport，Na+/Sugar Co-Transport以及水或尿素 运输。一旦发现蛋白质的部分显示出部分 运输反应，我们将使用分子，生物物理的组合 和生化技术以确定其结构以及如何确定它们 相互影响。初步研究表明SGLT1的C末端部分 可以用作葡萄糖单位剂，我们已经确定了几个 该结构域中的残留物与Na+/葡萄糖共糖相互作用 运输。计划是在糖运输中找到其他残留物 途径，并在NA+传输途径上进行类似的研究。这些 研究将提供较低的分辨率拓扑图。 有关Na+/糖/水偶联的运输蛋白和结构信息，以及 为获得更高分辨率结构的地面工作。