MOLECULAR ARCHITECTURE OF THE NA+/GLUCOSE COTRANSPORTER

共转运蛋白是一类重要的膜蛋白， 糖、氨基酸、肽、神经递质的积累 和细胞中的离子。这些蛋白质现在已知也共同运输水 （每循环200-500个分子）和小极性分子如尿素。的 基质传输的能量来自Na+（或H=） 跨质膜的电化学电位梯度。我们的目标是 了解协同转运体如何利用 Na+的下行运输驱动基质和水分的上行运输。 使用肠Na+/葡萄糖协同转运蛋白（SGLT 1）和细菌 同源物（弧菌SGLT），我们计划确定和确定的架构 Na+和底物转运途径，并确定Na+ 改变SGLT的构象，将Na+转运偶联到糖， 水上运输。实验策略是表达截断部分 非洲爪蟾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+ 单端口、糖单端口、Na+/糖共转运和水或尿素 运输一旦发现蛋白质的某些部分表现出 运输反应，我们将使用分子，生物物理 和生物化学技术来确定它们的结构， 互动.初步研究表明，SGLT 1的C-末端部分 可以作为葡萄糖单向转运体，我们已经确定了几个 在Na+/葡萄糖共-过程中与糖相互作用的残基。 运输我们的计划是找到糖运输过程中的其他残留物 途径，并对Na+转运途径进行类似的研究。这些 研究将提供一个低分辨率的拓扑图， 关于Na+/糖/水偶联的转运蛋白和结构信息，以及 为获得更高分辨率的结构奠定基础。

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.