MOLECULAR ARCHITECTURE OF THE NA+/GLUCOSE COTRANSPORTER

共转运蛋白是一类重要的膜蛋白 用于糖、氨基酸、多肽、神经递质的积累 以及细胞内的离子。现在已知这些蛋白质也可以共同运输水分。 (每周期200-500个分子)和尿素等极性小分子。这个 底物传输的能量来自于Na+(或H=) 跨质膜的电化学势梯度。我们的目标是 为了了解联合转运体如何利用从 下坡Na+运输驱动上坡基质和水分运输。 利用肠道Na+/葡萄糖共转运体(SGLT1)和一种细菌 同源(Vibrio SGLT)，我们计划识别和确定架构 Na+和底物的转运途径，并确定Na+如何 改变SGLTS的构象以将Na+转运偶联到糖和 水路运输。实验策略是表示被截断的部分 非洲爪哇卵母细胞和细菌中的共转运蛋白，以及 确定蛋白质的哪些部分保留部分反应，例如Na+ Uniport，糖Uniport，Na+/糖共转运，水或尿素 运输。一旦发现蛋白质的部分呈现部分 运输反应时，我们将使用分子、生物物理相结合的方法 和生化技术来确定它们的结构以及它们如何 互动。初步研究表明，SGLT1的C末端部分 可以作为葡萄糖单一转运体，我们已经确定了几个 在Na+/葡萄糖共同作用过程中，该结构域中与糖相互作用的残基 运输。计划是定位糖运输过程中的其他残留物 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/水偶联的转运体和结构信息，以及 为获得更高分辨率的结构奠定了基础。

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.