Molecular Architecture of the Na+/Glucose Cotransporter

描述(申请人提供)：钠/葡萄糖共转运体(SGLT1)是典型的共转运体，它利用Na+跨膜电化学势梯度中储存的能量将营养物质输送到细胞内。SGLT1主要表达在小肠的刷状缘膜上，在那里它负责每天吸收180g饲料中的葡萄糖和半乳糖。SGLT1对糖、盐、水的转运耦合为口服补液疗法治疗感染性腹泻提供了理论依据。SGLT1编码基因的突变会导致葡萄糖-半乳糖吸收不良。该项目的目标是确定SGLT1是如何结合钠、糖和水的运输的。目前的目标是1)使用ATR-FTIR、CD和荧光等光谱方法来监测和确定配体诱导的蛋白质构象变化的程度，2)利用光亲和标记和质谱学来确定底物结合部位，以及3)利用冷冻断裂电子显微镜来筛选2D和3D晶体来开始长期的研究来解析蛋白质的结构。这些实验将在人SGLT1和相关的转运蛋白上进行，在大肠杆菌中过度表达，纯化并重组为脂质体。这一结果将为钠/糖共转运的交替通道模型提供直接测试。

DESCRIPTION (provided by applicant): The Na+/glucose cotransporter (SGLT1) is the archetypical cotransporter that uses the energy stored in Na+ electrochemical potential gradients across membranes to drive nutrients into cells. SGLT1 is expressed mainly in the brush border membrane of the small intestine where it is responsible for the absorption of 180g of dietary glucose and galactose per day. The coupling of sugar, salt and water transport by SGLT1 provides the rational for the Oral Rehydration Therapy used to treat infectious diarrhea. Mutations in the gene coding for SGLT1 cause Glucose-Galactose-Malabsorption. The goal of this project is to determine how SGLT1 couples sodium, sugar and water transport. The present aims are to 1) use spectroscopic methods such as ATR-FTIR, CD and fluorescence to monitor and determine the extent of ligand induced conformational changes in the protein, 2) identify the substrate- binding sites using photo-affinity labeling and mass spectrometry, and 3) initiate long term studies to resolve the structure of the protein using freeze-fracture electron microscopy to screen for 2D and 3D crystals. These experiments will be carried out on human SGLT1 and related transporters over expressed in E. coli, purified and reconstituted into liposomes. The results will provide a direct test of the alternating access model for sodium/sugar cotransport.