Molecular Architecture of the Na+/Glucose Cotransporter

描述（由申请人提供）：Na+/葡萄糖共转运体（SGLT1）是一种典型的共转运体，它利用储存在膜上的Na+电化学电位梯度中的能量将营养物质驱动到细胞中。SGLT1主要在小肠刷状缘膜中表达，负责每天180g膳食葡萄糖和半乳糖的吸收。SGLT1对糖、盐和水运输的耦合作用为口服补液疗法治疗感染性腹泻提供了理论依据。编码SGLT1基因的突变导致葡萄糖-半乳糖-吸收不良。这个项目的目标是确定SGLT1如何耦合钠、糖和水的运输。目前的目标是1)使用光谱方法，如ATR-FTIR， CD和荧光来监测和确定配体诱导蛋白质构象变化的程度，2)使用光亲和标记和质谱识别底物结合位点，以及3)启动长期研究，以解决蛋白质结构使用冷冻断裂电子显微镜筛选二维和三维晶体。这些实验将在大肠杆菌中过表达的人SGLT1及相关转运体上进行，纯化并重组为脂质体。结果将为钠/糖共转运的交变通路模型提供直接检验。

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.