MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT

University Of California Los Angeles, Los Angeles CA

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Abstract

Our goal is to understand the mechanisms of intestinal ion, nutrient and water transport. Our approach is to express the brush border Na+/glucose contransporter (SGLT1) and fructose transporter (GLUT5) in Xenopus laevis oocytes, and to use a combination of molecular, biochemical and biophysical techniques to study glucose, fructose and water transport. We have demonstrated that SGLT1 transports Na+, glucose and water in a fixed stoichiometry, 2Na+/1 glucose/250 water, and we have proposed that cotransport is due to ligand and voltage- induced conformation changes. A major specific aim is to determine the structural basis of these conformational changes. SGLT1 will be expressed at high levels in oocytes, > 10/11 copies per cell, extrinsic fluorescent probes will be attached to sugar and ion binding sites, and optical methods will be used to probe the structure, and distance between , the sites. These measurements will be made on SGLT1 in different conformations, and during fast (us) changes from one conformation to another. The current model for contransport will be tested by measuring the kinetics and ligand specificity of the transporter working backwards, and comparing the results with novel predictions. A critical thermodynamic test of water cotransport will be to measure the effect of osmotic gradients of the energetics of Na+/ sugar co transport. These experiments will provide novel kinetic and structural information about Na+/sugar/water cotransport. We will examine the oligomeric structure and kinetic mechanism of fructose transport by GLUT5 and determine the role of GLUT5 in water transport. Experiments will be carried out GLUT5 expressed in oocytes, where we will measure fructose transport kinetics using radioactive tracer uptakes, the density and structure of GLUT5 in the plasma membrane using freeze fracture electron microscopy, and partial reactions using voltage-jump experiments. This data will be used to construct a kinetic model for fructose transport. Water transport by GLUT5 in the presence and absence of fructose will be measured by an optical method, and the result will be normalized to the number GLUT5 molecules expressed in the plasma membrane this will allow us to determine the role of GLUT5 in water transport across the brush border membrane.