Molecular Mechanisms of Intestinal Transport

University Of California Los Angeles, Los Angeles CA

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Abstract

DESCRIPTION (provided by applicant): The goal of this project is to understand how sodium, glucose and water are transported across the brush border membrane of the small intestine by the Na+/glucose cotransporter (hSGLT1). This membrane transport protein is responsible for the absorption of 180 g of glucose and galactose and more than 4 liters of fluid per day. Oral Rehydration Therapy, credited with saving thousands of infants a day from infectious diarrhea, is based on the coupling of glucose, salt and water transport by SGLT1. It is postulated that hSGLT1 couples Na+ transport to glucose and water transport by a series of ordered ligand induced conformational changes. Evidence suggests that sodium binding produces a reorganization of four transmemembranes helices near the C-terminus of the protein that permits sugar binding and transport. In order to accommodate the large glycosides that are transported (20 x 12 x 7 Angstroms) and the 300 water molecules that are also transported during one turnover of the transporter, we predict extensive changes in transmembrane helical packing during the transport cycle. To test this hypothesis we will use cysteine scanning mutagenesis along with thiol reactive fluorescent probes to identify which helices move, the order in which they move, and the distances they move during partial reactions of the transport cycle. This will be accomplished by expressing the cysteine mutant transporters in Xenopus laevis oocytes, and the cysteine residues will be labeled with fluorescent thiol reagents. The oocytes will be placed on the stage of an epifluorescence microscope and voltage clamped. Simultaneous recordings of charge movement and fluorescence will be obtained as a function of membrane voltage and the ligand concentrations. We will also use double cysteine mutants and fluorescence and luminescence energy transfer (FRET & LRET) to measure the distance between the probes on the two cysteines and how far they move as the transporter is moved from one conformation to another. This information will enable us to map the changes in helical packing, including the order, time course and magnitude of the helical motion during the transport cycle, and determine if they account for the transport of glucose and water. The impact of the work ranges from a molecular understanding of oral rehydration therapy to how large class of membrane transport proteins work.