Glucose uptake in absorptive epithelia has been demonstrated to occur by a Na- dependent mechanism at the brush border membrane. Identification of the protein responsible for this process, characterization of its two conformations and of the transition between these conformations, and isolation of the functional cotransporter, have led to this proposal. The long-term aim of this project is to examine the molecular mechanism of protein-mediated ion and organic solute transport across plasma membranes. The specific goals of this proposal are three-fold: 1) To examine cotransporter structure, including substrate sites, transport essential non-""""""""channel"""""""" regions (transport essential sulfydryl residue), and transport """"""""channels"""""""", using azido-glucose and TEMPO-glucose as observable glucose analogues and proteolytic digestion. 2) To directly observe transport steps using TEMPO- glucose as fluorescent quenching reagent for both native protein fluorescence and extrinsic probes, to determine energy transfer distance between substrate channels using TEMPO-glucose and fluorescent Na analogues, and to examine the kinetics of Na binding to the cotransporter (Na-induced conformational change, Na off rate, and Na on rate). 3) To combine the structural data with the transport steps to develop a three-dimensional model of Na-dependent glucose uptake. These studies will have a general impact on out understanding of the mechanism of transport of sugars, amino acids, and metabolic intermediates across plasma membranes of the kidney, intestine, liver, and brain. These studies offer a unique opportunity to examine transport at a molecular level.
