The PI has reported that both facilitative and Na+ dependent glucose transporters may transport their substrates through an aqueous pore. In addition, he has found that facilitative glucose transporters constitute the route of dehydroascorbic acid entry into cells, which may lead to a better understanding of the mechanisms that protect ocular tissues from oxidative damage. Glucose transporters appear to be gated channel proteins. Since solute transporters are essential to ocular function, he proposes to continue characterizing the structure-function relationship of facilitative glucose transporters (GLUTs) and other membrane proteins as archetypes of a number of solute transporters. He thus proposes: 1) To study GLUT structure by: a) cysteine scanning mutagenesis; function of mutants will be assayed by expression in oocytes; b) by 3-D modeling of proposed folds for GLUT1 such as the alpha-helical, beta-barrel, and mixed alpha-beta. We also propose to explore possible homologies between GLUT1 and a glucose transporter of known structure, maltoporin, as well as with segments of well-studied channel proteins such as aquaporins and potassium channels. 2) To investigate whether the passage of sugars and dehydroascorbic acid through GLUT1 can be ascribed to aromatic interactions with the transporter, in which case mutations that replace aromatic residues in GLUTs might affect the transporter selectivity and kinetics. 3) To characterize indirectly the GLUT channel cross section and binding properties by determining if molecules other than sugars traverse them, we have uncovered evidence that some amino acids (tyrosine, phenylalanine, leucine) traverse GLUT1. The PI proposes to characterize the kinetic parameters for each uptake mechanism, their sensitivity to inhibitors, and the degree to which these substrates can compete with each other, and with glucose and dehydroascorbate uptake. The hydrophobic envelopes of GLUT substrates will be determined by molecular dynamics simulations; this information will be used to deduce the shape and environmental characteristics of GLUT binding sites for these substrates. 4) To continue exploring the geometry and relative hydrophilicity of water permeable pathways in membrane proteins by comparing their water permeation characteristics with those obtained from molecular dynamics simulations and theoretical kinetic analysis of model channels.
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