The facilitative glucose transporter GLUT1 catalyzes the passive translocation of glucose across plasma membranes. Previous studies have demonstrated that regulation of GLUT1 activity is complex and subject to modulation by a variety of environmental and allosteric effectors. Mathematical models of GLUT1 kinetics have been derived which successfully account for subsets of these factors, though efforts to develop a single kinetic theory has not yet been achieved. Limitations in conventional experimental methodologies cannot provide kinetic data with the precision and continuity needed for further refinement of said models. We utilized a glucose-sensitive fluorescent protein (GlcSnFR) to develop novel experimental methodologies to facilitate elucidation of the GLUT1 kinetic mechanism. Characterization of the kinetic and optic properties of GlcSnFR and glucose binding (kon: 389.5 ± 12.1 M-1s-1, koff: 0.593 ± 0.103 s-1, kD: 1.52 ± 0.31 mM) allowed us to construct analyses which continuously estimate glucose flux through GLUT1 with subsecond temporal resolution and spacial resolution of a single human erythrocyte. We additionally revised the analytic methodology for interpreting the collected kinetic data sets to reduce the a priori assumptions regarding equilibrium states of the GLUT1 enzyme ensemble, which provided more flexible, model-independent characterizations of transporter activity. Using resealed human erythrocyte ghosts containing GlcSnFR, we applied techniques in confocal microscopy and fluorometry to achieve new insights into the kinetics of GLUT1. By microscopy, we observed transport in single cells to achieve conclusive evidence that populations of resealed ghosts are homogeneous in their composition, a previously unachieved result which retroactively validates to a great many previous studies. Further, we confirmed, unambiguously, that ATP induces directional asymmetry in GLUT1 through a reduction in rates of sugar efflux. By fluorometry, we demonstrated that previous measurements of the endofacial KM of GLUT1 for glucose are underestimated (~4 mM) and closer to ~20 mM. Finally, these new methods provide access to a new paradigm for measuring glucose transport in general and will serve to advance the field significantly in years to come.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1968 |
| Date | 23 February 2018 |
| Creators | Simon, Andrew H. |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Licensed under a Creative Commons license, http://creativecommons.org/licenses/by/4.0/ |
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