Glucose-derived glycolytic ATP is a crucial source of cellular fuel during hypoxia or ischemia. Some cells respond acutely to metabolic stress by activating the cell surface glucose transport protein - GluT1. The broad goal of this research program is to understand how GluT1 is activated and why only some GluT1-expressing cells respond in this way. Our efforts focus on 3 questions. 1) Which sugar transport steps are regulated? 2) What signals control sugar transport? 3) How do these signals control sugar transport? We address these questions in """"""""responder"""""""" cells (Clone 9 cells, cardiomyocytes, human red blood cells, K562 and HEK cells) where GluT1-mediated sugar transport is regulated and in """"""""nonresponders"""""""" (CHO cells) that lack regulation of GluT1-mediated sugar transport. Red cells and K562 cells contain a sugar binding complex that traps (occludes) newly imported sugars at the cytoplasmic membrane surface prior to release into cytosol. This complex is GluT1. ATP-GluT1 interactions promote substrate occlusion and inhibit sugar uniport but not antiport. We propose that ATP-dependent substrate occlusion causes sugar transport inhibition.
In Specific Aim 1, we compare the thermodynamics of sugar uniport, antiport and occlusion in cells containing or lacking ATP to test the hypothesis that ATP alters the transport pathway by way of ATP dependent sugar occlusion.
Specific Aim 2 measures transport over sub second intervals to test the hypothesis that ATP does not affect the sugar translocation step of transport.
Specific Aim 3 subjects GluT1 to differential chemical tagging/peptide mapping/sequence analysis to identify ATP-dependent substrate occlusion domains.
Specific Aim 4 monitors (by surface plasmon resonance) and reconstitutes GluT1 interactions with extracts from basal and metabolically stressed responder and nonresponder cells to answer the question: """"""""Do specific cellular factors modify GluT1 sensitivity to ATP?"""""""" Specific Aim 5 tests the hypothesis that GluT1 contains 2 classes of ATP binding sites (low affinity, occlusion-promoting sites and high affinity, H+-sensitive, occlusion-inhibiting sites) by measuring the pH dependence of GluT1 ATP-binding and ATP-dependent GluT1 substrate occlusion.
Specific Aim 6 tests the hypothesis that the AMP kinase agonist ZMP interacts directly with GluT1 by analysis of the effect of ZMP on GluT1 ATP binding.
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