GLUT2 plays a major role in the absorption of nutrient hexoses, being expressed constitutively in the BLM and transiently in the apical membrane. This study's objective was to characterize motifs within TM 7 of GLUT2 which determine substrate specificity. When expressed in Xenopus oocytes, hGLUT1 transports fructose (FRU) at a rate of only 0.1% relative to glucose (GLU), while in GLUT2 they are equivalent. Mutating the NAV motif to NAI in GLUT1 increased FRU transport to approximately 0.9% (2.5 pmoles/oocyte/30 min with 100 μM substrate) relative to that of GLU. Mutating the molecular filter QLS in hGLUT1 to HVA also increased the relative rate of FRU transport (up to 0.5%) but to a lesser extent than the NAI mutation. Further, the combined hGLUT1 HVA‐NAI mutant had a relative FRU transport rate of 1.1 % (6 pmoles/oocyte/30 min at 100 μM substrate). Kinetic analysis for the hGLUT1 HVA‐NAI revealed that the Km for GLU was comparable to that of hGLUT1wt (1.5mM), while the Km for FRU was comparable to that for hGLUT2 (~70 mM).Thus, we have successfully introduced hGLUT2‐like FRU kinetics into hGLUT1, without affecting the GLU transport by this isoform. These results point to the presence of, at least two, selectivity filters within class I hGLUTs. NAV/NAI is the exofacial access filter, while QLS/HVA seems to allow FRU translocation beyond the substrate binding site without influencing GLU binding and/or translocation.
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