Reconciling contradictory findings: Glucose transporter 1 (GLUT1) functions as an oligomer of allosteric, alternating access transporters

Abstract

Recent structural studies suggest that GLUT1 (glucose transporter 1)-mediated sugar transport is mediated by an alternating access transporter that successively presents exofacial (e2) and endofacial (e1) substrate-binding sites. Transport studies, however, indicate multiple, interacting (allosteric), and co-existent, exo- and endofacial GLUT1 ligand-binding sites. The present study asks whether these contradictory conclusions result from systematic analytical error or reveal a more fundamental relationship between transporter structure and function. Here, homology modeling supported the alternating access transporter model for sugar transport by confirming at least four GLUT1 conformations, the so-called outward, outward-occluded, inward-occluded, and inward GLUT1 conformations. Results from docking analysis suggested that outward and outward-occluded conformations present multiple β-d-glucose and maltose interaction sites, whereas inward-occluded and inward conformations present only a single β-d-glucose interaction site. Gln-282 contributed to sugar binding in all GLUT1 conformations via hydrogen bonding. Mutating Gln-282 to alanine (Q282A) doubled the <i>K_m</i>_(app) for 2-deoxy-d-glucose uptake and eliminated <i>cis</i>-allostery (stimulation of sugar uptake by subsaturating extracellular maltose) but not <i>trans</i>-allostery (uptake stimulation by subsaturating cytochalasin B). <i>cis</i>-Allostery persisted, but <i>trans</i>-allostery was lost in an oligomerization-deficient GLUT1 variant in which we substituted membrane helix 9 with the equivalent GLUT3 sequence. Moreover, Q282A eliminated <i>cis</i>-allostery in the oligomerization variant. These findings reconcile contradictory conclusions from structural and transport studies by suggesting that GLUT1 is an oligomer of allosteric, alternating access transporters in which 1) <i>cis</i>-allostery is mediated by intrasubunit interactions and 2) <i>trans</i>-allostery requires intersubunit interactions.