ACTIVATION OF GLUT1 GLUCOSE-TRANSPORTER IN HUMAN ERYTHROCYTES

Glut1, the only glucose transporter isoform expressed in the human red blood cell (RBC), binds to and is inhibited by cytochalasin B (CB). I n the present study we show that incubation of RBC ghost membranes wit h 10 mu M cytochalasin E (CE) results in a 1.8-fold increase in the nu mber of glucose-sensitive cytochalasin B binding sites. Moreover, trea tment with CE was associated with no observable change in the protein composition of RBC ghosts determined by SDS-PAGE. Removal of surface ( ''extrinsic'') proteins from RBC ghosts by treatment with 0.2 mM:EDTA (pH 12) for 30 min resulted in a similar 1.8-fold increase in the numb er of glucose-sensitive CB binding sites. Western blot analysis showed that treatment with CE or EDTA resulted in no change in the amount or mobility of Glut1 present in ghost membranes. Glucose transport, meas ured as CB-inhibitable 3-O-[H-3]methylglucose uptake by resealed ghost s, was stimulated in CE-treated resealed ghosts, with the t(1/2) to eq uilibrium decreasing from 6.8 +/- 0.5 to 3.9 +/- 0.3 s (P < 0.05). Tre atment of ghosts with CE or EDTA followed by Western blotting of sampl es in the presence or absence of beta-mercaptoethanol resulted in no c hange in immunoreactivity or mobility of the major Glut1 band. The abo ve results suggest that a significant fraction of Glut1 transporters e xists in an inactive form (''masked'') in RBC plasma membranes and tha t treatment of ghosts with CE or EDTA leads to an apparent activation of Glut1, (C) 1998 Academic Press.