Adenosine and adenosine triphosphate modulate the substrate binding affinity of glucose transporter GLUT1 in vitro

Evidence indicates that a large portion of the facilitative glucose transpo rter isoform GLUT1 in certain animal cells is kept inactive and activated i n response to acute metabolic stresses. A reversible interaction of a certa in inhibitor molecule with GLUT1 protein has been implicated in this proces s. In an effort to identify this putative GLUT1 inhibitor molecule, we stud ied here the effects of adenosine and adenosine triphosphate (ATP) on the b inding of D-glucose to GLUT1 by assessing their abilities to displace cytoc halasin B (CB), using purified GLUT1 in vesicles. At pH 7.4, adenosine comp etitively inhibited CB binding to GLUT1 and also reduced the substrate bind ing affinity by more than an order of magnitude, both with an apparent diss ociation constant (K-D) of 3.0 mM. ATP had no effect on CB and D-glucose bi nding to GLUT1, but reduced adenosine binding affinity to GLUT1 by 2-fold w ith a K-D of 30 mM. At pH 3.6, however, ATP inhibited the CB binding nearly competitively, and increased the substrate binding affinity by 4-5-fold, b oth with an apparent K-D of 1.22 mM. These findings clearly demonstrate tha t adenosine and ATP interact with GLUT1 in vitro and modulate its substrate binding affinity. They also suggest that adenosine and ATP may regulate GL UT1 intrinsic activity in certain cells where adenosine reduces the substra te-binding affinity while ATP increases the substrate-binding affinity by i nterfering with the adenosine effect and/or by enhancing the substrate-bind ing affinity at an acidic compartment. (C) 2001 Elsevier Science B.V. All r ights reserved.