Abstract

The human equilibrative nucleoside transporter 1 (hENT1) is an important modulator of the physiological action of adenosine. We identified amino acid residues involved in adenosine transport using a yeast-based assay to rapidly screen and identify randomly generated hENT1 mutants that exhibited decreased sensitivity to inhibition of adenosine transport by various hENT1 competitive inhibitors. We identified Met89 and Ser160 as important in the affinity of hENT1 for various substrates and inhibitors. Mutation to Met89Cys or Ser160Cys significantly (p < 0.05) increased the S⁶-(4-nitrobenzyl)-mercaptopurine riboside (NBMPR) IC₅₀ values by approximately 4- and 6-fold, respectively (42 ± 13 and 65 ± 1.6 nM) compared with the wild-type transporter (11 ± 0.7 nM). The double mutant Met89Cys/Ser160Cys synergistically increased the NBMPR IC₅₀ value to approximately 19-fold of that of the wild-type transporter. In contrast, compared with wild-type hENT1, the sensitivity to dipyridamole inhibition was significantly (p < 0.05) increased by only the Ser160Cys (∼2.6-fold) or the double mutant Met89Cys/Ser160Cys (∼4.7-fold) but not by the Met89Cys mutant. Mutation to Met89Cys or Ser160Cys increased the K_m of adenosine (∼8- and 3-fold) and the K_i of guanosine (∼6- and 2-fold). The double mutant increased both the K_m value of adenosine and the K_i value of guanosine by ∼8-fold and seemed to confer no additional reduction in adenosine or guanosine affinity than that by mutation of Met89 alone. Together, these data indicate that transmembrane domains (TMDs) 2 (Met89) and 4 (Ser160) of hENT1 interact and are important in conferring sensitivity to NBMPR. In contrast, Ser160 and Met89 of hENT1, respectively, play a dominant role in conferring sensitivity to dipyridamole and adenosine/guanosine affinity.