Received for publication October 12, 2004.
Revised November 19, 2004.
Accepted for publication November 19, 2004.

Residues Met89 and Ser160 in the Human Equilibrative Nucleoside Transporter 1 (hENT1) Affect hENT1's Affinity for Adenosine, Guanosine, NBMPR and Dipyridamole

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 hENT1's affinity for various substrates and inhibitors. Mutation to Met89Cys or Ser160Cys significantly (p<0.05) increased the NBMPR IC₅₀ by approximately 4 and 6-fold respectively (42 ± 13 and 65 ± 1.6 nM) when compared with the wild-type transporter (11 ± 0.7 nM). The double mutant, Met89Cys/Ser160Cys, synergistically increased NBMPR IC₅₀ to approximately 19-fold of the wild-type transporter. In contrast, when compared to wild-type hENT1, the sensitivity to dipyridamole inhibition was significantly (p<0.05) increased by only the Ser160Cys (~2.6-fold) or the double Met89Cys/Ser160Cys mutant (~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 of adenosine and K_i of guanosine by ~8-fold and appeared to confer no additional reduction in adenosine or guanosine affinity than that by mutation of Met89 alone. Collectively, these data indicate that TMD2 (Met89) and TMD4 (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.

Key words: Adenosine, Nucleoside/Nucleotide, Ischemia/Reperfusion