Mutation of Trp(29) of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity

[摘要]：hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved. Mutations at Trp(29) were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp(29) mutations significantly reduced the apparent V-max and/or increased the apparent K-m values for adenosine transport. Trp(29) mutations increased the IC50 values for hENT1 inhibition by dipyridamole, dilazep, NBMPR, soluflazine and draflazine. NBMPR and soluflazine displayed remarkably similar trends, with large aromatic substitutions at residue 29 resulting in the lowest IC50 values, suggesting that both drugs could interact via ring-stacking interactions with Trp(29). The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L4421 mutant that displayed a selective loss of purine nucleoside transport. W29T, L4421 and the double mutant W29T/L4421 were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp(29) is positioned close to Met(33), implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp(29) forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine.

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