Mutation of Trp29 of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity.
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 Trp29 were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp29 mutations significantly reduced the apparent V(max) and/or increased the apparent K(m) values for adenosine transport. Trp29 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 Trp29. The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L442I mutant that displayed a selective loss of purine nucleoside transport. W29T, L442I and the double mutant W29T/L442I were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp29 is positioned close to Met33, implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp29 forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine.
Paproski
Robert J
RJ
Membrane Protein Research Group, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.
Visser
Frank
F
Zhang
Jing
J
Tackaberry
Tracey
T
Damaraju
Vijaya
V
Baldwin
Stephen A
SA
Young
James D
JD
Cass
Carol E
CE
D000241:Adenosine / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D004109:Dilazep / Q000494:pharmacology; D004176:Dipyridamole / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D033721:Equilibrative Nucleoside Transporter 1 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D009705:Nucleosides / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011485:Protein Binding; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology*
D033721:Equilibrative Nucleoside Transporter 1; D009705:Nucleosides; D010879:Piperazines; D014665:Vasodilator Agents; C072040:draflazine; C054695:soluflazine; D013868:Thioinosine; D004176:Dipyridamole; D014364:Tryptophan; D004109:Dilazep; C001789:4-nitrobenzylthioinosine; D000241:Adenosine