title: Mutation of residue 33 of human equilibrative nucleoside transporters 1 and 2 alters sensitivity to inhibition of transport by dilazep and dipyridamole.
abstract: Human equilibrative nucleoside transporters (hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast expression plasmid containing the hENT1 cDNA was randomly mutated and screened by phenotypic complementation in Saccharomyces cerevisiae to identify mutants with reduced sensitivity to dilazep, clones with a point mutation that converted Met33 to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevis oocytes revealed that the mutant had less than one-tenth the sensitivity to dilazep and dipyridamole than wild type hENT1, with no change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity or apparent uridine affinity. To determine whether the reciprocal mutation in hENT2 (Ile33 to Met) also altered sensitivity to dilazep and dipyridamole, hENT2-I33M was created by site-directed mutagenesis. Although the resulting mutant (hENT2-I33M) displayed >10-fold higher dilazep and dipyridamole sensitivity and >8-fold higher uridine affinity compared with wild type hENT2, it retained insensitivity to NBMPR. These data established that mutation of residue 33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep and dipyridamole sensitivities in both proteins, suggesting that a common region of inhibitor interaction has been identified.
authors.affiliation: Canadian Institutes of Health Research Group in the Molecular Biology of Membrane Proteins, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada.
authors.forename: Frank
authors.initials: F
authors.lastname: Visser
authors.forename: Mark F
authors.initials: MF
authors.lastname: Vickers
authors.forename: Amy M L
authors.initials: AM
authors.lastname: Ng
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Baldwin
authors.forename: James D
authors.initials: JD
authors.lastname: Young
authors.forename: Carol E
authors.initials: CE
authors.lastname: Cass
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D004109:Dilazep / Q000494:pharmacology*; D004176:Dipyridamole / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014529:Uridine / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C438649:SLC29A1 protein, human; C438654:SLC29A2 protein, human; C438652:Slc29a1 protein, rat; C486681:Slc29a2 protein, mouse; C438655:Slc29a2 protein, rat; D013868:Thioinosine; D004176:Dipyridamole; D004109:Dilazep; C001789:4-nitrobenzylthioinosine; D014529:Uridine