Mutation of Trp29 of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity
Mutation of Trp29 of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity
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DOI：
10.1042/bj20080074
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发表时间：
2008-09-01
影响因子：
4.1
通讯作者：
Cass, Carol E.
中科院分区：
文献类型：
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作者：
Paproski, Robert J.;Visser, Frank;Cass, Carol E.
hENT 1（人平衡核苷转运蛋白1）被纳摩尔浓度的各种结构不同的冠状血管扩张剂药物抑制，包括双嘧达莫、地拉卓、德拉氟嗪、索拉氟嗪和NBMPR（硝基苄基巯基嘌呤核糖核苷）。当使用基于酵母的功能互补测定法筛选随机突变的hENT 1 cDNA文库时，鉴定出含有W29 G突变的克隆。多重序列比对显示该残基高度保守。产生Trp（29）处的突变并测试腺苷转运活性和抑制剂敏感性。Trp（29）突变显著降低腺苷转运的表观Vmax和/或增加表观Km值。Trp（29）突变增加了双嘧达莫、地拉卓、NBMPR、solufrazine和draflazine对hENT 1抑制的IC 50值。NBMPR和solufrazine显示出非常相似的趋势，在残基29处具有大的芳族取代，导致最低的IC 50值，表明两种药物可以通过与Trp的环堆积相互作用相互作用（29）。W29 T突变体显示嘧啶核苷转运活性的选择性丧失，这与先前鉴定的显示嘌呤核苷转运选择性丧失的L4421突变体形成对比。对W29 T、L4421和双突变体W29 T/L4421的核苷转运活性进行了动力学表征。TM（跨膜片段）1的螺旋轮投影表明，Trp（29）位于靠近Met（33）的位置，先前涉及核苷和抑制剂识别，并且这两个残基排列在渗透性易位途径中。数据还表明，Trp（29）形成了双嘧达莫、地拉卓、NBMPR、soloflazine和draflazine的结合位点的一部分或接近这些结合位点。
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.