Specific mutations in transmembrane helix 8 of human concentrative Na+/nucleoside cotransporter hCNT1 affect permeant selectivity and cation coupling.
Specific mutations in transmembrane helix 8 of human concentrative Na+/nucleoside cotransporter hCNT1 affect permeant selectivity and cation coupling.
复制标题
人浓缩 Na/核苷协同转运蛋白 hCNT1 跨膜螺旋 8 的特定突变影响渗透选择性和阳离子偶联。
DOI：
10.1021/bi061692s
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
J. Young
中科院分区：
文献类型：
--
作者：
Melissa D. Slugoski;Shauna Loewen;A. Ng;Kyla M. Smith;S. Yao;E. Karpinski;C. Cass;S. Baldwin;J. Young
Na+/核苷共转运蛋白hCNT 1（650个残基）和hCNT 2（658个残基）在氨基酸序列上72%相同，并且含有13个推定的跨膜螺旋（TM）。两者都转运尿苷和腺苷，但分别对嘧啶核苷（cit系统）和嘌呤核苷（cif系统）有选择性。以前，我们使用定点诱变和功能表达在非洲爪蟾卵母细胞中，以确定两对相邻的残基在TM 7和8的hCNT 1（Ser 319-Gln 320和Ser 353-Leu 354），当转换为相应的残基在hCNT 2（Gly-Met和Thr-Val，分别），改变了渗透选择性的转运从cit到CIF。我们现在报告的TM 8单独相应的突变的影响的调查，并证明独特的S353 T和L354 V诱导的变化，核苷特异性和阳离子耦合，分别。hCNT 1突变S353 T导致胞苷转运效率（Vmax/Km比值）显著降低，并与L354 V（S353 T/L354 V）结合，导致新的尿苷优先转运表型。此外，L354 V突变显著增加了hCNT 1对Na+和Li+的表观亲和力。两个hCNT 1 TM 8残基表现出尿苷保护抑制对氯汞苯磺酸盐转化为半胱氨酸时，表明它们占据的位置内或紧邻一个共同的阳离子/核苷易位孔。
The Na+/nucleoside cotransporters hCNT1 (650 residues) and hCNT2 (658 residues) are 72% identical in amino acid sequence and contain 13 putative transmembrane helices (TMs). Both transport uridine and adenosine but are otherwise selective for pyrimidine (system cit) and purine (system cif) nucleosides, respectively. Previously, we used site-directed mutagenesis and functional expression in Xenopus oocytes to identify two pairs of adjacent residues in TMs 7 and 8 of hCNT1 (Ser319-Gln320 and Ser353-Leu354) that, when converted to the corresponding residues in hCNT2 (Gly-Met and Thr-Val, respectively), changed the permeant selectivity of the transporter from cit to cif. We now report an investigation of the effects of corresponding mutations in TM 8 alone and demonstrate unique S353T- and L354V-induced changes in nucleoside specificity and cation coupling, respectively. hCNT1 mutation S353T produced a profound decrease in cytidine transport efficiency (Vmax/Km ratio) and, in combination with L354V (S353T/L354V), resulted in a novel uridine-preferring transport phenotype. In addition, the L354V mutation markedly increased the apparent affinity of hCNT1 for Na+ and Li+. Both hCNT1 TM 8 residues exhibited uridine-protectable inhibition by p-chloromercuribenzene sulfonate when converted to Cys, suggesting that they occupy positions within or closely adjacent to a common cation/nucleoside translocation pore.