Slugoski Melissa D, Ng Amy M L, Yao Sylvia Y M, Smith Kyla M, Lin Colin C, Zhang Jing, Karpinski Edward, Cass Carol E, Baldwin Stephen A, Young James D
Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada.
J Biol Chem. 2008 Mar 28;283(13):8496-507. doi: 10.1074/jbc.M710433200. Epub 2008 Jan 16.
The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 mediates transport of a broad range of purine and pyrimidine nucleosides and nucleoside drugs, whereas hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively. All three hCNTs are Na(+)-coupled. Unlike hCNT1/2, however, hCNT3 is also capable of H(+)-mediated nucleoside cotransport. Using site-directed mutagenesis in combination with heterologous expression in Xenopus oocytes, we have identified a C-terminal intramembranous cysteine residue of hCNT3 (Cys-561) that reversibly binds the hydrophilic thiol-reactive reagent p-chloromercuribenzene sulfonate (PCMBS). Access of this membrane-impermeant probe to Cys-561, as determined by inhibition of hCNT3 transport activity, required H(+), but not Na(+), and was blocked by extracellular uridine. Although this cysteine residue is also present in hCNT1 and hCNT2, neither transporter was affected by PCMBS. We conclude that Cys-561 is located in the translocation pore in a mobile region within or closely adjacent to the nucleoside binding pocket and that access of PCMBS to this residue reports a specific H(+)-induced conformational state of the protein.
人类中的浓缩核苷转运体（CNT）蛋白家族由三个成员代表，即hCNT1、hCNT2和hCNT3。hCNT3属于一个在系统发育上与hCNT1/2不同的CNT亚家族，它介导多种嘌呤和嘧啶核苷以及核苷类药物的转运，而hCNT1和hCNT2分别对嘧啶核苷和嘌呤核苷具有选择性。所有这三种hCNT均与Na(+)偶联。然而，与hCNT1/2不同的是，hCNT3还能够进行H(+)介导的核苷共转运。通过在非洲爪蟾卵母细胞中进行定点诱变并结合异源表达，我们确定了hCNT3的一个C末端膜内半胱氨酸残基（Cys-561），它可逆地结合亲水性硫醇反应试剂对氯汞苯磺酸盐（PCMBS）。通过抑制hCNT3转运活性来确定这种膜不透性探针与Cys-561的结合，需要H(+)，而不是Na(+)，并且被细胞外尿苷阻断。尽管这个半胱氨酸残基在hCNT1和hCNT2中也存在，但这两种转运体都不受PCMBS的影响。我们得出结论，Cys-561位于核苷结合口袋内或紧邻其的一个可移动区域的转运孔中，并认为PCMBS与该残基的结合反映了蛋白质特定的H(+)诱导的构象状态。
