Mulinta Ras, Yao Sylvia Y M, Ng Amy M L, Cass Carol E, Young James D
From the Membrane Protein Disease Research Group, Departments of Physiology and.
Oncology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and.
J Biol Chem. 2017 Jun 9;292(23):9505-9522. doi: 10.1074/jbc.M116.743997. Epub 2017 Apr 6.
The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na and/or H electrochemical gradients. CNT family member NupC resembles hCNT1 in permeant selectivity but is H-coupled. Using heterologous expression in oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C-), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent -chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C-) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C-) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function.
人类浓缩核苷转运蛋白（CNT）的SLC28家族有三个成员：hCNT1、hCNT2和hCNT3。与钠偶联的hCNT1和hCNT2分别转运嘧啶和嘌呤核苷，而hCNT3利用钠和/或氢的电化学梯度转运嘧啶和嘌呤核苷。CNT家族成员NupC在通透选择性上与hCNT1相似，但与氢偶联。利用卵母细胞中的异源表达以及工程化的无半胱氨酸hCNT3蛋白hCNT3（C-），使用膜不透性硫醇反应试剂对氯汞苯磺酸盐对该蛋白转运结构域（界面螺旋2、发夹1、假定跨膜结构域（TM）7和TM8）以及支架结构域的TM9进行了取代半胱氨酸可及性方法分析。对hCNT3（C-）整个C端后半部分的这种系统扫描，以及对野生型hCNT1转运结构域和无半胱氨酸NupC（C-）相应跨膜结构域的平行研究，得出的结果验证了新开发的人类CNT的CNT膜结构的结构同源模型，揭示了转运结构域跨膜结构域内延伸的构象可移动区域，确定了具有功能重要性的孔衬残基，并提供了转运蛋白功能新出现的新型电梯式机制的证据。
