a Membrane Protein Disease Research Group, Department of Physiology , University of Alberta , Edmonton , Canada.
Channels (Austin). 2018;12(1):291-298. doi: 10.1080/19336950.2018.1506665.
The human SLC28 family of concentrative (Na-dependent) nucleoside transporters has three members, hCNT1, hCNT2 and hCNT3. Previously, we have used heterologous expression in Xenopus laevis oocytes in combination with an engineered cysteine-less hCNT3 protein hCNT3(C-) to undertake systematic substituted cysteine accessibility method (SCAM) analysis of the transporter using the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate (PCMBS). A continuous sequence of more than 300 individual amino acid residue positions were investigated, including the entire transport domain of the protein, as well as important elements of the corresponding hCNT3 structural domain. We have now constructed 3D structural homology models of hCNT3 based upon inward-facing, intermediates and outward-facing crystal structures of the bacterial CNT Neisseria wadsworthii CNT to show that all previously identified PCMBS-sensitive residues in hCNT3 are located above (ie on the extracellular side of) the key diagonal barrier scaffold domain TM9 in the transporter's outward-facing conformation. In addition, both the Na and permeant binding sites of the mobile transport domain of hCNT3 are elevated from below the scaffold domain TM9 in the inward-facing conformation to above TM9 in the outward-facing conformation. The hCNT3 homology models generated in the present study validate our previously published PCMBS SCAM data, and confirm an elevator-type mechanism of membrane transport.
人类 SLC28 家族的摄取（Na 依赖性）核苷转运体有三个成员，hCNT1、hCNT2 和 hCNT3。之前，我们使用非洲爪蟾卵母细胞中的异源表达，并结合无半胱氨酸的 hCNT3 蛋白 hCNT3(C-)，使用不可渗透膜的硫醇反应试剂对转运体进行系统取代的半胱氨酸可及性方法（SCAM）分析氯汞苯磺酸（PCMBS）。我们已经研究了超过 300 个单独的氨基酸残基位置的连续序列，包括蛋白质的整个转运结构域，以及相应 hCNT3 结构域的重要元素。我们现在已经基于细菌 CNT Neisseria wadsworthii CNT 的内向、中间体和外向晶体结构构建了 hCNT3 的 3D 结构同源模型，以表明 hCNT3 中所有先前确定的 PCMBS 敏感残基都位于转运体外向构象的关键对角屏障结构域 TM9 之上（即在细胞外侧面）。此外，hCNT3 可移动转运结构域的 Na 和通透结合位点均从内向构象中 TM9 下方升高到外向构象中的 TM9 上方。本研究生成的 hCNT3 同源模型验证了我们之前发表的 PCMBS SCAM 数据，并证实了一种提升型膜转运机制。
