The Human ZIP4 Transporter Has Two Distinct Binding Affinities and Mediates Transport of Multiple Transition Metals
The Human ZIP4 Transporter Has Two Distinct Binding Affinities and Mediates Transport of Multiple Transition Metals
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DOI：
10.1021/bi201553p
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发表时间：
2012-02-07
期刊：
影响因子：
2.9
通讯作者：
Dempski, Robert E.
中科院分区：
文献类型：
--
作者：
Antala, Sagar;Dempski, Robert E.
锌是体内第二丰富的过渡金属。尽管数以百计的生物分子需要锌来维持正常的功能和/或结构，但锌转运到细胞中的机制尚不清楚。ZIP （Zrt-和irt样蛋白；SLC39A）蛋白家族的作用是增加细胞内锌的浓度。ZIP蛋白家族成员之一，人类ZIP4 （hZIP4; SLC39A4）蛋白的突变可导致肠病性肢端皮炎（AE）。AE的特点是生长迟缓和腹泻，以及行为和神经障碍。虽然hZIP4蛋白表达的细胞分布已经被阐明，但阳离子特异性、锌转运的动力学参数以及参与阳离子易位的残基仍是未解决的问题。因此，我们在非洲爪蟾卵母细胞中异种表达hZIP4，建立了高信噪比的锌摄取实验。实验结果表明，当阳离子浓度在微摩尔范围内时，hZIP4可以传输锌、铜（II）和镍。我们还确定了一种纳米摩尔结合亲和力，其中铜（II）和锌可以运输。相反，在这些条件下，镍可以结合，但不能通过hZIP4运输。最后，用马来酰亚胺或焦碳酸二乙基标记hZIP4表明，细胞外可接近的组氨酸残基，而不是半胱氨酸残基，直接或间接地需要阳离子摄取。我们的实验结果确定了至少两个二价阳离子的配位位点，并为研究ZIP蛋白家族提供了一个新的框架。
Zinc is the second most abundant transition metal in the body. Despite the fact that hundreds of biomolecules require zinc for proper function and/or structure, the mechanism of zinc transport into cells is not well-understood. The ZIP (Zrt- and Irt-like proteins; SLC39A) family of proteins acts to increase cytosolic concentrations of zinc. Mutations in one member of the ZIP family of proteins, the human ZIP4 (hZIP4; SLC39A4) protein, can result in the disease acrodermatitis enteropathica (AE). AE is characterized by growth retardation and diarrhea, as well as behavioral and neurological disturbances. While the cellular distribution of hZIP4 protein expression has been elucidated, the cation specificity, kinetic parameters of zinc transport, and residues involved in cation translocation are unresolved questions. Therefore, we have established a high signal-to-noise zinc uptake assay following heterologous expression of hZIP4 in Xenopus laevis oocytes. The results from our experiments have demonstrated that zinc, copper(II), and nickel can be transported by hZIP4 when the cation concentration is in the micromolar range. We have also identified a nanomolar binding affinity where copper(II) and zinc can be transported. In contrast, under these conditions, nickel can bind but is not transported by hZIP4. Finally, labeling of hZIP4 with maleimide or diethylpyrocarbonate indicates that extracellularly accessible histidine, but not cysteine, residues are required, either directly or indirectly, for cation uptake. The results of our experiments identify at least two coordination sites for divalent cations and provide a new framework for investigating the ZIP family of proteins.