Entry to "HCO3- tunnel" revealed by SLC4A4 human mutation and structural model
Entry to "HCO3- tunnel" revealed by SLC4A4 human mutation and structural model
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DOI：
10.1074/jbc.m709819200
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发表时间：
2008-06-27
影响因子：
4.8
通讯作者：
Romero, Michael F.
中科院分区：
文献类型：
--
作者：
Chang, Min-Hwang;DiPiero, Jennifer;Romero, Michael F.
青光眼、白内障和近端肾小管酸中毒是由人产电Na+碳酸氢盐协同转运蛋白（NBCe 1/SLC 4A 4）点突变引起的疾病（1，2）。一个这样的突变，R298 S，位于NBCe 1的胞质N-末端结构域，只有中等（75%）的功能。由于SLC转运蛋白在其膜和N末端一级序列中具有高度相似性，因此我们将NBCe 1同源建模到带3（AE 1）的晶体结构坐标上（3）。预测Arg-298位于溶剂不可接近的表面下口袋中，并通过H-键合和电荷-电荷相互作用与Glu-91或Glu-295缔合。我们通过定点突变扰乱了Glu-91和Arg-298之间的这些假定的相互作用，并使用在非洲爪蟾卵母细胞中的表达来测试我们的结构模型。任一残基的突变导致运输功能降低。功能通过电荷反转（E91 R/R298 E）“修复”，这意味着这两个残基是可互换的和相互依赖的。这些结果对比目前的理解AE 1 N末端作为蛋白质结合位点，并提出hkNBCe 1（和其他SLC 4）细胞质N末端在控制HCO 3渗透中发挥作用。
Glaucoma, cataracts, and proximal renal tubular acidosis are diseases caused by point mutations in the human electrogenic Na+ bicarbonate cotransporter (NBCe1/SLC4A4) (1, 2). One such mutation, R298S, is located in the cytoplasmic N-terminal domain of NBCe1 and has only moderate (75%) function. As SLC transporters have high similarity in their membrane and N-terminal primary sequences, we homology-modeled NBCe1 onto the crystal structure coordinates of Band 3(AE1) (3). Arg-298 is predicted to be located in a solvent-inaccessible subsurface pocket and to associate with Glu-91 or Glu-295 via H-bonding and charge- charge interactions. We perturbed these putative interactions between Glu-91 and Arg-298 by site-directed mutagenesis and used expression in Xenopus oocyte to test our structural model. Mutagenesis of either residue resulted in reduced transport function. Function was "repaired" by charge reversal (E91R/R298E), implying that these two residues are interchangeable and interdependent. These results contrast the current understanding of the AE1 N terminus as protein-binding sites and propose that hkNBCe1 (and other SLC4) cytoplasmic N termini play roles in controlling HCO3- permeation.