Critical amino acid residues involved in the electrogenic sodium-bicarbonate cotransporter kNBC1-mediated transport
Critical amino acid residues involved in the electrogenic sodium-bicarbonate cotransporter kNBC1-mediated transport
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DOI：
10.1113/jphysiol.2005.084988
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发表时间：
2005-06-15
影响因子：
5.5
通讯作者：
Kurtz, I
中科院分区：
文献类型：
--
作者：
Abuladze, N;Azimov, R;Kurtz, I
我们以前已经报道了一个拓扑模型的产电Na+-HCO 3-协同转运蛋白（NBC 1），其中协同转运蛋白跨越质膜10倍的N-和C-末端定位在细胞内。SLC 4超家族成员之间的跨膜段（TM）和细胞内/细胞外环（IL/EL）的保守氨基酸残基的分析提供了在本研究中采取的诱变方法，以确定参与NBC 1介导的离子转运的氨基酸的基础。使用大规模诱变，参与由肾脏中表达的NBC 1的主要变体（kNBC 1）介导的离子转运的酸性和碱性氨基酸pu 13突变为中性和/或带相反电荷的氨基酸。所有突变体kNBC 1共转运蛋白在HEK-293 T细胞中表达，并使用2 '，7'-双-（羧乙基）-5（6）-羧基荧光素（BCECF）的细胞内pH测量来确定突变体的Na+依赖性碱通量。关键的谷氨酸，天冬氨酸，赖氨酸，精氨酸和组氨酸残基的IL/EL和TM的检测是必不可少的kNBC 1介导的Na+依赖的基础运输。此外，还检测了TM和IL/EL中的关键苯丙氨酸、丝氨酸、酪氨酸、苏氨酸和丙氨酸残基。此外，IL/EL和TM中的几个氨基酸残基被证明是膜靶向所必需的。这些数据表明，在假定的面向外和面向内的构象中，参与离子识别的kNBC 1带电氨基酸的分布不对称。一个模型，总结关键氨基酸残基参与kNBC 1介导的离子转运。
We have previously reported a topological model of the electrogenic Na+-HCO3- cotransporter (NBC1) in which the cotransporter spans the plasma membrane 10 times with N- and C-termini localized intracellularly. An analysis of conserved amino acid residues among members of the SLC4 superfamily in both the transmembrane segments (TMs) and intracellular/extracellular loops (ILs/ELs) provided the basis for the mutagenesis approach taken in the present study to determine amino acids involved in NBC1-mediated ion transport. Using large-scale mutagenesis, acidic and basic amino acids putatively involved in ion transport mediated by the predominant variant of NBC1 expressed in the kidney (kNBC1) were mutated to neutral and/or oppositely charged amino acids. All mutant kNBC1 cotransporters were expressed in HEK-293T cells and the Na+-dependent base flux of the mutants was determined using intracellular pH measurements with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Critical glutamate, aspartate, lysine, arginine and histidine residues in ILs/ELs and TMs were detected that were essential for kNBC1-mediated Na+-dependent base transport. In addition, critical phenylalanine, serine, tyrosine, threonine and alanine residues in TMs and ILs/ELs were detected. Furthermore, several amino acid residues in ILs/ELs and TMs were shown to be essential for membrane targeting. The data demonstrate asymmetry of distribution of kNBC1 charged amino acids involved in ion recognition in putative outward-facing and inward-facing conformations. A model summarizing key amino acid residues involved in kNBC1-mediated ion transport is presented.