Cloning and functional expression of a human kidney Na+:HCO3- cotransporter
Cloning and functional expression of a human kidney Na+:HCO3- cotransporter
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DOI：
10.1074/jbc.272.31.19111
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发表时间：
1997-08-01
影响因子：
4.8
通讯作者：
Soleimani, M
中科院分区：
文献类型：
--
作者：
Burnham, CE;Amlal, H;Soleimani, M
肾脏中存在几种HCO3-转运方式，包括Na+非依赖的Cl/HCO3-交换(由一系列的Cl-/HCO3-交换器介导)、钠依赖的Cl-/HCO3-交换和Na+：HCO3-共转运。Na+偶联的HCO3-转运体和AE异构体之间的功能相似性(即HCO3-的转运体和对4，4‘-二异硫氰基二苯乙烯-2，2’-二磺酸抑制的敏感性)建议了一种基于与AE家族的结构相似性来克隆其他转运体的策略。在GenBank(TM)表达序列标签数据库中鉴定了编码与已知阴离子交换器相关的蛋白质的部分的表达序列标签并用于设计寡核苷酸探针，该探针用于筛选人肾cDNA文库，对几个克隆进行了鉴定、分离和测序，将两个重叠的克隆拼接在一起，形成一个7.6kb的cDNA，它包含了一个新蛋白质的完整编码区，根据推导的氨基酸序列，该cDNA编码一个M-r为116,040的蛋白质。该蛋白与人脑AE3有29%的同源性。Northern印迹分析表明，该7.6kb的mRNA在肾脏和胰腺中高度表达，在脑中有检测到的水平。瞬时转染HEK-293细胞的功能研究表明，克隆的转运蛋白介导Na+：HCO3-共转运。
Several modes of HCO3- transport occur in the kidney, including Na+-independent Cl/HCO3- exchange (mediated by the AE family of Cl-/HCO3- exchangers), sodium-dependent Cl-/HCO3-, exchange, and Na+:HCO3- cotransport. The functional similarities between the Na+ coupled HCO3- transporters and the AE isoforms (i.e. transport of HCO3- and sensitivity to inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) suggested a strategy for cloning the other transporters based on structural similarity with the AE family, An expressed sequence tag encoding part of a protein that is related to the known anion exchangers was identified in the GenBank(TM) expressed sequence tag data base and used to design an oligonucleotide probe, This probe was used to screen a human kidney cDNA library, Several clones were identified, isolated, and sequenced, Two overlapping cDNA clones were spliced together to form a 7.6-kilobase cDNA that contained the entire coding region of a novel protein, Based on the deduced amino acid sequence, the cDNA encodes a protein with a M-r of 116,040. The protein has 29% identity with human brain AE3. Northern blot analysis reveals that the 7.6-kilobase mRNA is highly expressed in kidney and pancreas, with detectable levels in brain. Functional studies in transiently transfected HEK-293 cells demonstrate that the cloned transporter mediates Na+:HCO3- cotransport.