Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain
Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain
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DOI：
10.1074/jbc.274.7.4377
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发表时间：
1999-02-12
影响因子：
4.8
通讯作者：
Shull, GE
中科院分区：
文献类型：
--
作者：
Baird, NR;Orlowski, J;Shull, GE
为了分离编码Na+/H+交换异构体5(NHE5)的基因，我们利用NHE5基因的外显子序列筛选了一个人脾文库。分离到了跨度为2.9kb的克隆，但它们含有多个内含子，并且在5‘和3’端都缺少编码序列。缺失的5‘端序列通过5’端快速扩增和对NHE5基因组克隆的分析获得，缺失的3‘端序列通过3’端快速扩增获得。聚合酶链式反应扩增脑内含子得到的产物中，每个内含子都被正确地切除，而内含子在脾和睾丸中被保留，这表明在这些器官中表达的NHE5转录本并不编码功能转运体。对NHE5基因的内含子/外显子组织进行了分析，发现其与NHE3基因非常相似。NHE5基因编码与NHE3关系最密切的896个氨基酸的蛋白质，在Na+/H+交换缺陷的成纤维细胞中表达，并具有介导Na+/H+交换活性的作用。Northern印迹分析表明，编码NHE5的mRNA在大脑的多个区域表达，包括海马区，这与它调节海马区和其他神经元细胞内pH的可能性一致。
To isolate a cDNA encoding Na+/H+ exchanger isoform 5 (NHE5), we screened a human spleen library using exon sequences of the NHE5 gene. Clones spanning 2.9 kilobase pairs were isolated; however, they contained several introns and were missing coding sequences at both the 5' and 3' ends. The missing 5' sequences were obtained by 5'-rapid amplification of cDNA ends and by analysis of an NHE5 genomic clone, and the missing 3' sequences were obtained by 3'-rapid amplification of cDNA ends. Polymerase chain reaction amplification of brain cDNA yielded products in which each of the introns had been correctly excised, whereas the introns were retained in products from spleen and testis, suggesting that the NHE5 transcripts expressed in these organs do not encode a functional transporter. The intron/exon organization of the NHE5 gene was analyzed and found to be very similar to that of the NHE3 gene. The NHE5 cDNA, which encodes an 896-amino acid protein that is most closely related to NHE3, was expressed in Na+/H+ exchanger-deficient fibroblasts and shown to mediate Na+/H+ exchange activity. Northern blot analysis demonstrated that the mRNA encoding NHE5 is expressed in multiple regions of the brain, including hippocampus, consistent with the possibility that it regulates intracellular pH in hippocampal and other neurons.