Cloning, tissue distribution, and functional analysis of the human Na+/N+ exchanger isoform, NHE3.
We previously isolated a 1.4-kb partial cDNA from a human kidney cortex library. Using both library screening and reverse transcription-polymerase chain reaction of human kidney RNA, we obtained the entire coding region of the human NHE3 cDNA. The human NHE3 cDNA encoded a protein of 834 amino acids with a calculated relative molecular weight of 92,906. It exhibited 89 and 88% amino acid identity with rat and rabbit NHE3, respectively. The stable transfection of a composite human NHE3 cDNA into Na+/H+ exchanger-deficient PS120 cells established Na+/H+ exchange. Functionally, human NHE3 was similar to the rabbit and rat NHE3 homologues, being relatively resistant to inhibition by amiloride, half-maximal inhibition (IC50) = 49.0 microM, and ethylisopropylamiloride, IC50 = 6.6 microM, and being stimulated by fibroblast growth factor but inhibited by phorbol 12-myristate 13-acetate. However, unlike the rabbit or rat NHE3, human NHE3 message was not restricted to kidney, intestine, stomach, and brain. Northern analysis of multiple human tissues detected NHE3 message, in descending order, as follows: kidney >> small intestine >> testes > ovary > colon = prostate > thymus > peripheral leukocyte = brain > spleen > placenta. Message in the kidney, small intestine, and colon was primarily of 6.7 kb, whereas both 6.7- and 8.9-kb bands were expressed nearly equivalently in the other tissues. No NHE3 message was detected in the human heart, lung, liver, skeletal muscle, or pancreas.
Brant
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Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195, USA.
Yun
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Donowitz
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Tse
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D014018:Tissue Distribution
D018076:DNA, Complementary; D012333:RNA, Messenger; C000613385:SLC9A3 protein, human; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers