Cloning and expression of a rabbit cDNA encoding a serum-activated ethylisopropylamiloride-resistant epithelial Na+/H+ exchanger isoform (NHE-2)

• Published in: The Journal of biological chemistry Vol. 268; no. 16; pp. 11917 - 11924
• Main Authors: CHUNG-MING TSE, LEVINE, S. A, YUN, C. H. C, MONTROSE, M. H, LITTLE, P. J, POUYSSEGUR, J, DONOWITZ, M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.06.1993
• Subjects: Adenocarcinoma; Amiloride - analogs & derivatives; Amiloride - pharmacology; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Blotting, Northern; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cloning, Molecular; Colonic Neoplasms; DNA - genetics; Drug Resistance; Epithelium - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Humans; Intestinal Mucosa - metabolism; Molecular Sequence Data; Molecular Weight; Organ Specificity; Poly A - genetics; Poly A - isolation & purification; Proteins; Rabbits; Rats; Restriction Mapping; RNA - genetics; RNA - isolation & purification; RNA, Messenger; Sequence Homology, Amino Acid; Sodium - metabolism; Sodium-Hydrogen Exchangers; Transfection; Tumor Cells, Cultured; Vertebrata; Mammalia; Sodium; Transcription; Rabbit; Proton; Tissue specificity; Primary structure; Lagomorpha; Molecular cloning; Carrier protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)50287-3

A unique Na+/H+ exchanger isoform, NHE-2, was cloned and characterized. NHE-2 is a protein of 809 amino acids with a calculated size of 90,787. It exhibits overall amino acid identity of 50, 44, and 60% with other cloned mammalian Na+/H+ exchangers NHE-1, NHE-3, and NHE-4, respectively. Northern blot analysis of poly(A+) RNA isolated from rabbit ileum, kidney cortex, and kidney medulla using NHE-2 cDNA as a probe revealed messages of 5.2, 4.2, and 3.2 kilobases with relative abundance (in descending order) kidney medulla > kidney cortex > ileum. More detailed tissue distribution of message was performed by ribonuclease protection assay. NHE-2 was predominantly expressed in kidney, intestine, and adrenal gland with a small amount in skeletal muscle and trachea. Stable expression of NHE-2 in PS120 fibroblasts confirmed that NHE-2 is a functional Na+/H+ exchanger which is defined by amiloride-sensitive Na+-dependent alkalinization of acid-loaded cells. NHE-2 has the same Ki for amiloride inhibition as NHE-1 (1 microM) but is 25-fold more resistant to ethylisopropylamiloride inhibition than is NHE-1 (500 versus 20 nM). Like NHE-1, NHE-2 can be activated by serum. Expression of NHE-2 in a polarized human intestinal epithelial cell line, Caco-2 cells, results in functional expression of NHE-2 in the apical membrane. Thus, we conclude that NHE-2 is a candidate to be an apical membrane Na+/H+ exchanger in intestinal and renal epithelial cells.