Cloning and expression of a rabbit cDNA encoding a serum-activated ethylisopropylamiloride-resistant epithelial Na+/H+ exchanger isoform (NHE-2).
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.
Tse
C M
CM
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Levine
S A
SA
Yun
C H
CH
Montrose
M H
MH
Little
P J
PJ
Pouyssegur
J
J
Donowitz
M
M
D000230:Adenocarcinoma; D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003110:Colonic Neoplasms; D004247:DNA / Q000235:genetics*; D004351:Drug Resistance; D004848:Epithelium / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009928:Organ Specificity; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D011817:Rabbits; D051381:Rats; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D012333:RNA, Messenger; C081517:SLC9A2 protein, human; C513110:Slc9a2 protein, rat; D017923:Sodium-Hydrogen Exchangers; D011061:Poly A; D012313:RNA; D000584:Amiloride; D004247:DNA; D012964:Sodium; C039614:ethylisopropylamiloride