Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.

Abstract

A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5′‐untranslated region, 2451 bp of open reading frame and 1163 bp of 3′‐untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8‐Br‐cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29‐18 and Caco‐2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta‐galactosidase and the C‐terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI‐AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the ‘housekeeping’ Na+/H+ exchanger.

Basic Fibroblast Growth Factor Stimulates Surface Expression and Activity of Na+/H+ Exchanger NHE3 via Mechanism Involving Phosphatidylinositol 3-Kinase

“…Cells were transfected with NHE3-GFP cDNA (or with pEGFP-N3 vector) using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. The cell population expressing NHE3-GFP was initially enriched using the acid selection technique (18). Stable clones expressing functional NHE3-GFP (PS120-E3G cells) were subsequently isolated using dilutional cloning, propagated in a large scale culture, and frozen in liquid nitrogen.…”

Structure/function studies of mammalian Na‐H exchangers‐‐an update.

“…Since the cloning of NHE1 by Sardet et al (1989), three additional members of the mammalian gene family have been identified by our group (Tse et al 1994a;Tse et al 1993b;Tse et al 1991) and by Orlowski and Shull (Orlowski et al 1992;Wang et al 1993). We named these clones in the order of their molecular identification as NHE1, NHE2, NHE3, NHE4, etc.…”

Molecular cloning, sequencing, tissue distribution, and functional expression of a Na+/H+ exchanger (NHE-2).

“…Orlowski et al (7) have identified three closely related Na+/H+ exchangers in rat tissues, NHE-1, NHE-3, and NHE-4, by screening cDNA libraries from the stomach, colon, brain, and spleen. Similarly, Tse et al (8,9) have identified two isoforms, NHE-1 and NHE-3, in rabbit by screening an ileal cDNA library. These isoforms exhibit differences in sequence homology, tissue expression, and molecular mass (7)(8)(9).…”

“…Similarly, Tse et al (8,9) have identified two isoforms, NHE-1 and NHE-3, in rabbit by screening an ileal cDNA library. These isoforms exhibit differences in sequence homology, tissue expression, and molecular mass (7)(8)(9).…”