Cloning and Expression of a Chloride-dependent Na+-H+ Exchanger

Abstract

Electroneutral Na؉ -H ؉ exchange is present in virtually all cells, mediating the exchange of extracellular Na ؉ for intracellular H ؉ and, thus, plays an important role in the regulation of intracellular pH, cell volume, and transepithelial Na ؉ absorption. Recent transport studies demonstrated the presence of a novel chloridedependent Na ؉ -H ؉ exchange in the apical membrane of crypt cells of rat distal colon. We describe the cloning of a 2.5-kb full-length cDNA from rat distal colon that encodes 438 amino acids and has six putative transmembrane spanning domains. Of the 438 amino acids 375 amino acids at the N-terminal region are identical to Na ؉ -H ؉ exchange (NHE)-1 isoform with the remaining 63 amino acids comprising a completely novel C terminus. In situ hybridization revealed that this transcript is expressed in colonic crypt cells, whereas Northern blot analysis established the presence of its 2.5-kb mRNA in multiple tissues. Despite its much smaller size compared with all other known Na ؉ -H ؉ exchange isoforms, NHEdeficient PS120 fibroblasts stably transfected with this cDNA exhibited Na ؉ -dependent intracellular pH recovery to an acid load that was chloride-dependent and inhibited both by 5-ethylisopropylamiloride, an amiloride analogue, and by 5-nitro-2-(3-phenylproplyamino)-benzoic acid, a Cl ؊ channel blocker, but only minimally affected by 25 M 3-methylsulfonyl-4piperidonbenzoyl-guanidine, an NHE-1 and NHE-2 isoform inhibitor.

PTH stimulates a Cl−-dependent and EIPA-sensitive current in chick proximal tubule cells in culture

“…Although the transport mechanism behind the PTHinduced I sc response is unknown, an intriguing possibility is suggested by recent studies of a novel Cl Ϫ -dependent NHE found in rat distal colon crypt cells (33,34,39). As determined by pH gradient-stimulated 22 Na uptake by apical membrane vesicles, this "Cl-NHE" requires Cl Ϫ and is sensitive to both NPPB and EIPA.…”

“…Moreover, an antibody to CFTR also partially blocked Cl-NHE activity (33). Recently, this transporter was cloned from rat distal colon crypt cells and shown to have homology with NHE1, but with a markedly shortened and novel COOH-terminal domain (39). Of particular interest, cDNA probes for this transporter revealed widespread distribution of specific mRNA in several rat tissues, including kidney.…”

The Na+/H+exchanger isoform 2 is the predominant NHE isoform in murine colonic crypts and its lack causes NHE3 upregulation

“…A Cl Ϫ -dependent Na ϩ /H ϩ exchanger has been functionally (24) and molecularly (26) identified in rat colonic epithelium. To assess its potential presence in murine colon, we performed pH i recovery experiments in the absence of Cl Ϫ (Fig.…”

“…By using NHE2-deficient mice, we also ascertained that the NHE2-inhibitory concentration of HOE-642 does not inhibit something else. We ruled out the presence of NHE4 (Rossmann, unpublished observation) and of a Cl Ϫ -dependent, amiloride-sensitive Na ϩ /H ϩ exchanger as found in the rat distal colon (24,26). Because we measure ϩ /H ϩ exchange in NHE2 (ϩ/ϩ) mice only.…”

A vH+-ATPase is present in cultured sheep ruminal epithelial cells

“…In other tissues Cl Ϫ ions have been reported to modulate basal NHE activity as well as NHE activity induced by hyperosmolality (2,22,35,38), and a distinct Cl Ϫ -dependent Na ϩ /H ϩ exchange has been identified in the apial membrane of colonic crypt cells (38,39 has no effect on the butyrate-induced pH i recovery (41). In contrast, in the HEPES-buffered medium used in this study lowering of [Cl Ϫ ] e impairs cell pH recovery from an acid load by ϳ33% and, in special, its HOE 694-sensitive part.…”