Cloning and expression of a chloride-dependent Na+-H+ exchanger

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 chloride-dependent 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, NHE-deficient 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 microm 3-methylsulfonyl-4piperidonbenzoylguanidine, an NHE-1 and NHE-2 isoform inhibitor. In contrast to other Na(+)-H(+) exchange isoforms in colonic epithelial cells, chloride-dependent Na(+)-H(+) exchange mRNA abundance was increased by dietary sodium depletion. Based on these results we predict that chloride-dependent Na(+)-H(+) exchange represents a new class of Na(+)-H(+) exchangers that may regulate ion transport in several organs.