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 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 μM 3-methylsulfonyl-4piperidonbenzoyl-guanidine, 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.