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Papers In Press, published online ahead of print December 28, 2001
J. Biol. Chem, 10.1074/jbc.M110852200
Submitted on November 12, 2001
Revised on December 20, 2001
Accepted on December 28, 2001

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

Pitchai Sangan, Carsten A. Wagner, Vazhaikkurichi M. Rajendran, John P. Geibel, and Henry J. Binder

Department of Internal Medicine, Yale University, New Haven, CT 06520-8019

Corresponding Author: henry.binder{at}yale.edu

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, while northern blot analysis established the presence of its 2.5 kb mRNA in multiple tissues. Despite its much smaller size compared to 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 Cl dependent and inhibited both by 5-ethylisopropylamiloride (EIPA), an amiloride analogue, and by 5'-nitro-2-(3-phenylproplyamino)-benzoic acid (NPPB), a Cl channel blocker, but only minimally affected by 25 {um}M 3-methylsulfonyl-4piperidonbenzoylguanidine (HOE694), a NHE-1 and NHE-2 isoform inhibitor. In contrast to other Na-H exchange isoforms in colonic epithelial cells, Cl-dependent Na-H exchange mRNA abundance was increased by dietary Na depletion. Based on these results we predict that Cl-dependent Na-H exchange represents a new class of Na-H exchanges that may regulate ion transport in several organs.


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