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.
Sangan
Pitchai
P
Department of Internal Medicine, Yale University, New Haven, Connecticut 06520-8019, USA.
Rajendran
Vazhaikkurichi M
VM
Geibel
John P
JP
Binder
Henry J
HJ
D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D002713:Chlorine / Q000378:metabolism; D003001:Cloning, Molecular; D003106:Colon / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017403:In Situ Hybridization; D008297:Male; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics*; D013997:Time Factors; D014018:Tissue Distribution
D018076:DNA, Complementary; D004232:Diuretics; D012333:RNA, Messenger; C452850:Slc9a1 protein, rat; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D002713:Chlorine; D000584:Amiloride; D012964:Sodium