Rat brain and kidney cDNA libraries were constructed and screened with a cDNA insert corresponding to the mRNA for the sheep kidney Na+,K+-ATPase catalytic subunit. The alpha-subunit cDNAs isolated from the kidney library were derived from a single class of messenger RNA, and the brain cDNAs were derived from three classes of messenger RNA. The most abundant brain cDNA, which spans 5.1 kilobases, encodes the alpha(+) form of the enzyme. The second most abundant brain cDNA, which spans 3.65 kilobases, is identical with that of the kidney form and therefore encodes the alpha isoform. The third class of cDNA, which spans 3.55 kilobases, was present at low abundance and encodes an isoform of the alpha-subunit, designated alpha III, which has not been identified previously. The complete nucleotide sequence and deduced amino acid sequence for each of the brain and kidney cDNAs have been determined. In addition, we have identified a lysine-rich sequence that may function as a movable, ion-selective gate during cation binding and occlusion and have also identified several amino acid sequence variations that appear to explain some of the well-known species and tissue differences in cardiac glycoside sensitivity.

Previous studies using isolated tissues suggest that the colonic H, K-ATPase (cHKA), expressed in the colon and kidney, plays an important role in K+ conservation. To test the role of this pump in K+ homeostasis in vivo, we generated a cHKA-deficient mouse and analyzed its ability to retain K+ when fed a control or K+-free diet. When maintained on a control diet, homozygous mutant (cHKA-/-) mice exhibited no deficit in K+ homeostasis compared to wild-type (cHKA+/+ greater, similar mice. Although fecal K+ excretion in cHKA-/- mice was double that of cHKA+/+ mice, fecal K+ losses were low compared with urinary K+ excretion, which was similar in both groups. When maintained on a K+-free diet for 18 d, urinary K+ excretion dropped over 100-fold, and to similar levels, in both cHKA-/- and cHKA+/+ mice; fecal K+ excretion was reduced in both groups, but losses were fourfold greater in cHKA-/- than in cHKA+/+ mice. Because of the excess loss of K+ in the colon, cHKA-/- mice exhibited lower plasma and muscle K+ than cHKA+/+ mice. In addition, cHKA-/- mice lost twice as much body weight as cHKA+/+ mice. These results demonstrate that, during K+ deprivation, cHKA plays a critical role in the maintenance of K+ homeostasis in vivo.

The NHE4 Na؉ /H ؉ exchanger is abundantly expressed on the basolateral membrane of gastric parietal cells. To test the hypothesis that it is required for normal acid secretion, NHE4-null mutant (NHE4 ؊/؊ ) mice were prepared by targeted disruption of the NHE4 (Slc9a4) gene. NHE4؊/؊ mice survived and appeared outwardly normal. Analysis of stomach contents revealed that NHE4 ؊/؊ mice were hypochlorhydric. The reduction in acid secretion was similar in 18-day-old, 9-week-old, and 6-month-old mice, indicating that the hypochlorhydria phenotype did not progress over time, as was observed in mice lacking the NHE2 Na ؉ /H ؉ exchanger. Histological abnormalities were observed in the gastric mucosa of 9-week-old NHE4 ؊/؊ mice, including sharply reduced numbers of parietal cells, a loss of mature chief cells, increased numbers of mucous and undifferentiated cells, and an increase in the number of necrotic and apoptotic cells. NHE4؊/؊ parietal cells exhibited limited development of canalicular membranes and a virtual absence of tubulovesicles, and some of the microvilli had centrally bundled actin. We conclude that NHE4, which may normally be coupled with the AE2 Cl ؊ /HCO 3 ؊ exchanger, is important for normal levels of gastric acid secretion, gastric epithelial cell differentiation, and development of secretory canalicular and tubulovesicular membranes. Naϩ /H ϩ exchanger (NHE) 1 isoform 4 is encoded by Slc9a4, a member of the Slc9a gene family, comprising at least 9 genes encoding transporters that catalyze the electroneutral exchange of Na ϩ and H ϩ across cell membranes (1, 2). NHE4 exhibits a limited tissue distribution, with very high expression in stomach and low levels in kidney, pancreas, salivary glands, and hippocampus (3-6).2 In the stomach, NHE4 protein has been localized to the basolateral membranes of epithelial cells lining the gastric glands (7). Its mRNA is expressed at particularly high levels in parietal and chief cells and at somewhat lower levels in mucous cells (8).In gastric parietal cells, basolateral Na ϩ /H ϩ exchange is involved in intracellular pH and volume homeostasis and has been proposed to play a critical role in acid secretion (8 -10). There is evidence that at least part of this Na ϩ /H ϩ exchange activity may be coupled with basolateral Cl Ϫ /HCO 3 Ϫ exchange (11). If this were the case, it would provide a mechanism for uptake of both Na ϩ and Cl Ϫ , which would contribute to maintenance of cell volume and, in combination with Na ϩ ,K ϩ -ATPase activity, maintain the intracellular ion concentrations and membrane potential required for HCl and KCl secretion. Studies using AE2 knockout mice have shown that AE2 is the predominant basolateral Cl Ϫ /HCO 3 Ϫ exchanger in gastric parietal cells; AE2-null mutants were achlorhydric and their gastric parietal cells exhibited a severe deficiency in the development of secretory canaliculi (12). These findings indicated that the loss of AE2 disrupted the electrochemical driving forces for gastric HCl and KCl secretion and suggested that basolateral...

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We have isolated and analyzed the genes encoding the human and rat gastric H,K-ATPase catalytic subunits. The complete sequence of the human gene, including 2.2 kb of 5'-flanking sequence, and the 5' end of the rat gene, including exons 1-4 and 2.5 kb of 5'-flanking sequence, have been determined. The human gene contains 22 exons. Its intron-exon organization is identical to that of the Na,K-ATPase gene, except that exon 6 corresponds to a fusion of exons 6 and 7 of the Na,K-ATPase gene. The transcription initiation sites of both the human and rat genes were determined by primer extension and S1 nuclease protection analyses. Comparison of the 5'-flanking regions of the human and rat genes revealed three extended regions of high sequence similarity, one of which includes a potential TATA box and other basic promoter elements beginning about 30 nucleotides upstream of the transcription start site. Other conserved sequences, including possible response elements for Ca2+ and cAMP, which are known intracellular mediators of acid secretion, are located up to 2 kb 5' to the transcription initiation site.