Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice.

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.

Mouse chromosomal location of three epithelial sodium channel subunit genes and an apical sodium chloride cotransporter gene.

The amiloride-sensitive epithelial sodium channel alpha, beta, and gamma subunit genes, Scnn1a, Scnn1b, and Scnn1g, and the thiazide-sensitive sodium chloride cotransporter gene, Slc12a1, have been mapped in the mouse using an interspecific backcross panel. These loci map to previously defined homologous regions between human and mouse chromosomes and provide additional information regarding human/mouse comparative mapping.

Structure of the human gastric H,K-ATPase gene and comparison of the 5'-flanking sequences of the human and rat genes.

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.

Molecular cloning of a third isoform of the calmodulin-sensitive plasma membrane Ca2+-transporting ATPase that is expressed predominantly in brain and skeletal muscle.

A complementary DNA for a third isoform of the calmodulin-sensitive plasma membrane Ca-ATPase has been isolated from a rat brain cDNA library. The nucleotide sequence of the 5.1-kilobase pair cDNA has been determined, and the amino acid sequence of the protein, designated PMCA3, has been deduced. PMCA3 is 1159 amino acids in length and has an Mr of 127,300. It exhibits 81% and 85% amino acid identity, respectively, to isoforms 1 and 2 (PMCA1 and PMCA2) of the plasma membrane Ca-ATPase. The transcript encoding PMCA3 is similar to that of PMCA1 in that it contains a sequence in the 3'-untranslated region that has the potential to encode an alternative calmodulin binding domain and carboxyl terminus. The tissue distribution of mRNAs encoding isoforms 1, 2, and 3 has been determined by Northern blot hybridization analyses. PMCA1 mRNAs are expressed in all tissues examined, suggesting that this protein may serve as a housekeeping form of the enzyme. However, PMCA2 and PMCA3 mRNAs exhibit a high degree of tissue specificity. PMCA2 mRNAs are expressed predominantly in brain and heart, whereas PMCA3 mRNAs are expressed predominantly in brain and skeletal muscle.

A novel Ca2+ pump expressed in brain, kidney, and stomach is encoded by an alternative transcript of the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase gene. Identification of cDNAs encoding Ca2+ and other cation-transporting ATPases using an oligonucleotide probe derived from the ATP-binding site.

We describe the results of a study designed to identify cDNAs encoding Ca2+-transporting ATPases and other cation-transporting ATPases of the aspartylphosphate class. Rat brain, kidney, and stomach cDNA libraries were screened with an oligonucleotide hybridization probe corresponding to a 23-amino acid sequence from part of the ATP-binding site of the sarcoplasmic reticulum Ca-ATPase. This procedure resulted in the isolation of cDNAs encoding (i) the plasma membrane Ca-ATPase, (ii) an apparent Ca-ATPase that exhibits high amino acid similarity to the sarcoplasmic reticulum Ca2+ pumps, (iii) a transport ATPase of unknown ion specificity and (iv) two Ca-ATPase isoforms encoded by the gene for the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase. Several isoforms of the Na,K-ATPase and gastric H,K-ATPase that had been characterized previously were also identified. The complete nucleotide sequences have been determined for the two classes of cDNA derived from alternatively spliced transcripts of the slow-twitch muscle sarcoplasmic reticulum Ca-ATPase gene. One of these cDNAs, isolated from the stomach library, encodes a Ca-ATPase that is identical to the skeletal muscle enzyme. The second class of cDNA, found in brain, kidney, and stomach libraries, is identical to that of the slow-twitch isoform throughout much of its length but encodes an alternative C terminus and has a different 3'-untranslated sequence. Whereas the muscle isoform consists of 997 amino acids and terminates with the sequence Ala-Ile-Leu-Glu, the second isoform is 1043 amino acids in length due to the replacement of these last 4 amino acids with a 50-amino acid sequence that contains a potential transmembrane domain followed by a consensus sequence for an N-linked glycosylation site.

Molecular cloning of two isoforms of the plasma membrane Ca2+-transporting ATPase from rat brain. Structural and functional domains exhibit similarity to Na+,K+- and other cation transport ATPases.

Complementary DNAs for two isoforms of the plasma membrane Ca2+-ATPase from rat brain have been isolated. The cDNAs were identified using an oligonucleotide probe derived from a conserved amino acid sequence of the ATP binding site of the aspartylphosphate family of transport ATPases. The complete nucleotide sequences have been determined, and the primary structures for both isoforms have been deduced. The Mr of isoform 1, which has 1,176 amino acids, is 129,500 and that of isoform 2, which has 1,198 amino acids, is 132,605. A region of isoform 1 exhibits perfect amino acid identity with a fragment consisting of 17 amino acids from the phosphorylation domain of the human erythrocyte calmodulin-sensitive Ca2+-ATPase (James, P., Zvaritch, E.I., Shakhparonov, M.I., Penniston, J.T., and Carafoli, E. (1987) Biochem. Biophys. Res. Commun. 149, 7-12). A comparison of transport ATPases from diverse species has allowed the identification of a sequence that may form part of a second ATP binding site. Amino acid similarity and hydropathy profile comparisons suggest that the transmembrane organization of the plasma membrane Ca2+-ATPase is the same as that of the Na+,K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase; the data indicate that each of these enzymes has an even number of transmembrane domains and that their C termini are located on the cytoplasmic side of the membrane. An arginine-rich sequence that is highly characteristic of the "A" domain of a calmodulin binding site is located near the C termini of both isoforms. This putative A domain is identical in isoforms 1 and 2 but their "B" domains, and the remaining C-terminal sequences, are very different. However, 3'-untranslated sequences of the isoform 1 transcript have the potential to encode a calmodulin binding B domain and a C terminus that is very similar to that of isoform 2. This suggests the possibility that additional diversity may occur via alternative processing of the primary transcript.

Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha-subunit from rat brain.

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.

Histidinol dehydrogenase (his D) mutants of Salmonella typhimurium.

A multidisciplinary analysis has been applied to over 150 hisD mutants of Salmonella typhimurium in a study of gene-enzyme relationship. The mutants were examined for production of immunologically cross-reacting material by using antibody to purified histidinol dehydrogenase, and for genetic complementation by using a set of F' factors bearing Escherichia coli hisD complementing mutants. Classifications as to missense, nonsense, frameshift, or deletion mutant are proposed on the basis of mutagenesis and suppression tests. For the suppression tests the mutants were examined both by a simultaneous suppression technique and by testing for response to E. coli F' factors bearing a recessive lethal amber and a recessive lethal ochre suppressor. The data are interpreted in relation to the position of the mutations in the recombination and complementation maps and in relation to the known composition of histidinol dehydrogenase. The gene hisD appears to be single cistron for the production of a single biosynthetic polypeptide.