NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

In chloride-secretory epithelia, the basolateral Na-K2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl ؊ and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K ؉ that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1 ؊/؊ ) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl ؊ channel, were reduced in jejunum, cecum, and trachea of Nkcc1 ؊/؊ mice, indicating that NKCC1 contributes to cAMP-induced Cl ؊ secretion. In contrast, secretion of gastric acid in adult Nkcc1 ؊/؊ stomachs and enterotoxin-stimulated fluid secretion in the intestine of suckling Nkcc1 ؊/؊ mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K ؉ movements involved in generation of the K ؉ -rich endolymph and the endocochlear potential.

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.

The relative importance of plasma membrane Ca 2؉ -ATPase (PMCA) 1 and PMCA4 was assessed in mice carrying null mutations in their genes (Atp2b1 and Atp2b4). Loss of both copies of the gene encoding PMCA1 caused embryolethality, whereas heterozygous mutants had no overt disease phenotype. Despite widespread and abundant expression of PMCA4, PMCA4 null (Pmca4 ؊/؊ ) mutants exhibited no embryolethality and appeared outwardly normal. Loss of PMCA4 impaired phasic contractions and caused apoptosis in portal vein smooth muscle in vitro; however, this phenotype was dependent on the mouse strain being employed. Pmca4 ؊/؊ mice on a Black Swiss background did not exhibit the phenotype unless they also carried a null mutation in one copy of the Pmca1 gene. Pmca4 ؊/؊ male mice were infertile but had normal spermatogenesis and mating behavior. Pmca4؊/؊ sperm that had not undergone capacitation exhibited normal motility but could not achieve hyperactivated motility needed to traverse the female genital tract. Ultrastructure of the motility apparatus in Pmca4 ؊/؊ sperm tails was normal, but an increased incidence of mitochondrial condensation indicated Ca 2؉ overload. Immunoblotting and immuno-histochemistry showed that PMCA4 is the most abundant isoform in testis and sperm and that it is localized to the principle piece of the sperm tail, which is also the location of the major Ca 2؉ channel (CatSper) required for sperm motility. These results are consistent with an essential housekeeping or developmental function for PMCA1, but not PMCA4, and show that PMCA4 expression in the principle piece of the sperm tail is essential for hyperactivated motility and male fertility.

We have isolated and characterized a complementary DNA for the catalytic subunit of the sheep kidney sodium/potassium-dependent ATPase. The 1,016-amino-acid protein seems to have eight transmembrane domains. The apparent ouabain binding site is located at the extracellular junction of two transmembrane domains and is linked to the phosphorylation site by a 60-amino-acid conserved sequence that may be a major channel for energy transduction.