It is well accepted that inhibition of the Na,K-ATPase in the heart, through effects on the Na/Ca exchanger, raises the intracellular Ca2+ concentration and strengthens cardiac contraction. However, the contribution that individual isoforms make to this calcium regulatory role is unknown. Assessing the phenotypes of mouse hearts with genetically reduced levels of Na,K-ATPase alpha 1 or alpha 2 isoforms clearly demonstrates different functional roles for these isoforms in vivo. Heterozygous alpha 2 hearts are hypercontractile as a result of increased calcium transients during the contractile cycle. In contrast, heterozygous alpha 1 hearts are hypocontractile. The different functional roles of these two isoforms are further demonstrated since inhibition of the alpha 2 isoform with ouabain increases the contractility of heterozygous alpha 1 hearts. These results definitively illustrate a specific role for the alpha 2 Na,K-ATPase isoform in Ca2+ signaling during cardiac contraction.

The Na,K-ATPase, a member of the P-type ATPases, is composed of two subunits, ␣ and ␤, and is responsible for translocating Na ؉ out of the cell and K ؉ into the cell using the energy of hydrolysis of one molecule of ATP. The electrochemical gradient it generates is necessary for many cellular functions, including establishment of the plasma membrane potential and transport of sugars and ions in and out of the cell. Families of isoforms for both the ␣ and ␤ subunits have been identified, and specific functional roles for individual isoforms are just beginning to emerge. The ␣4 isoform is the most recently identified Na,K-ATPase ␣ isoform, and its expression has been found only in testis. Here we show that expression of the ␣4 isoform in testis is localized to spermatozoa and that inhibition of this isoform alone eliminates sperm motility. These data describe for the first time a biological function for the ␣4 isoform of the Na,KATPase, revealing a critical role for this isoform in sperm motility.

The NBC1 Na ؉ /HCO 3 ؊ cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO 3 ؊ absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO 3 ؊ secretion in the intestinal tract, null mutant (NBC1 /HCO 3 Ϫ cotransporters (1-3). NBC1 has two protein variants, which localize to basolateral membranes (4) and mediate electrogenic Na ϩ /HCO 3 Ϫ cotransport (2, 3). The kNBC1 variant is expressed in kidney epithelia and eye (4, 5), and the pNBC1 variant is expressed in pancreas, duodenum, colon, and several other tissues (4 -8). The stoichiometry of the transporter can be altered from 1NaϪ by phosphorylation of a residue near the carboxyl terminus (9). In the kidney, the ion stoichiometry and electrochemical driving forces for NBC1 result in Na ϩ and HCO 3 Ϫ extrusion across the basolateral membrane (2, 3, 9, 10); thus, in the kidney, NBC1 functions in HCO 3 Ϫ reabsorption in the proximal tubule (2, 3). In pancreas and the intestinal tract, the ion stoichiometry and driving forces for NBC1 appear to result in Na ϩ and HCO 3 Ϫ entry into the cell (2, 8); thus, in intestine and colon, NBC1 has been proposed to mediate HCO 3 Ϫ uptake across the basolateral membrane to support transepithelial anion secretion (8, 11).Human patients with proximal renal tubular acidosis resulting from mutations in NBC1 have been reported (12-16), thereby confirming a bicarbonate-absorptive role for NBC1 in kidney. The primary mutations were single amino acid substitutions (R298S, T485S, R510H, A799V, R881C, and S427L), which appeared to cause decreased function of the cotransporter rather than loss of function (12)(13)(14). One patient had an inactivating mutation in the unique N terminus of the kidney NBC1 variant (Q29X), but the pancreatic variant, which is expressed in many other tissues and at low levels in kidney (4), was intact (15). Only a single patient has been identified with a complete inactivating mutation, a nucleotide deletion that causes a frameshift at codon 721 (16). The pRTA resulting from NBC1 mutations clearly shows that this transporter is essential for renal HCO 3 Ϫ absorption; however, clinically significant intestinal disease has not been reported.NBC1 has been localized to the basolateral membrane of epithelial cells lining both the small and large intestine (8,17,18). In the colon, its expression was greatest in crypt cells, consist-* This work was supported by National Institutes of Health (NIH) Grants DK50594 and HL61974 (to G. E. S.), DK67749 (to L. R. G.), DK57552 (to J. N. L.), DK48816 (to L. L. C.), and T32-RR-07004 (to J. E. S.) and NIEHS, NIH, Grant ES06096 (to the Center for Environmental Genetics, Alvaro Puga PI). The costs of publication of this article were defrayed in part by the payment of page charges. This article mus...

The AE2 Cl ؊ /HCO 3 ؊ exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for HCO 3 ؊ efflux and Cl ؊ influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2 ؊/؊ ) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2 ؊/؊ mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2 ؊/؊ mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2 ؊/؊ gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.

Sodium/proton exchangers [Na(+)/H(+) (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na(+) and Cl(-) flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(-)], and NHE3 knockout [NHE3(-)] mice. Under basal conditions, NHE(+) and NHE2(-) jejuna had similar rates of net Na(+) (approximately 6 microeq/cm(2) x h) and Cl(-) (approximately 3 microeq/cm(2) x h) absorption. In contrast, NHE3(-) jejuna had reduced net Na(+) absorption (approximately 2 microeq/cm(2) x h) but absorbed Cl(-) at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net Na(+) and Cl(-) absorption in all genotypes. Studies of the Na(+) absorptive flux (J) indicated that J in NHE(+) jejunum was not sensitive to 1 microM EIPA, whereas J in NHE3(-) jejunum was equally sensitive to 1 and 100 microM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMP(i)) abolished net NaCl absorption and stimulated electrogenic Cl(-) secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(-) and NHE3(-) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na(+) absorption; 2) an amiloride-sensitive Na(+) transporter partially compensates for Na(+) absorption in NHE3(-) jejunum; 3) cAMP(i) stimulation abolishes net Na(+) absorption in NHE(+), NHE2(-), and NHE3(-) jejunum; and 4) electroneutral Cl(-) absorption is not directly dependent on either NHE2 or NHE3.