Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice.

The amiloride-sensitive epithelial sodium channel, ENaC, is a heteromultimeric protein made up of three homologous subunits (alpha, beta and gamma) (1,2). In vitro, assembly and expression of functional active sodium channels in the Xenopus oocyte is strictly dependent on alpha-ENaC--the beta and gamma subunits by themselves are unable to induce an amiloride-sensitive sodium current in this heterologous expression system (2). In vivo, ENaC constitutes the limiting step for sodium absorption in epithelial cells that line the distal renal tubule, distal colon and the duct of several exocrine glands. The adult lung expresses alpha, beta and gamma ENaC (3,4), and an amiloride-sensitive electrogenic sodium reabsorption has been documented in upper and lower airways (3-7), but it is not established whether this sodium transport is mediated by ENaC in vivo. We inactivated the mouse alpha-ENaC gene by gene targeting. Amiloride-sensitive electrogenic Na+ transport was abolished in airway epithelia from alpha-ENaC(-/-) mice. Alpha-ENaC(-/-) neonates developed respiratory distress and died within 40 h of birth from failure to clear their lungs of liquid. This study shows that ENaC plays a critical role in the adaptation of the newborn lung to air breathing.

Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1.

Autosomal recessive pseudohypoaldosteronism type I is a rare life-threatening disease characterized by severe neonatal salt wasting, hyperkalaemia, metabolic acidosis, and unresponsiveness to mineralocorticoid hormones. Investigation of affected offspring of consanguineous union reveals mutations in either the alpha or beta subunits of the amiloride-sensitive epithelial sodium channel in five kindreds. These mutations are homozygous in affected subjects, co-segregate with the disease, and introduce frameshift, premature termination or missense mutations that result in loss of channel activity. These findings demonstrate the molecular basis and explain the pathophysiology of this disease.

Maturation of the catalytic alpha-subunit of Na,K-ATPase during intracellular transport.

The protease sensitivity of the catalytic alpha-subunit of Na,K-ATPase during intracellular transport along the exocytic pathway has been investigated in two amphibian epithelial cell lines. Controlled trypsinolysis followed by immunoprecipitation of cell homogenates or microsomal fractions from [35S]methionine pulse-chased A6 kidney cells revealed distinct cleavage patterns by SDS-PAGE. Shortly after synthesis (7-min pulse), the 98-kD alpha-subunit is fully sensitive to trypsin digestion and is cleaved into a 35-kD membrane-bound and a 27.5-kD soluble peptide. With a 15-min pulse, 10% of the newly synthesized polypeptide becomes resistant to trypsin digestion. With longer chase time, the proportion of protease-resistant alpha-subunit further increases. Concomitantly, the alpha-subunit acquires the ability to undergo cation-dependent conformational transitions, as reflected by distinct tryptic digest patterns in the presence of Na+ or K+. Similar results were obtained in TBM cells, a toad bladder cell line. Our data indicate that the catalytic subunit of Na,K-ATPase is structurally rearranged during intracellular transport from its site of synthesis to its site of action at the cell surface, a modification which might mark the functional maturation of the enzyme.

Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of a novel H,K-ATPase.

The kidney plays an essential role in regulating potassium and acid balance. A major site for these regulations is in the collecting tubule. In the present study, we report the primary sequence of a novel alpha subunit of the P-ATPase gene family, which we isolated from the urinary bladder epithelium of the toad Bufo marinus, the amphibian equivalent of the mammalian collecting tubule. The cDNA encodes a protein of 1,042 amino acids which shares approximately 67% identity with the alpha 1 subunit of the ouabain-inhibitable Na,K-ATPase and approximately 69% identity with the alpha subunit of the SCH28080-inhibitable gastric H,K-ATPase. When coexpressed in Xenopus oocytes with a beta subunit isolated from the same cDNA library, the ATPase is able to transport rubidium (a potassium surrogate) inward, and hydrogen outward, leading to alkalization of the intracellular compartment and acidification of the external medium. The novel ATPase has a unique pharmacological profile showing intermediate sensitivity to both ouabain and SCH28080. Our findings indicate that the bladder ATPase is a member of a new ion motive P-ATPase subfamily. The bladder ATPase is expressed in the urinary tract but not in the stomach or the colon. This H,K-ATPase may be one of the molecules involved in H+ and K+ homeostasis, mediating the transport of these ions across urinary epithelia and therefore regulating their urinary excretion.

Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.

Role of the transmembrane and extracytoplasmic domain of beta subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps.

The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an alpha and a beta subunit. The H,K-ATPase beta subunit (beta HK) can partially act as a surrogate for the Na,K-ATPase beta subunit (beta NK) in the formation of functional Na,K-pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of beta NK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase alpha subunits (alpha NK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus beta NK and rabbit beta HK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (beta NK/HK, beta HK/NK). We have expressed these constructs with or without coexpression of alpha NK in the Xenopus oocyte. In the absence of alpha NK, Xenopus beta NK and all chimera that contained the ectodomain of beta NK were retained in the ER while beta HK and all chimera with the ectodomain of beta HK could leave the ER suggesting that ER retention of unassembled Xenopus beta NK is mediated by a retention signal in the ectodomain. When coexpressed with alpha NK, only beta NK and beta NK/HK chimera assembled efficiently with alpha NK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. beta HK or chimera with the transmembrane domain of beta HK assembled less efficiently with alpha NK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of beta NK is important for efficient assembly with alpha NK and that both the transmembrane and the ectodomain of beta subunits play a role in modulating the transport activity of Na,K-pumps.

Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity.

High resistance epithelia express on their apical side an amiloride-sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride-sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613-10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function.

Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells.

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosterone-induced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K+-ATPase was mainly mediated by a two- to fourfold accumulation of both alpha- and beta-subunit mRNAs. The specific competitor spironolactone decreased basal Na+ transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosterone-dependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.

CFTR as a cAMP-dependent regulator of sodium channels.

Cystic fibrosis transmembrane regulator (CFTR), the gene product that is mutated in cystic fibrosis (CF) patients, has a well-recognized function as a cyclic adenosine 3',5'-monophosphate (cAMP)-regulated chloride channel, but this property does not account for the abnormally high basal rate and cAMP sensitivity of sodium ion absorption in CF airway epithelia. Expression of complementary DNAs for rat epithelial Na+ channel (rENaC) alone in Madin Darby canine kidney (MDCK) epithelial cells generated large amiloride-sensitive sodium currents that were stimulated by cAMP, whereas coexpression of human CFTR with rENaC generated smaller basal sodium currents that were inhibited by cAMP. Parallel studies that measured regulation of sodium permeability in fibroblasts showed similar results. In CF airway epithelia, the absence of this second function of CFTR as a cAMP-dependent regulator likely accounts for abnormal sodium transport.

Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system.

Liddle syndrome is an autosomal dominant form of hypertension resulting from deletion or missense mutations of a PPPxY motif in the cytoplasmic COOH terminus of either the beta or gamma subunit of the epithelial Na channel (ENaC). These mutations lead to increased channel activity. In this study we show that wild-type ENaC is downregulated by intracellular Na+, and that Liddle mutants decrease the channel sensitivity to inhibition by intracellular Na+. This event results at high intracellular Na+ activity in 1.2-2.4-fold higher cell surface expression, and 2.8-3.5-fold higher average current per channel in Liddle mutants compared with the wild type. In addition, we show that a rapid increase in the intracellular Na+ activity induced downregulation of the activity of wild-type ENaC, but not Liddle mutants, on a time scale of minutes, which was directly correlated to the magnitude of the Na+ influx into the oocytes. Feedback inhibition of ENaC by intracellular Na+ likely represents an important cellular mechanism for controlling Na+ reabsorption in the distal nephron that has important implications for the pathogenesis of hypertension.

Liddle disease caused by a missense mutation of beta subunit of the epithelial sodium channel gene.

Mutations in beta or gamma subunit of the epithelial sodium channel (ENaC) have been found to cause a hereditary form of human hypertension, Liddle syndrome. Most of the mutations reported are either nonsense mutations or frame shift mutations which would truncate the cytoplasmic carboxyl terminus of the beta or gamma subunits of the channel, suggesting that these domains are important for the normal regulation of this channel. We sequenced ENaC in a family with Liddle syndrome and found a missense mutation in beta subunit which predicts substitution of Tyr by His at codon 618, 2 bp downstream from a missense mutation (P616L) that has been reported recently. Presence of this mutation correlates with the clinical manifestations (hypertension, hypokalemia, suppressed aldosterone secretion) in this kindred. Functional expression studies in the Xenopus oocytes revealed constitutive activation of the Y618H mutant indistinguishable from that observed for the deletion mutant (R564stop) identified in the original pedigree of Liddle. Our data suggest that the region between Pro616 and Tyr618 is critically important for regulation of ENaC activity.

Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its alpha subunit.

The autosomal recessive form of type I pseudohypoaldosteronism (PHA-I) is an inherited salt-losing syndrome resulting from diminution-of-function mutations in the 3 subunits of the epithelial Na+ channel (ENaC). A PHA-I stop mutation (alpha(R508stop)) of the ENaC alpha subunit is predicted to lack the second transmembrane domain and the intracellular COOH-terminus, regions of the protein involved in pore function. Nonetheless, we observed a measurable Na+ current in Xenopus laevis oocytes that coexpress the beta and gamma subunits with the truncated alpha subunit. The mutant alpha was coassembled with beta and gamma subunits and was present at the cell surface at a lower density, consistent with the lower Na+ current seen in oocytes with the truncated alpha subunit. The single-channel Na+ conductance for the mutant channel was only slightly decreased, and the appearance of the macroscopic currents was delayed by 48 hours with respect to wild-type. Our data suggest novel roles for the alpha subunit in the assembly and targeting of an active channel to the cell surface, and suggest that channel pores consisting of only the beta and gamma subunits can provide significant residual activity. This activity may be sufficient to explain the absence of a severe pulmonary phenotype in patients with PHA-I.

Purification and characterization of (Na+ + K+)-ATPase from toad kidney.

This report describes the partial purification and the characteristics of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from an amphibian source. Toad kidney microsomes were solubilized with sodium deoxycholate and further purified by sodium dodecyl sulphate treatment and sucrose gradient centrifugation, according to the methods described by Lane et al. [(1973) J. Biol. Chem. 248, 7197--7200], Jørgensen [(1974) Biochim. Biophys. Acta 356, 36--52] and Hayashi et al. [(1977) Biochim. Biophys. Acta 482, 185--196]. (Na+ + K+)-ATPase preparations with specific activities up to 1000 mumol Pi/mg protein per h were obtained. Mg2+-ATPase only accounted for about 2% of the total ATPase activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed three major protein bands with molecular weights of 116 000, 62 000 and 26 000. The 116 000 dalton protein was phosphorylated by [gamma-32P]ATP in the presence of sodium but not in the presence of potassium. The 62 000 dalton component stained for glycoproteins. The Km for ATP was 0.40 mM, for Na+ 12.29 mM and for K+ 1.14 mM. The Ki for ouabain was 35 micron. Temperature activation curves showed two activity peaks at 37 degrees C and at 50 degrees C. The break in the Arrhenius plot of activity versus temperature appeared at 15 degrees C.

Structural organization of alpha-subunit from purified and microsomal toad kidney (Na+ + K+)-ATPase as assessed by controlled trypsinolysis.

The membrane organization of the alpha-subunit of purified (Na+ + K+)-ATPase ((Na+ + K+)-dependent adenosine triphosphate phosphorylase, EC 3.6.1.3) and of the microsomal enzyme of the kidney of the toad Bufo marinus was compared by using controlled trypsinolysis. With both enzyme preparations, digestions performed in the presence of Na+ yielded a 73 kDa fragment and in the presence of K+ a 56 kDa, a 40 kDa and small amounts of a 83 kDa fragment from the 96 kDa alpha-subunit. In contrast to mammalian preparations (Jørgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415), trypsinolysis of the purified amphibian enzyme led to a biphasic loss of (Na+ + K+)-ATPase activity in the presence of both Na+ and K+. These data could be correlated with an early rapid cleavage of 3 kDa from the alpha-subunit in both ionic conditions and a slower degradation of the remaining 93 kDa polypeptide. On the other hand, in the microsomal enzyme, a 3 kDa shift of the alpha-subunit could only be produced in the presence of Na+. Our data indicate that (1) purification of the amphibian enzyme with detergent does not influence the overall topology of the alpha-subunit but produces a distinct structural alteration of its N-terminus and (2) the amphibian kidney enzyme responds to cations with similar conformational transitions as the mammalian kidney enzyme. In addition, anti alpha-serum used on digested enzyme samples revealed on immunoblots that the 40 kDa fragment was better recognized than the 56 kDa fragment. It is concluded that the NH2-terminal of the alpha-subunit contains more antigenic sites than the COOH-terminal domain in agreement with the results of Farley et al. (Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906).

Aldosterone-induced glycoproteins: electrophysiological-biochemical correlation.

Aldosterone induces the synthesis of a group of glycoproteins (GP65,70) in toad urinary bladders which are potential effectors of the natriferic action of this hormone. In the present study we have confirmed that aldosterone produces a two-phase electrophysiological response. During the early phase (less than 3 h) short-circuit current and transepithelial conductance increase in parallel, while during the late phase (greater than 3 h) short-circuit current continues to increase without any further change in conductance. By biosynthetically labeling aldosterone-treated toad bladders with [35S]methionine either during the early (h 0-2 or 1-3) or the late (h 4-6 or 7-9) phases of the natriferic response, we have demonstrated that GP65,70 is synthesized as a late effect of aldosterone. Since synthesis of GP65,70 occurs at a time when the electromotive force of the Na+ pump is increasing, and since GP65,70 biochemically resembles the beta subunit of Na+/K+-ATPase, studies were undertaken to examine whether GP65,70 is the beta subunit. Purified amphibian renal beta subunit was analyzed by two-dimensional polyacrylamide gel electrophoresis and was found to have an isoelectric point and Mr value similar to those of GP65,70. However, when nitrocellulose blots containing wheat germ agglutinin-purified proteins from aldosterone-treated bladders were stained with monospecific polyclonal antibodies developed against the beta subunit, GP65,70 was not recognized, whereas a group of slightly more acidic proteins of similar Mr were recognized. Thus, GP65,70 is not the beta subunit of Na+/Ka+-ATPase. Further studies are needed to determine the cellular function of GP65,70.

Modulation of the Na,K-pump function by beta subunit isoforms.

To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K-pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.

Aldosterone induces a rapid increase in the rate of Na,K-ATPase gene transcription in cultured kidney cells.

Aldosterone (300 nM) induces a 4-fold increase over a 6-hr stimulation in A6 kidney cells from Xenopus laevis in the abundance of mRNA beta 1 and a 2-fold in that of mRNA alpha 1 coding for each Na,K-ATPase subunit, which is in agreement with a previous report. After a 3-hr stimulation already, aldosterone elicited a significant increase of mRNA beta 1 (2.51-fold +/- 0.47, n = 3, P less than 0.05) and a nonsignificant increase of mRNA alpha 1 (1.26-fold +/- 0.30, n = 3, NS). Increasing doses of cycloheximide up to 3 micrograms ml-1 led to 90% inhibition of protein synthesis, but failed to block the differential effect of aldosterone on mRNA abundance over a 6-h incubation period. The rate of transcription was measured by a nuclear run-on assay. The basal rate of mRNA alpha 1 transcription exceeded that of mRNA beta 1 by 2.8-fold. Aldosterone (300 nM) stimulated the transcription of the two subunit genes. Fifteen minutes after aldosterone addition there was a significant and parallel increase in the rate of transcription of the alpha 1 subunit (1.98-fold +/- 0.20, n = 3, P less than 0.02) and that of the beta 1 subunit (2.13 +/- 0.32, n = 3, P less than 0.04). After a 45-min stimulation period the transcription rate of the alpha 1 subunit remained at the level observed at 15 min (1.84-fold +/- 0.14, n = 4, P less than 0.01), while the transcription rate of the beta 1 subunit increased further (2.89-fold +/- 0.38, n = 4, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Rat liver 11 beta-hydroxysteroid dehydrogenase complementary deoxyribonucleic acid encodes oxoreductase activity in a mineralocorticoid-responsive toad bladder cell line.

The mineralocorticoid receptor displays equal affinity for aldosterone and corticosterone. It has been proposed that aldosterone selectivity in vivo is achieved by the conversion of corticosterone into its inactive metabolite 11-dehydrocorticosterone by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). To test this hypothesis, we transfected rat liver 11 beta HSD cDNA into TBM cells, a sodium-transporting cell line. These cells respond equally well to aldosterone and corticosterone, indicating that endogenous 11 beta HSD is expressed at low levels in TBM cells. Although exogenous rat liver 11 beta HSD was expressed at high levels in transfected cells, mineralocorticoid selectivity was not observed. By contrast, the biologically inactive 11-dehydrocorticosterone was readily converted into corticosterone, a potent agonist for sodium transport. Our results indicate that rat liver 11 beta HSD behaves predominantly as a reductase in TBM cells. Another 11 beta HSD isoform is likely to be responsible for the dehydrogenase reaction in aldosterone-responsive cells.

Aldosterone regulation of gene transcription leading to control of ion transport.

Aldosterone, like other steroid hormones, initiates its effects by binding to intracellular receptors; these receptors are then able to control the transcription of several genes. The products of these genes eventually modulate the activity of ionic transport systems located in the apical and the basolateral membrane of specialized epithelial cells, thereby modulating the excretion of Na+ and K+ ions. Considerable progress has been made recently in understanding these mechanisms and the structure of the proteins involved in these processes. A novel principle has been discovered to explain the selective effect of aldosterone on its target epithelia. These tissues exclude competing glucocorticoid hormones by the activity of the 11 beta-hydroxysteroid dehydrogenase to allow aldosterone, an enzyme-resistant steroid, to bind to its receptors. Aldosterone induces numerous changes in the activity of membrane ion transport systems and enzymes and cell morphology. Although the enhancement of Na,K-ATPase synthesis and the increase of the number of active Na+ channels in the apical membrane appear as both direct and primary effects, the mechanisms of the other effects remain to be determined. The knowledge of the primary structure of several elements of the aldosterone response system (e.g., mineralocorticoid receptor and Na,K-ATPase) allows us to understand abnormal regulation of Na+ balance at the molecular level and, potentially, to identify genetic alterations responsible for these defects.

Functional expression of N-terminal truncated alpha-subunits of Na,K-ATPase in Xenopus laevis oocytes.

N-terminal deletion mutants of Na,K-ATPase alpha 1 isoforms initiating translation at Met34 (alpha 1T1) or at Met43 (alpha 1T2) were expressed in X. laevis oocytes. Compared to beta 3 cRNA injected controls, the co-expression of alpha 1wt, alpha 1T1, alpha 1T2 with beta 3 subunits results in a 2- to 3-fold increase of ouabain binding sites, parallelled by a concomitant increase in Na,K-pump current. The apparent K1/2 for potassium activation of the alpha 1T2/beta 3 Na,K-pumps is significantly higher than that of the alpha 1wt/beta 3 or alpha 1T1/beta 3 Na,K-pumps expressed at the cell surface. Total deletion of the lysine-rich N-terminal domain thus allows the expression of active Na,K-pump but with distinct cation transport properties.