Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Previous work suggested that the structural gene for the A system transporter and the mRNA for the alpha subunit of the Na+,K(+)-ATPase in Chinese hamster ovary cells CHO-K1 [wild type (WT)] are coordinately controlled by regulatory gene R1. This conclusion was based on analysis of a mutant for the A system, alar4. This mutant had a constitutive level of A system transport activity equal to the level found in derepressed WT cells and a 4 times increase in abundance of the alpha 1 subunit of Na+,K(+)-ATPase mRNA over that found in repressed WT. The level of Na+ per cell in alar4 was not significantly greater than that found in the WT. To further characterize the likely coregulation of both genes, we have studied the A system activity and Na+,K(+)-ATPase mRNA alpha 1-subunit levels in cells grown under various conditions that result in repression or derepression of the A system in the WT. System A activity increased up to 2-3 times the basal transport rate (repressed state) and Na+,K(+)-ATPase mRNA alpha 1-subunit levels showed a 3-fold increase after amino acid starvation (derepressed state). These changes occurred along with a decrease in intracellular Na+ levels. N-Methyl-alpha-aminoisobutyric acid and beta-alanine, previously shown to be corepressors for the A system, prevented to a similar extent A system derepression and Na+,K(+)-ATPase mRNA alpha 1-subunit accumulation. On the other hand, phenylalanine and lysine, amino acids that are not corepressors of the A system, failed to significantly prevent derepression of both genes. Hybrids between the WT and alar4 have the phenotype of the WT when grown under repressed conditions. These results give further support to the proposition that both the A system transporter and mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase are coordinately controlled by regulatory gene R1 and elevated Na+ concentrations are not involved. No Na+,K(+)-ATPase activity was detected in derepressed cells. Activity was restored by the addition of monensin. However, this activity was no greater than that obtained in repressed cells. Indications are that the reduced Na+ content in derepressed cells inhibits Na+,K(+)-ATPase activity and that conditions that favored derepression do not allow for de novo synthesis of the Na+,K(+)-ATPase.     

Multiple genes encode the human Na+,K+-ATPase catalytic subunit.

A human genomic library was constructed and screened with hybridization probes derived from sheep and rat cDNAs encoding the alpha and alpha(+) isoforms, respectively, of the Na+,K+-ATPase catalytic subunit. Genomic sequences spanning 150 kilobases were isolated. Four genes, designated alpha A, alpha B, alpha C, and alpha D, each 20-25 kilobases in length, were identified by restriction mapping, Southern blot hybridization analysis, and limited DNA sequencing. We present evidence that two of these genes, alpha A and alpha B, encode the alpha and alpha(+) isoforms, respectively. The other genes, alpha C and alpha D, one of which is physically linked to the alpha(+) gene, exhibit nucleotide and amino acid homology to Na+,K+-ATPase catalytic subunit cDNA sequences but do not correspond to any previously identified isoforms.     

The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.

Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity, although the activity was lower than that of the wild-type SR Ca(2+)-ATPase. Moreover, this Ca(2+)-sensitive ATPase activity was inhibited by ouabain. The chimera NCC, in which Met1-Gly354 of the SR Ca(2+)-ATPase were replaced with the corresponding portion of the Na+,K(+)-ATPase, lost the thapsigargin-sensitive Ca(2+)-ATPase activity seen in CCC and [n/c]CC. [3H]Ouabain binding to [n/c]CC and NCC demonstrated that the affinity for this inhibitor seen in the wild-type chicken Na+,K(+)-ATPase was restored in these chimeric molecules. Thus, the ouabain-binding domains are distinct from the thapsigargin sites; ouabain binds to the amino-terminal portion (Met1 to Asp200) of the Na+,K(+)-ATPase alpha 1 subunit, whereas thapsigargin interacts with the regions after Asp162 of the Ca(2+)-ATPase. Moreover, the amino-terminal 200 amino acids of the Na+,K(+)-ATPase alpha 1 subunit are sufficient to exert ouabain-dependent inhibition even after incorporation into the corresponding portion of the Ca(2+)-ATPase, and the segment Ile163 to Gly354 of the SR Ca(2+)-ATPase is critical for thapsigargin- and Ca(2+)-sensitive ATPase activity.     

High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit.

Recently, a beta subunit for the rat gastric H+,K(+)-ATPase (HK beta), which is structurally similar to the beta subunit of Na+, K(+)-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta subunit assembly with different isoforms of the alpha subunit of Na+, K(+)-ATPase. Coexpression in yeast cells of the HK beta with both the sheep alpha 1 subunit and the rat alpha 3 subunit isoforms of Na+, K(+)-ATPase (alpha 1 and alpha 3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha 1 plus HK beta, alpha 3 plus HK beta) have a high affinity for ouabain (Kd, 5-10 nM) and are expressed at levels similar to those formed with the rat beta 1 subunit of Na+, K(+)-ATPase (beta 1) (alpha 1 plus beta 1 or alpha 3 plus beta 1). Potassium acts as a specific antagonist of ouabain binding by alpha 1 plus HK beta and alpha 3 plus HK beta just like sodium pumps formed with beta 1. Sodium pumps formed with the HK beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta subunit may play a role in sodium pump function.     

Human placental Na+,K+-ATPase alpha subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization.

A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na+,K+-ATPase alpha subunit was cloned from human placenta and its sequence was identical to that encoding the alpha subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na+,K+-ATPase gene from various human tissues and cell lines revealed only one band (approximately 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine greater than placenta greater than liver greater than pancreas, and in the cell lines the levels were human erythroleukemia greater than butyrate-induced colon greater than colon greater than brain greater than HeLa cells. mRNA was undetectable in reticulocytes, consistent with our failure to detect positive clones in a size-selected (greater than 2 kilobases) lambda gt11 reticulocyte cDNA library. DNA analysis revealed a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the alpha subunit is on the short arm (band p11-p13) of chromosome 1.     

Phosphorylation of the catalytic subunit of Na+,K(+)-ATPase inhibits the activity of the enzyme.

We have examined two distinct protein kinases, cAMP-dependent protein kinase and protein kinase C, for their ability to phosphorylate and regulate the activity of three different types of Na+,K(+)-ATPase preparation. cAMP-dependent protein kinase phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 1 mol of phosphate per mol of alpha subunit. Protein kinase C phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 2 mol of phosphate per mol of alpha subunit. The phosphorylation by each of the kinases was associated with an inhibition of Na+,K(+)-ATPase activity of about 40-50%. These two protein kinases also inhibited the activity of a partially purified preparation of Na+,K(+)-ATPase from rat renal cortex and the activity of Na+,K(+)-ATPase present in preparations of basolateral membrane vesicles from rat renal cortex.     

Phosphoinositide-3 kinase binds to a proline-rich motif in the Na+, K+-ATPase alpha subunit and regulates its trafficking

Endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I(A) phosphoinositide-3 kinase (PI3K-I(A)) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I(A), through its p85alpha subunit-SH3 domain, binds to a proline-rich region in the Na(+),K(+)-ATPase catalytic alpha subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na(+),K(+)-ATPase alpha subunit and results in increased PI3K-I(A) activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na(+),K(+)-ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I(A) and its activation during Na(+),K(+)-ATPase endocytosis in response to G protein-coupled receptor signals.     

Gene transfer of the Na+,K+-ATPase beta1 subunit using electroporation increases lung liquid clearance

The development of nonviral methods for efficient gene transfer to the lung is highly desired for the treatment of several pulmonary diseases. We have developed a noninvasive procedure using electroporation to transfer genes to the lungs of rats. Purified plasmid (100-600 microg) was delivered to the lungs of anesthetized rats through an endotracheal tube, and a series of square-wave pulses were delivered via electrodes placed on the chest. Relatively uniform gene expression was observed in multiple cell types and layers throughout the lung, including airway and alveolar epithelial cells, airway smooth muscle cells, and vascular endothelial cells, and this finding was dose- and pulse length-dependent. Most important, no inflammatory response was detected. To demonstrate efficacy of this approach, the beta1 subunit of the Na(+),K(+)-ATPase was transferred to the lungs of rats with or without electroporation, and 3 days later, alveolar fluid clearance was measured. Animals electroporated with the beta1 subunit plasmid showed a twofold increase in alveolar fluid clearance and Na(+),K(+)-ATPase activity as compared with animals receiving all other plasmids, with or without electroporation. These results demonstrate that electroporation is an effective method to increase clearance by introducing therapeutic genes (Na(+),K(+)-ATPase) into the rat lung.     

High-affinity ouabain binding by a chimeric gastric H+,K+-ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the alpha 1-subunit of rat Na+,K+-ATPase

Na(+),K(+)-ATPase and gastric H(+),K(+)-ATPase are two related enzymes that are responsible for active cation transport. Na(+), K(+)-ATPase activity is inhibited specifically by ouabain, whereas H(+),K(+)-ATPase is insensitive to this drug. Because it is not known which parts of the catalytic subunit of Na(+),K(+)-ATPase are responsible for ouabain binding, we prepared chimeras in which small parts of the alpha-subunit of H(+),K(+)-ATPase were replaced by their counterparts of the alpha(1)-subunit of rat Na(+),K(+)-ATPase. A chimeric enzyme in which transmembrane segments 5 and 6 of H(+), K(+)-ATPase were replaced by those of Na(+),K(+)-ATPase could form a phosphorylated intermediate, but hardly showed a K(+)-stimulated dephosphorylation reaction. When transmembrane segments 3 and 4 of Na(+),K(+)-ATPase were also included in this chimeric ATPase, K(+)-stimulated dephosphorylation became apparent. This suggests that there is a direct interaction between the hairpins M3-M4 and M5-M6. Remarkably, this chimeric enzyme, HN34/56, had obtained a high-affinity ouabain-binding site, whereas the rat Na(+), K(+)-ATPase, from which the hairpins originate, has a low affinity for ouabain. The low affinity of the rat Na(+),K(+)-ATPase previously had been attributed to the presence of two charged amino acids in the extracellular domain between M1 and M2. In the HN34/56 chimera, the M1/M2 loop, however, originates from H(+),K(+)-ATPase, which has two polar uncharged amino acids on this position. Placement of two charged amino acids in the M1/M2 loop of chimera HN34/56 results in a decreased ouabain affinity. This indicates that although the M1/M2 loop affects the ouabain affinity, binding occurs when the M3/M4 and M5/M6 hairpins of Na(+),K(+)-ATPase are present.     

Inhibition of Na+,K+-ATPase by interferon gamma down-regulates intestinal epithelial transport and barrier function

BACKGROUND & AIMS: To determine how interferon (IFN)-gamma inhibits epithelial barrier and ion transport functions, intestinal T84 cells were studied. METHODS: Acute and chronic effects of IFN-gamma on T84 barrier function, Na+,K+-adenosine triphosphatase (ATPase) activity, and certain ion transport and tight junctional proteins were determined. To assess the role of Na+,K+-ATPase and intracellular Na+, similar studies with the Na+,K+-ATPase inhibitor ouabain and Na+ ionophore monensin were performed. To determine the role of nitric oxide (NO), the NO donor SPER-NO was used. RESULTS: IFN-gamma acutely (<6 hour) decreased cellular Na+,K+-ATPase activity, followed later (>24 hours) by decreases in expression of Na/K/2Cl, the alpha subunit of Na+,K+-ATPase, occludin, and ZO-1. In contrast, cystic fibrosis transmembrane conductance regulator or the Na+ pump beta subunit were unchanged. Ouabain and monensin caused nearly identical changes to IFN-gamma. Incubation in low Na+ media significantly blunted the chronic effects of IFN-gamma. Hypotonic-induced cell swelling, in contrast, had effects similar to IFN-gamma but did not alter the expression of the Na+ pump alpha subunit. The NO donor SPER-NO rapidly inhibited Na+,K+-ATPase and also down-regulated transport and barrier proteins. CONCLUSIONS: IFN-gamma inhibition of Na+,K+-ATPase activity acutely causes increases in intracellular Na(i) concentration and cell volume, which are distinct signaling events that ultimately result in a leaky and dysfunctional epithelium associated with chronic inflammation.     

Membrane disposition of the M5-M6 hairpin of Na+,K(+)-ATPase alpha subunit is ligand dependent.

Extensive proteolytic digestion of Na+,K(+)-ATPase (EC 3.6.1.37) by trypsin produces a preparation where most of the extramembrane portions of the alpha subunit have been digested away and the beta subunit remains essentially intact. The fragment Gln-737-Arg-829 of the Na+,K(+)-ATPase alpha subunit, which includes the putative transmembrane hairpin M5-M6, is readily, selectively, and irreversibly released from the posttryptic membrane preparation after incubation at 37 degrees C for several minutes. Once released from the membrane, the fragment aggregates but remains water soluble. Occlusion of K+ or Rb+ specifically prevents release of the Gln-737-Arg-829 fragment into the supernatant. Labeling of the posttryptic membrane preparation with cysteine-directed reagents revealed that Cys-802 (which is thought to be located within the M6 segment) is protected against the modification by Rb+ while this fragment is in the membrane but can be readily modified upon release. Cation occlusion apparently alters the folding and/or disposition of the M5-M6 fragment in the membrane in a way that does not occur when the fragment migrates to the aqueous phase. The ligand-dependent disposition of the M5-M6 hairpin in the membrane along with recent labeling studies suggest a key role for this segment in cation pumping by Na+,K(+)-ATPase.     

Electroporation-mediated gene transfer of the Na+,K+ -ATPase rescues endotoxin-induced lung injury

RATIONALE: Acute lung injury and acute respiratory distress syndrome are common clinical syndromes resulting largely from the accumulation of and inability to clear pulmonary edema, due to injury to the alveolar epithelium. Gene therapy may represent an important alternative for the treatment and prevention of these diseases by restoring alveolar epithelial function. We have recently developed an electroporation strategy to transfer genes to the lungs of mice, with high efficiency and low inflammation. OBJECTIVES: We asked whether electroporation-mediated transfer of genes encoding subunits of the Na+,K+ -ATPase could protect from LPS-induced lung injury or be used to treat already injured lungs by up-regulating mechanisms of pulmonary edema clearance. METHODS: Plasmids were delivered to the lungs of mice using transthoracic electroporation. Lung injury was induced by intratracheal administration of LPS (4 mg/kg body weight). Biochemical, cellular, and physiologic measurements were taken to assess gene transfer and lung injury. MEASUREMENTS AND MAIN RESULTS: Improvements in wet-to-dry ratios, pulmonary effusions, bronchoalveolar lavage protein levels and cellularity, alveolar fluid clearance, and respiratory mechanics were seen after delivery of plasmids expressing Na+,K+ -ATPase subunits, but not control plasmids, in LPS-injured lungs. Delivery of plasmids expressing Na+,K+ -ATPase subunits both protected from subsequent lung injury and partially reversed existing lung injury by these measures. CONCLUSIONS: These results demonstrate that electroporation can be used effectively in healthy and injured lungs to facilitate gene delivery and expression. To our knowledge, this is the first successful use of gene delivery to treat existing lung injury, and may have future clinical potential.     

Alanine Prevents the Decrease of Na+,K+-ATPase Activity in Experimental Phenylketonuria

Our objective was to investigate the effect of alanine administration on Na⁺,K⁺-ATPase activity in cerebral cortex of rats subjected to chemically-induced phenylketonuria. Wistar rats were treated from the 6th to the 28th day of life with subcutaneous injections of either 2.6 mol alanine or 5.2 mol phenylalanine plus 2.6 mol -methylphenylalanine per g body weight or phenylalanine plus -methylphenylalanine plus alanine in the same doses or equivalent volumes of 0.15 M saline. The animals were killed on the 29th or 60th day of life. Synaptic plasma membrane from cerebral cortex was prepared for Na⁺,K⁺-ATPase activity determination. The results showed that alanine injection prevents the decrease of Na⁺,K⁺-ATPase activity in animals subjected to experimental phenylketonuria. Therefore, in case the same effects are achieved with ingested alanine, it is possible that alanine supplementation may be an important dietary adjuvant for phenylketonuric patients.     

Inhibition of Na+, K+-ATPase Activity by the Metabolites Accumulating in Homocystinuria

Homocystinuria is an inborn error of sulfur amino acid metabolism characterized predominantly by vascular and nervous system dysfunction. In this study we determined the in vitro effects of homocysteine and methionine, metabolites which accumulate in homocystinuria, on Na⁺, K⁺-ATPase, and Mg²⁺-ATPase activities in synaptic membranes from the hippocampus of rats. The results showed that both metabolites significantly inhibit Na⁺, K⁺-ATPase but not Mg²⁺-ATPase activity at concentrations usually observed in plasma of homocystinuric patients. Furthermore, incubation of hippocampal homogenates with homocysteine also elicited an inhibition of the enzyme activity which was however prevented by the simultaneous addition of cysteine to the medium. In addition, cysteine or methionine per se did not modify the two enzymatic activities. These findings indicate that oxidation of critical groups in the enzyme may possibly be involved in homocysteine inhibitory effect. Moreover, kinetic studies performed to investigate the interaction between homocysteine and methionine on Na⁺, K⁺-ATPase inhibition suggested a common site for the two amino acids in the enzyme. Considering the critical role exerted by Na⁺, K⁺-ATPase in brain, it is proposed that the inhibition provoked by homocysteine and methionine on the enzyme activity may be possibly related to the brain dysfunction characteristic of homocystinuria.     

The cardiac conduction system in the rat expresses the alpha 2 and alpha 3 isoforms of the Na+,K(+)-ATPase.

The sodium pump is crucial for the function of the heart and of the cardiac conduction system, which initiates the heartbeat. The alpha (catalytic) subunit of this pump has three isoforms; the alpha 1 isoform is ubiquitous, but the alpha 2 and alpha 3 isoforms are localized to excitable tissue. Because rodent alpha 2 and alpha 3 isoforms are relatively sensitive to ouabain, which also slows cardiac conduction, we studied heart-cell-specific expression of pump isoform genes. Multiple conduction-system structures, including sinoatrial node, bundle branches, and Purkinje strands, had prominent, specific hybridization signal for alpha 2 and alpha 3 isoforms compared with adjacent working myocytes. This gene-expression approach may be useful for labeling conduction tissue and also for localizing specific membrane channels and receptors in this system.     

Tissue specificity, localization in brain, and cell-free translation of mRNA encoding the A3 isoform of Na+,K+-ATPase.

The isolation of multiple Na+,K+-ATPase cDNAs from rat brain has led to the discovery of a family of alpha-isoform genes. Using A1 (alpha), A2 (alpha+), and A3 (alpha III) Na+,K+-ATPase gene probes, we have analyzed the distribution of Na+,K+-ATPase mRNAs in adult and fetal rat tissues by RNA blot and hybridization histochemistry. A1 Na+,K+-ATPase mRNA was found ubiquitously among various tissues, with highest levels in transport epithelial and neural tissues. A2 mRNA was found in adult neural and muscle tissues, and A3 mRNA was found only in neural tissues and fetal heart muscle. Both A1 and A2 mRNAs were less abundant in fetal brain than in adult brain; in contrast, A3 mRNA was abundant at both stages. In situ mapping of brain areas that contain A3 mRNA suggests that this Na+,K+-ATPase isoenzyme is expressed predominantly by neural cells. Analysis of Na+,K+-ATPase proteins generated by cell-free translation of synthetic mRNAs suggests that the A3 protein has properties similar to A2 (alpha+).     

Dopamine inhibits mammalian photoreceptor Na+,K+-ATPase activity via a selective effect on the alpha3 isozyme.

The rat retina contains dopaminergic interplexiform cells that send processes to the outer plexiform layer where dopamine is released in a light-dependent manner. We report herein that physiologically relevant concentrations of dopamine inhibited ouabain-sensitive photoreceptor oxygen consumption in dark- and light-adapted rat retinas and inhibited Na+,K+-ATPase specific activity (EC 3.6.1.37) in a rat rod outer-inner segment preparation. Experiments with the selective D1 agonist fenoldopam or D2 agonist quinpirole and experiments with dopamine plus either the D1 antagonist SCH23390 or D2/D4 antagonist clozapine showed that the inhibition of oxygen consumption and enzyme activity were mediated by D2/D4-like receptors. The amphetamine-induced release of dopamine, monitored by the inhibition of oxygen consumption, was blocked by L-2-amino-4-phosphonobutyric acid and kynurenic acid. Pharmacological and biochemical experiments determined that the IC50 values of ouabain for the alpha1-low and alpha3-high ouabain affinity isozymes of photoreceptor Na+,K+-ATPase were approximately 10(-5) and approximately 10(-7) M, respectively, and that the D2/D4-like mediated inhibition of Na+,K+-ATPase was exclusively selective for the alpha3 isozyme. The dopamine-mediated inhibition of alpha3 first occurred at 5 nM, was maximal at 100 microM (-47%), had an IC50 value of 382 +/- 23 nM, and exhibited negative cooperativity (Hill coefficient, 0.27). Prior homogenization of the rod outer-inner segment completely prevented the long-lasting inhibition, suggesting that the effect was coupled to a second messenger. Although the physiological significance of our findings to photoreceptor function is unknown, we hypothesize that these results may have relevance for the temporal tuning properties of rods.     

Regulation of endocytic pH by the Na+,K+-ATPase in living cells.

Acidification of endocytosed ligands destined for lysosomes is biphasic, with a rapid drop to pH 6, followed by a slow decrease to pH 5. Continuous measurements of transferrin acidification have confirmed that the pH minimum in early (presorting) endosomes is approximately pH 6. On the basis of measurements of endosomal acidification in vitro, it has been proposed that the pH in the early endosome is limited by the internalization of the Na+,K+-ATPase, which generates an interior-positive membrane potential in this compartment [Fuchs, R., Schmid, S. & Mellman, I. (1989) Proc. Natl. Acad. Sci. USA 86, 539-543]. We present two lines of evidence that strongly implicate the Na+,K+-ATPase as a major regulatory element of endocytic pH in vivo. First, ouabain, a specific inhibitor of the Na+,K+-ATPase, interferes with the regulation of acidification in early endocytic compartments. Transferrin is normally rapidly acidified to pH 6.0-6.2, followed by alkalinization during recycling. In the presence of ouabain, the minimum pH of transferrin-containing endosomes decreases from 6.0-6.2 to less than 5.3. Second, ouabain eliminates the resistance to both the growth inhibitory and vacuologenic effects of chloroquine in the lysosomal acidification defective cell line CHL60-64. The phenotype of this cell line is consistent with a defect in the removal or inactivation of the early acidification regulatory elements from the late endocytic compartments. The ouabain data suggest that the defect in this cell line is due to improper localization of the Na+,K+-ATPase. A model for pH regulation and vacuolation by weak bases is discussed.