Synthesis and assembly of functional mammalian Na,K-ATPase in yeast.

The yeast Saccharomyces cerevisiae was investigated as an in vivo protein expression system for mammalian Na,K-ATPase. Unlike animal cells, yeast cells lack endogenous Na,K-ATPase. Expression of high affinity ouabain binding sites, ouabain-sensitive ATPase activity, or ouabain-sensitive p-nitrophenylphosphatase activity in membrane fractions of yeast cells was observed to require the expression of both alpha subunit and beta subunit polypeptides of Na,K-ATPase in the same cell. High affinity ouabain binding sites are also expressed at the cell surface of intact yeast cells containing both the alpha subunit and the beta subunit of Na,K-ATPase. These observations demonstrate that both the alpha subunit and the beta subunit of Na,K-ATPase are required for the expression of functional Na,K-ATPase activity and that yeast cells can correctly assemble this oligomeric membrane protein and transport it to the cell surface.     

CV-1 cell recipients of the mouse ouabain resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids

The cation-transporting activity and Na,K-ATPase activity of CV-1 cell recipients of the mouse ouabain resistance gene (ouaR6, or OR6 cells; see Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493) have been further characterized. OR6 cells grown in strophanthidin (a cardiac aglycon which may be removed rapidly from the Na,K-ATPase) possess both ouabain-sensitive and -insensitive 86Rb+ uptake activities. The ouabain-sensitive 86Rb+ uptake activity of these cells (OR6-S cells) exhibits the same Ki for ouabain as that of the CV-1 parent cells (Ki(app) = 3 x 10(-7) M ouabain), but accounts for only approximately 30% of total 86Rb+ uptake into Na+-loaded OR6-S cells, compared to 80% for CV-1 cells. Most of the ouabain-resistant 86Rb+ uptake in OR6-S cells is dependent on internal Na+ and is insensitive to furosemide, suggesting that it is due to an ouabain-resistant Na,K pump. In OR6-S cell lysates, 50% of Na+-dependent ATPase activity is insensitive to 1 mM ouabain, compared to less than 5% in CV-1 cell lysates. In addition, purified plasma membranes from OR6-S cells contain a 100-kDa protein which is transiently phosphorylated by ATP in an Na+-dependent, K+-sensitive manner, like the alpha subunit of the CV-1 Na,K-ATPase and the canine renal Na,K-ATPase, but which is unaffected by preincubation in 1 mM ouabain. All of these data suggest that OR6-S cells possess a ouabain-insensitive Na,K pump with characteristics similar to the ouabain-sensitive pump of CV-1 parent cells. Since the mouse ouabain resistance gene does not encode either subunit of the Na,K-ATPase, these results suggest that the ouabain resistance gene product may modify the ouabain sensitivity of the endogenous CV-1 Na,K pump.     

Extracellular domains,transmembrane segments,and intracellular domains interact to determine the cation selectivity of Na,K- and gastric H,K-ATPase

We have previously reported that three residues of the fourth transmembrane segment (TM4) of the Na,K- and gastric H,K-ATPase alpha-subunits appear to play a major role in the distinct cation selectivities of these pumps [Mense, M., et al. (2000) J. Biol. Chem. 275, 1749-1756]. Substituting these three residues in the Na,K-ATPase sequence with their H,K-ATPase counterparts (L319F, N326Y, T340S) and replacing the TM3-TM4 ectodomain sequence with that of the H,K-ATPase alpha-subunit result in a pump that exhibits 50% of its maximal ATPase activity in the absence of Na(+) when the assay is performed at pH 6.0. This effect is not seen when the ectodomain alone is replaced. To gain more insight into the contributions of the three residues to establishing the selectivity of these pumps for Na(+) ions versus protons, we generated Na,K-ATPase constructs in which these residues are replaced by their H,K-ATPase counterparts either singly or in combinations. Surprisingly, none of the point mutants nor even the triple mutant was able to hydrolyze ATP at pH 6.0 at a rate greater than 20% of their respective V(max)s. For the point mutants L319F and N326Y, protons seem to competitively inhibit ATP hydrolysis at pH 6.0, based on the low apparent affinity for Na(+) ions at pH 6.0 compared to pH 7.5. It would appear, therefore, that the cation selectivity of Na,K- and H,K-ATPase is generated through a cooperative effort between residues of transmembrane segments and the flanking loops that connect these transmembrane domains. This view is further supported by homology modeling of the Na,K-ATPase based on the crystal structure of the SERCA pump.     

Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras.

Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na, K-ATPase were constructed and expressed in LLC-PK1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356-519N (the N-terminal half of the cytoplasmic M4-M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K+. The 356-519 domain and, to a greater extent, the H519N replacement confer increased apparent selectivity for protons relative to Na+ at cytoplasmic sites as shown by the persistence of K+ influx when the proton concentration is increased and the Na+ concentration decreased. The pH and K+ dependence of ouabain-inhibitable ATPase of membranes derived from the transfected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at putative cytoplasmic H+ activation sites. Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+ exchange is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Na+ dependence of pump and ATPase activities suggest relatively active H+/K+ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).     

Residues of the fourth transmembrane segments of the Na,K-ATPase and the gastric H,K-ATPase contribute to cation selectivity

We have generated protein chimeras to investigate the role of the fourth transmembrane segments (TM4) of the Na,K- and gastric H, K-ATPases in determining the distinct cation selectivities of these two pumps. Based on a helical wheel analysis, three residues of TM4 of the Na,K-ATPase were changed to their H,K-counterparts. A construct carrying three mutations in TM4 (L319F, N326Y, and T340S) and two control constructs were heterologously expressed in Xenopus laevis oocytes and in the pig kidney epithelial cell line LLC-PK(1). Biochemical ATPase assays demonstrated a large sodium-independent ATPase activity at pH 6.0 for the pump carrying the TM4 substitutions, whereas the control constructs exhibited little or no activity in the absence of sodium. Furthermore, at pH 6.0 the K(1/2)(Na(+)) shifted to 1.5 mM for the TM4 construct compared with 9.4 and 5.9 mM for the controls. In contrast, at pH 7.5 all three constructs had characteristics similar to wild type Na,K-ATPase. Large increases in K(1/2)(K(+)) were observed for the TM4 construct compared with the control constructs both in two-electrode voltage clamp experiments in Xenopus oocytes and in ATPase assays. ATPase assays also revealed a 10-fold shift in vanadate sensitivity for the TM4 construct. Based on these findings, it appears that the three identified TM4 residues play an important role in determining both the specific cation selectivities and the E(1)/E(2) conformational equilibria of the Na,K- and H,K-ATPase.     

Functional Relationship between Na/K-ATPase and NMDA-Receptors in Rat Cerebellum Granule Cells

Activation of rat cerebellum granule cells by N-methyl-D-aspartate (NMDA, 10(-4)-10(-3) M) results in progressive increase in reactive oxygen species (ROS) and suppression of the ouabain-sensitive part of Na/K-ATPase activity. When Na/K-ATPase was inhibited by high ouabain concentrations (10(-5)-5 x 10(-4) M), an increase in stationary ROS level in neuronal cells was noted, this effect being attenuated by NMDA antagonists, MK-801 and D-AP5. It is concluded that in cerebellum neurons, ouabain-resistant Na/K-ATPase is responsible for suppression of intracellular level of ROS, which, in turn, inhibit ouabain-sensitive Na/K-ATPase.     

Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant.

From a continuous spent sulfite liquor fermentation plant, two species of yeast were isolated, Saccharomyces cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3, was heavily flocculating and produced a higher ethanol yield from spent sulfite liquor than did commercial baker's yeast. The greatest difference between isolate 3 and baker's yeast was that of galactose fermentation, even when galactose utilization was induced, i.e., when they were grown in the presence of galactose, prior to fermentation. Without acetic acid present, both baker's yeast and isolate 3 fermented glucose and galactose sequentially. Galactose fermentation with baker's yeast was strongly inhibited by acetic acid at pH values below 6. Isolate 3 fermented galactose, glucose, and mannose without catabolite repression in the presence of acetic acid, even at pH 4.5. The xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were determined in some of the isolates as well as in two strains of S. cerevisiae (ATCC 24860 and baker's yeast) and Pichia stipitis CBS 6054. The S. cerevisiae strains manifested xylose reductase activity that was 2 orders of magnitude less than the corresponding P. stipitis value of 890 nmol/min/mg of protein. The xylose dehydrogenase activity was 1 order of magnitude less than the corresponding activity of P. stipitis (330 nmol/min/mg of protein).     

Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant.

From a continuous spent sulfite liquor fermentation plant, two species of yeast were isolated, Saccharomyces cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3, was heavily flocculating and produced a higher ethanol yield from spent sulfite liquor than did commercial baker's yeast. The greatest difference between isolate 3 and baker's yeast was that of galactose fermentation, even when galactose utilization was induced, i.e., when they were grown in the presence of galactose, prior to fermentation. Without acetic acid present, both baker's yeast and isolate 3 fermented glucose and galactose sequentially. Galactose fermentation with baker's yeast was strongly inhibited by acetic acid at pH values below 6. Isolate 3 fermented galactose, glucose, and mannose without catabolite repression in the presence of acetic acid, even at pH 4.5. The xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were determined in some of the isolates as well as in two strains of S. cerevisiae (ATCC 24860 and baker's yeast) and Pichia stipitis CBS 6054. The S. cerevisiae strains manifested xylose reductase activity that was 2 orders of magnitude less than the corresponding P. stipitis value of 890 nmol/min/mg of protein. The xylose dehydrogenase activity was 1 order of magnitude less than the corresponding activity of P. stipitis (330 nmol/min/mg of protein).     

Analysis of mutant origin recognition complex with reduced ATPase activity in vivo and in vitro

In eukaryotes, ORC (origin recognition complex), a six-protein complex, is the most likely initiator of chromosomal DNA replication. ORC belongs to the AAA(+) (ATPases associated with a variety of cellular activities) family of proteins and has intrinsic ATPase activity derived from Orc1p, one of its subunits. To reveal the role of this ATPase activity in Saccharomyces cerevisiae (baker's yeast) ORC, we mutated the Orc1p sensor 1 and sensor 2 regions, which are important for ATPase activity in AAA(+) proteins. Plasmid-shuffling analysis revealed that Asn(600), Arg(694) and Arg(704) are essential for the function of Orc1p. In yeast cells, overexpression of Orc1R694Ep inhibited growth, caused inefficient loading of MCM (mini-chromosome maintenance complex of proteins) and slowed the progression of S phase. In vitro, purified ORC-1R [ORC with Orc1R694Ep (Orc1p Arg(694)-->Glu mutant)] has decreased ATPase activity in the presence or absence of origin DNA. However, other activities (ATP binding and origin DNA binding) were indistinguishable from those of wild-type ORC. The present study showed that Arg(694) of the Orc1p subunit is important for the ATPase activity of ORC and suggests that this ATPase activity is required for efficient MCM loading on to origin DNA and for progression of S phase.     

Interaction of cofilin with triose-phosphate isomerase contributes glycolytic fuel for Na,K-ATPase via Rho-mediated signaling pathway

We reported previously that cofilin, an actin-binding protein, interacts with Na,K-ATPase and enhances its activity (Lee, K., Jung, J., Kim, M., and Guidotti, G. (2001) Biochem. J. 353, 377-385). To understand the nature of this interaction and the role of cofilin in the regulation of Na,K-ATPase activity, we searched for cofilin-binding proteins in the rat skeletal muscle cDNA library using the yeast two-hybrid system. Several cDNA clones were isolated, some of which coded for triose-phosphate isomerase, a glycolytic enzyme. The interaction of cofilin with triose-phosphate isomerase as well as Na,K-ATPase was confirmed by immunoprecipitation and confocal microscopy in HeLa cells. Cofilin was translocated to the plasma membrane along with triose-phosphate isomerase by the Rho activator lysophosphatidic acid but not by the p160 Rho-associated kinase inhibitor Y-27632, suggesting that the phosphorylated form of cofilin bound to TPI interacts with Na,K-ATPase. Ouabain-sensitive (86)Rb(+) uptake showed that Na,K-ATPase activity was increased by the overexpression of cofilin and lysophosphatidic acid treatment, but not by the overexpression of mutant cofilin S3A and Y-27632 treatment. Pretreatment with the glycolytic inhibitor iodoacetic acid caused a remarkable reduction of Na,K-ATPase activity, whereas pretreatment with the oxidative inhibitor carbonyl cyanide m-chlorophenylhydrazone caused no detectable changes, suggesting that the phosphorylated cofilin is involved in feeding glycolytic fuel for Na,K-ATPase activity. These findings provide a novel molecular mechanism for the regulation of Na,K-ATPase activity and for the nature of the functional coupling of cellular energy transduction.     

Catalytic function of nongastric H,K-ATPase expressed in Sf-21 insect cells

We previously demonstrated that the alpha-subunit of human nongastric H,K-ATPase (Atp1al1) can assemble with the gastric H,K-ATPase beta-subunit (betaHK) into an active ion pump upon coexpression in Xenopus oocytes. To gain insight into enzymatic functions, we have analyzed the Atp1al1-betaHK complex using a baculovirus expression system. The efficient formation of the functional Atp1al1-betaHK complex in membranes of Sf-21 insect cells was obtained upon co-infection with recombinant baculoviruses expressing Atp1al1 and betaHK. Expression of either protein alone did not produce active ATPase. The effects of K(+), Na(+), pH, and ATP and inhibitors on ATPase activity of the recombinant Atp1al1-betaHK complex were analyzed. The Atp1al1-betaHK complex was shown to exhibit significant ATPase activity in nominally K(+)-free medium. The addition of K(+) stimulated the ATP hydrolysis up to 3-fold with K(m) approximately 116 microM K(+). The ATPase activity was moderately sensitive to ouabain and to SCH 28080 with apparent K(i) values in K(+)-free medium of approximately 64 microM and approximately 93 microM, respectively. Potassium exhibited strong antagonism toward both inhibitors. Assays of the ouabain-sensitive ATPase activity revealed inhibitory effects of Na(+) with the apparent K(i) of approximately 24 mM in the absence of added K(+) and with K(i) within the range of 60-70 mM in the presence of > or = 1 mM K(+). Thus, the human nongastric H,K-ATPase represented by the recombinant Atp1al1-betaHK complex exhibits enzymatic properties of K(+)-dependent ATPase sensitive to ouabain, SCH 28080, and Na(+). It differs from Na,K-ATPase in cation dependence and differs from gastric H,K-ATPase and Na,K-ATPase in sensitivity to inhibitors.     

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.     

Acetylcholine induced reciprocal changes in the Na, K-ATPase and acetylcholinesterase activity of nerve and membrane preparations of Na, K-ATF-azy]

ATPase and cholinesterase activities in the homogenate of the frog nerve and membrane Na,K-ATPase preparation of the bovine brain were investigated. Preliminary treatment of the nerve and the preparation by acetylcholine solution (10(-6)--10(-7) M) enhanced their Na,K-ATPase activity and reduced their cholinesterase activity. Possible mechanisms of this phenomenon are discussed.     

Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase.

Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site. In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells. The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis. The binding of [3H]ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi. The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM. The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480. The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM). These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate. It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase.     

Biosynthesis of lysine in Rhodotorula glutinis: role of pipecolic acid.

Glutamate-alpha-ketoadipate transaminase, saccharopine reductase, and saccharopine dehydrogenase activities were demonstrated in extracts of Rhodotorula glutinis but alpha-aminoadipate reductase activity could not be measured in whole cells or in extracts. Lysine auxotroph lys1 grew in the presence of L-lysine or DL-alpha-aminoadipate and incorporated radioactivity from DL-alpha-amino-[I-14C]adipate into lysine during growth. Growing wild-type cells converted L-[U-14C]lysine into alpha-amino-[14C]adipate, suggesting both biosynthetic and degradative roles for alpha-aminoadipate. Lysine auxotrophs lys1, lys2 and lys3 of R. glutinis, unlike lysine auxotrophs of Saccharomyces cerevisiae, satisfied their growth requirement with L-pipecolate. Moreover, extracts of wild-type R. glutinis catalysed the conversion of L-pipecolate to alpha-aminoadipate-delta semialdehyde. These results suggest a biosynthetic role for L-pipecolate in R. glutinis but not in S. cerevisiae.     

Physiological properties of some yeast strains

Twenty yeast strains have recently been isolated in pure cultures from natural and industrial sources and identified based mainly on physiological properties. The majority of the strains (15) are alcohologenic belonging to the genus Saccharomyces and comprise two brewer's (beer) yeast strains (S. carlsbergensis= S. uvarum A and B), two baker's yeast strains (S. cerevisiae CA and CP), one spirit yeast strain (S. cerevisiae CF) and ten wine yeast strains (S. cerevisiae var. ellipsoideus = S. ellipsoideus 1, 3, 4, 6, 8 and 9; S. oviformis 2, 5 and 7; and S. uvarum 10). The other 5 yeast strains belong to different species: Kloeckera apiculate, Candida mycoderma (Mycoderma vini), Pichia membranaefaciens, Rhodotorula glutinis and Torulopsis holmii, respectively.     

Attempts to detect lycopersene formation in yeast

1. beta-Ionone vapour has been shown to cause an increase in the more saturated carotenes and a decrease in the less saturated carotenes of Rhodotorula glutinis. Lycopersene (dihydrophytoene) has been proposed as a precursor to phytoene. Attempts were made to isolate lycopersene from beta-ionone-treated cultures of R. glutinis. 2. Large samples of beta-ionone-treated cultures were examined for the presence of lycopersene. Spots were detected on silicic acid plates that could not be differentiated from synthetic lycopersene on the basis of column and thin-layer chromatographic separations and staining techniques. The lycopersene-like substance could be obtained from non-treated pigmented yeast as well as baker's yeast. 3. An extraction of bacterial-grade yeast extract also yielded a lycopersene-like substance. The extracts of R. glutinis cells cultured on media not containing yeast extract did not contain the lycopersene-like compound. 4. No significant carbon was incorporated into the lycopersene zone from (14)C-labelled mevalonate, acetate and glucose by R. glutinis and baker's yeast. 5. These results indicate that compounds may exist with chromatographic properties similar to lycopersene, but that lycopersene could not be detected in either a pigmented or a non-pigmented yeast.     

Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) are involved in Na+ transport, and Gln-923 (M8) is essential for Na,K-ATPase activity

The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), were increased by around 2 approximately 8-fold in the case of T774A, V920E, and E954A. The apparent K0.5 values for K+, as estimated by the Na,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparent K0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6 approximately 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.     

Ethyl isopropylamiloride downregulates Na,K-ATPase gene expression which confers cytotoxicity in primary proximal tubule cell cultures

Our original attempt was to examine whether inhibition of Na/H exchange in proximal tubule would affect the expression of basolateral membrane protein Na,K-ATPase. Three amiloride analogues were tested within the range of 10(-6) M to 10(-4) M in primary cultures of proximal tubule cells. Only ethylisopropyl amiloride (EIPA) dose-dependently downregulated Na,K-ATPase activity in cultured proximal tubule cells. The time course study revealed that EIPA (10(-4) M) significantly decreased Na,K-ATPase alpha- and alpha-mRNA abundance within 4 hr and suppressed Na,K-ATPase alpha- and beta-mRNA levels by 76.3 +/- 4.5% and 85.5 +/- 5.8%, respectively, within 24 hr. The decrease in Na,K-ATPase mRNA was followed by a decrease in Na,K-ATPase activity by 22.5 +/- 10.8% and 48.8 +/- 5.9% within 12 and 24 hr, respectively, which could be reflected by a coordinate decrease in levels of both alpha- and mature beta-protein. The cell viability was not affected until 20 hr of EIPA treatment, when an increase in LDH release and cell detachment was observed. Because EIPA rapidly decreased intracellular pH (pHi) to 6.7 within 2 hr and raising pHi to 6.6 by metabolic acidosis could not elicit changes in Na,K-ATPase activity, EIPA-induced downregulation of Na,K-ATPase should not be mediated through H+. In view of the time course of EIPA effects on Na,K-ATPase subunit mRNA, protein, activity and cell toxicity, the cytotoxic effect is likely resulted from a decrease in Na,K-ATPase activity. Take together, we conclude that EIPA induces downregulation of Na,K-ATPase expression via both pre- and post-translational mechanisms, which confers cytotoxic effects on proximal tubule cells.