A Hybrid between Na+,K+-ATPase and H+,K+-ATPase Is Sensitive to Palytoxin, Ouabain, and SCH 28080*

Robert A. FarleyDagger §, Silvia Schreiber, Shyang-Guang WangDagger , and Georgios Scheiner-Bobis

From the Dagger  Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California 90033 and the  Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Frankfurter Strasse 100, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany

Received for publication, September 26, 2000, and in revised form, October 17, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Na+,K+-ATPase is inhibited by cardiac glycosides such as ouabain, and palytoxin, which do not inhibit gastric H+,K+-ATPase. Gastric H+,K+-ATPase is inhibited by SCH28080, which has no effect on Na+,K+-ATPase. The goal of the current study was to identify amino acid sequences of the gastric proton-potassium pump that are involved in recognition of the pump-specific inhibitor SCH 28080. A chimeric polypeptide consisting of the rat sodium pump alpha 3 subunit with the peptide Gln905-Val930 of the gastric proton pump alpha  subunit substituted in place of the original Asn886-Ala911 sequence was expressed together with the gastric beta  subunit in the yeast Saccharomyces cerevisiae. Yeast cells that express this subunit combination are sensitive to palytoxin, which interacts specifically with the sodium pump, and lose intracellular K+ ions. The palytoxin-induced K+ efflux is inhibited by the sodium pump-specific inhibitor ouabain and also by the gastric proton pump-specific inhibitor SCH 28080. The IC50 for SCH 28080 inhibition of palytoxin-induced K+ efflux is 14.3 ± 2.4 µM, which is similar to the Ki for SCH 28080 inhibition of ATP hydrolysis by the gastric H+,K+-ATPase. In contrast, palytoxin-induced K+ efflux from cells expressing either the native alpha 3 and beta 1 subunits of the sodium pump or the alpha 3 subunit of the sodium pump together with the beta  subunit of the gastric proton pump is inhibited by ouabain but not by SCH 28080. The acquisition of SCH 28080 sensitivity by the chimera indicates that the Gln905-Val930 peptide of the gastric proton pump is likely to be involved in the interactions of the gastric proton-potassium pump with SCH 28080.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The sodium pump (Na+,K+-ATPase)1 and the gastric proton pump (H+,K+-ATPase) are ion-transporting ATPases, both hydrolyzing ATP to actively pump ions against their electrochemical gradients (1, 2). Unlike other ion-transporting ATPases, they both require a highly glycosylated smaller peptide, the beta  subunit, for their function, in addition to a larger ATP-recognizing alpha  subunit. The alpha  subunits of proton and sodium pumps have ~60% identical primary sequences and form 10 membrane-spanning domains, denoted M1-M10, with their amino and carboxyl termini localized in the cytosol (3-9). The beta  subunits are 30% identical and form a single membrane span with the amino terminus on the extracellular side of the membrane. Despite this limited identity of the primary structure, the tertiary structure of both types of beta  subunits must be very similar, since the proton pump beta  subunit was shown in numerous investigations to form functional complexes with the alpha  subunit of the sodium pump when these proteins were expressed together (10-14).

Both enzymes are pharmacological receptors for clinically relevant drugs. Thus, the pumping activity of the gastric proton pump is specifically inhibited by imidazopyridines like 8-benzyloxy-3-cyanomethyl-2-methyl-imidazo[1,2-a]pyridine (SCH 28080) or omeprazole (15-17). Derivatives of the latter are widely used clinically for controlling hyperacidity or peptic ulcer (18, 19). The sodium pump is specifically inhibited by a series of naturally occurring steroids, such as ouabain and digitalis (20, 21). Based on their clinical use, these substances are also referred to as cardiac glycosides or cardioactive steroids; their application helps to increase muscular contractility of the failing heart (22-24).

In recent years, the work of many investigators has been focused on the identification of amino acids or peptides of the proton and sodium pumps involved in interaction with omeprazole and SCH 28080, or ouabain and related cardiac glycosides, respectively. Analysis of a series of sodium pump mutants helped to identify several amino acids involved in the recognition of ouabain (25-28), but little is known about amino acids or peptides of the gastric proton pump involved in the recognition of omeprazole or SCH 28080. Amino acids in both the amino- and carboxyl-terminal halves of the alpha  subunit of gastric H+,K+-ATPase have been suggested to participate in the binding sites for these inhibitors. Blostein et al. (29) expressed a chimeric polypeptide consisting of the amino-terminal 519 amino acids of gastric H+,K+-ATPase and the COOH-terminal 507 amino acids of Na+,K+-ATPase in LLC-PK1 cells and reported inhibition of potassium influx by SCH 28080. Munson et al. (30) labeled a peptide from the M1-M2 region of gastric H+,K+-ATPase with a photoactive analog of SCH 28080. Labeling experiments have also identified Cys892 within the extracellular loop connecting the M7 and M8 membrane spans of the gastric proton-potassium pump alpha  subunit as a component of the binding site for omeprazole (17). Since omeprazole binding to the proton pump can be inhibited by SCH 28080 (31), this result raises the possibility that amino acids within the COOH-terminal half of the gastric pump alpha  subunit might also participate in interactions of the gastric proton pump with SCH 28080.

To test this hypothesis, we made use of the highly specific interactions between the sodium pump and palytoxin to investigate whether the M7/M8 extracellular peptide of the gastric proton pump alpha  subunit is involved in SCH 28080 recognition. Palytoxin from marine corals of the genus Palythoa, like the cardioactive steroids, is a specific inhibitor of the sodium pump (32, 33). Unlike the cardioactive steroids, however, which inhibit ion flow through the pump, palytoxin converts the enzyme into an open channel that allows ions to flow down their concentration gradient (34). The palytoxin-induced channels have been studied with electrophysiological tools (35, 36) and have been found to have a single-channel conductance of about 10 picosiemens in crayfish giant axons. Single-channel recordings have also been obtained from Na+,K+-ATPase incorporated into planar lipid bilayers (36). Expression of Na+,K+-ATPase in the yeast Saccharomyces cerevisiae causes the yeast cells to lose intracellular K+ when exposed to palytoxin, whereas yeast cells without Na+,K+-ATPase are not affected by palytoxin (11, 37, 38). Taking advantage of the high specificity of the palytoxin/sodium pump interaction, we used the expression of Na+,K+-ATPase in yeast to address the following questions. Does palytoxin also induce a K+ efflux from yeast cells that express a hybrid sodium pump consisting of a sodium pump alpha  subunit and a gastric proton pump beta  subunit? Does a similar phenomenon occur when yeast cells express a chimeric sodium pump alpha  subunit containing the extracellular sequence Gln905-Val930 from the M7/M8 loop of the gastric proton pump alpha  subunit? Finally, is any palytoxin-induced K+ efflux observed sensitive to ouabain or SCH 28080?


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Vectors-- The yeast expression vectors used in this study and the various combinations used for transformation of yeast cells are summarized in Table I. The methods applied for their construction have been described previously. Therefore, a brief summary of the most important features is given here. The yeast expression plasmid YEp1PT was used as a vector for the cDNA coding for the rat sodium pump alpha 3 subunit. This vector is denoted YEpralpha 3 (39). The same expression plasmid was also used for the insertion of the cDNA coding for a chimera of the sodium pump alpha 3 subunit that was made to contain Gln905-Val930 of the rat gastric proton pump alpha  subunit in place of the original Asn886-Ala911 (13). This vector is named YEpNGH26. Vectors pG1T-rbeta 1 and pG1T-HKbeta were used for the expression of the rat sodium pump beta 1 subunit and the rat gastric proton pump beta  subunit, respectively (39).

Yeast Cells Used for Expression-- The S. cerevisiae strain 30-4 (MAT-alpha , trp1, ura3, Vn2, GAL+) was used for transformation with either of the yeast expression vectors YEpNGH26 (coding for the chimeric sodium pump alpha  subunit) or YEpralpha 3 (coding for the wild-type sodium pump alpha 3 subunit of the rat) together with either of the vectors pG1T-rbeta 1 or pG1T-HKbeta coding for the beta  subunits of rat Na+,K+-ATPase and gastric H+,K+-ATPase, respectively. Thus, coexpression of the various vectors results in the four different combinations of subunits given in Table I. The conditions for the selection of transformants are described elsewhere (40). Microsomes from transformed or nontransformed yeast cells were prepared as described previously (40). Protein content of the microsomal preparations was determined by the method of Lowry (41).

Detection of NKalpha 3, NGH26, and HKbeta Subunits in a Western Blot-- The conditions for electrophoresis and immunoblot detection of alpha 3 or NGH26 subunits have been described previously in detail (13). Briefly, 100 µg of microsomal protein isolated from yeast expressing either the NKalpha 3/NKbeta or NGH26/HKbeta heterodimers was run on a 7.5% SDS-polyacrylamide gel and was then electrotransferred onto nitrocellulose membranes by semidry blotting at 0.8 mA/cm2. The alpha 3 and NGH26 subunits were visualized by using monoclonal antibody 5 against sodium pump alpha  subunits (1:400 dilution) and the commercially available enhanced chemiluminescence kit (ECL) following the protocol of the provider.

HKbeta subunits in membrane preparations from cells expressing either NKalpha 3/HKbeta or NGH26/HKbeta were detected in a similar way. Here, however, 35 µg of microsomal protein were sufficient for electrophoresis and semidry blotting. The monoclonal antibody 2/2E6 served as a primary antibody against the gastric pump beta  subunit (1:2000 dilution). All other conditions were the same as above.

Estimation of the Gastric Proton Pump beta  Subunits in Yeast Membrane Preparations by an Antibody Capture Assay-- An antibody capture assay (42) was used with some minor modifications (43) to estimate the expression level of the HKbeta in membrane preparations from yeast expressing either NKalpha 3/HKbeta or NGH26/HKbeta . Microsomal proteins from nontransformed cells served as a control.

A total of 35 µg of yeast microsomal protein in 50 µl of phosphate-buffered saline containing 0.1% Tween 20 (PBS-T) was added to the wells of a microtiter plate. After incubation for 2 h at room temperature, the microtiter wells were washed twice with PBS-T. Afterward, 150 µl of blocking buffer composed of 3% bovine serum albumin in PBS-T with 0.02% sodium azide were added overnight to saturate the remaining protein binding sites of the well. Then, the wells were washed once again as described before. To each of the wells 50 µl of a monoclonal antibody (2/2E6) against the gastric proton pump beta  subunit were added at a 1:2000 dilution in PBS-T and incubation continued for 2 h at room temperature. Unbound antibody was then removed by four washes with PBS-T, and afterward 50 µl of an alkaline phosphatase-conjugated anti-mouse IgG were added to the wells. After 2 h of incubation at room temperature, the wells were washed four times with PBS-T, and then twice with 10 mM diethanolamine, pH 9.5, containing 5 mM MgCl2. All further steps concerning the time course of the formation of p-nitrophenolate from p-nitrophenyl phosphate were carried out as described (42).

Ouabain Binding to Yeast Microsomal Preparations-- A total of 250 µg of yeast microsomal protein was incubated for 1 h at 30 °C with 5 mM PO4 (Tris form), 5 mM MgCl2, 10 mM Tris/HCl, pH 7.5, and various concentrations of [3H]ouabain. The incubation volume was 250 µl. Thereafter, the protein was pelleted by centrifugation for 5 min at 12,000 × g. After washing twice with 1 ml of ice-cold water, the pellet was dissolved in 250 µl of 1 M NaOH by incubation for 15 min at 80 °C. The NaOH was neutralized by 250 µl of 1 M HCl, and the radioactivity was determined by scintillation counting after the addition of scintillation fluid.

ATP-promoted Binding of [3H]Ouabain-- A total of 125 µg of microsomal protein was incubated for 5 min at 30 °C in 10 mM Tris/HCl, pH 7.5, 50 nM [3H]ouabain, 5 mM MgCl2, 50 mM NaCl, and various concentrations of ATP (Tris form). The total volume of each sample was 250 µl. Thereafter, the protein was pelleted by centrifugation at 12,000 × g for 5 min, washed twice with H2O at 4 °C, dissolved in 250 µl of 1 M NaOH, and processed as described at the end of the preceding paragraph.

Effect of SCH 28080 on the ATP-promoted Binding of [3H]Ouabain-- To investigate whether SCH 28080 inhibits binding of [3H]ouabain to yeast membranes, the above experiment was repeated at 0, 1, and 10 µM ATP in the presence of 25 µM SCH 28080. All other conditions were the same as described above.

Palytoxin-induced K+ Efflux from Yeast Cells Expressing Wild-type and Chimeric Na+,K+-ATPase-- Single yeast colonies were incubated at 30 °C in a cell incubator with vigorous shaking overnight in 5 ml of SD growth medium (6.7 g/liter Bacto Yeast nitrogen base without amino acids) supplemented with d-galactose (20 g/liter SD medium) as the only carbon source. The cell suspension was then transferred to 200 ml of SD medium and incubation continued for an additional 24 h under the same conditions. Thereafter, cells were collected by centrifugation for 5 min at 3000 × g, washed twice with 30 ml of NaGHBC (200 mM NaCl, 100 mM glucose, 10 mM HEPES, 0.5 mM boric acid, 1 mM CaCl2, adjusted to pH 7.5 with Tris), and suspended in NaGHBC to a final concentration of 106 cells/ml (A600 = 6).

For the measurement of the palytoxin-induced K+ loss from yeast cells, 450 µl of the cell suspension were incubated with various concentrations of palytoxin in the absence or presence of ouabain or SCH 28080 for 2 h at 30 °C. Since SCH 28080 was dissolved in dimethyl sulfoxide:ethanol (1:1, v/v), the content of the solvent was kept constant in all samples; the final volume of each sample was 500 µl. To determine the total cellular K+ content (100% value), 450 µl of the yeast cell suspension were incubated for 1 h at 70 °C with 50 µl of 1% lithium dodecyl sulfate (w/v). Afterward, cells were centrifuged at 12,000 × g for 2 min in a bench-top centrifuge. The supernatants were collected and measured for their K+ content in a flame photometer.

Initial rates of palytoxin-induced K+ efflux were measured in a flow ionometer. Yeast cells were suspended at a concentration of 15 × 106 cells/ml in NaGHBC buffer containing 0.2, 0.5, or 5 µM palytoxin for cells with NKalpha 3/NKbeta 1; 0.5, 2, or 5 µM palytoxin for cells expressing NKalpha 3/HKbeta ; and 1, 2.5, or 5 µM palytoxin for cells expressing the NGH26/HKbeta hybrid. The total volume of the solution was 1 ml. Using a peristaltic pump, the reaction mixture was transported to a chamber containing a K+-sensitive electrode. The time elapsed between palytoxin addition and signal recording was 7 min, and recording was continued for an additional 5 min. The electric signals derived from the K+-sensitive electrode were digitized and analyzed by a computer program provided by the supplier of the flow ionometer.

Data Analysis-- Data are plotted in the figures as mean values ± standard deviations. The lines through the data are the best fits of appropriate equations obtained using the programs InPlot 4.0 or Prism (GraphPad Software, San Diego, CA).

Materials-- Growth media were purchased from Difco (Detroit, MI). Palytoxin was obtained from Dr. L. Bèress (Christian-Albrechts-Universität, Kiel, Germany). The yeast strain 30-4 was obtained from Dr. R. Hitzeman (Genentech, CA). Restriction endonucleases were from MBI Fermentas (Vilnius, Lithuania) or Amersham Buchler (Braunschweig, Germany). Amersham Bucher was also the provider of [3H]ouabain (28 Ci/mmol) and of the enhanced chemiluminescence Western blot analysis system. The nitrocellulose membranes were from Schleicher & Schuell (Dassel, Germany). The microtiter plates MaxiSorb were from Nunc (Wiesbaden, Germany). Alkaline phosphatase-conjugated anti-mouse IgG was purchased from SeroTec (Oxford, United Kingdom). The hybridoma cells producing monoclonal antibody 5 were purchased from Developmental Studies Hybridoma Bank Project, University of Iowa (Iowa City, IA). The antibody 2/2E6 against the gastric proton pump beta  subunit was a gift of Dr. C. Okomoto (University of Southern California, Los Angeles, CA). The flow ionometer is a product of ZABS GmbH (Marburg an der Lahn, Germany).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of [3H]Ouabain-- In the presence of phosphate (P) and Mg2+, Na+,K+-ATPase (E2) becomes phosphorylated (E2-P) and binds ouabain with high affinity [E2*-P·ouabain]. Use of radioactive ouabain enables one to determine the affinity of the enzyme for ouabain under these conditions. Thus, as expected, the rat wild-type sodium pump alpha 3 and beta 1 subunits (NKalpha 3/NKbeta 1) in microsomal yeast membranes bind [3H]ouabain with high affinity (Fig. 1). The Kd determined from a Scatchard plot is 7.7 ± 0.3 nM (Table II). A similar affinity for ouabain was obtained with membranes from cells expressing the NKalpha 3/HKbeta subunits (Kd of 6.4 ± 1.4 nM). The Bmax for ouabain binding to the NKalpha 3/HKbeta hybrid (0.85 ± 0.07 pmol/mg) is slightly reduced compared with that of the wild-type sodium pump (1.1 ± 0.02 pmol/mg) (Table II). Ouabain binding to microsomal membranes from cells expressing the chimeric sodium pump alpha  subunit (NGH26) together with the sodium pump beta 1 subunit (NKbeta 1) is almost undetectable (data not shown).



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Fig. 1.   Scatchard analysis of [3H]ouabain binding. Membranes isolated from yeast cells expressing the NKalpha 3/NKbeta 1 () or either of the chimeric proteins NKalpha 3/HKbeta (black-down-triangle ) and NGH26/HKbeta (*) were incubated in the presence of 5 mM phosphate, 5 mM MgCl2, and various concentrations of [3H]ouabain for 60 min at 30 °C as described under "Experimental Procedures." Afterward, bound [3H]ouabain was measured by scintillation counting. Nonspecifically bound [3H]ouabain was determined by including 3 mM nonradioactive ouabain in the assay. The figure shows one representative experiment.

Replacement of the sodium pump beta 1 by the proton pump beta  subunit results in the formation of NGH26/HKbeta complexes capable of binding ouabain (Fig. 1). The equilibrium dissociation constant Kd for ouabain binding to the NGH26/HKbeta heterodimer (21.1 ± 2.5 nM; Table II) is slightly increased compared with complexes assembled with NKalpha 3, indicating a reduction in affinity. In addition, the maximum amount of ouabain bound by NGH26/HKbeta is also reduced (0.18 ± 0.01 pmol/mg), accounting for only a fraction (17%) of the total binding obtained with the wild-type sodium pump (Fig. 1; Table II).

Expression of NKalpha 3, NGH26, and HKbeta Subunits in Yeast-- To investigate the relative level of expression of the alpha 3 and NGH26 subunits, microsomal proteins isolated from transformed yeast were probed in a Western blot with a monoclonal antibody raised against the sodium pump alpha  subunit. Fig. 2 shows that both subunits are expressed in the yeast in comparable quantities. As shown in the same figure, the antibody does not recognize any protein of ~100 kDa corresponding to the alpha 3 or NGH26 subunit in microsomes isolated from nontransformed yeast cells.



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Fig. 2.   Immunodetection of NKalpha 3 and NGH26 subunits. Microsomal proteins from yeast expressing either NGH26/HKbeta (lane 1) or NKalpha 3/NKbeta (lane 2) heterodimers were first separated by SDS-polyacrylamide gel electrophoresis and then electrotransferred onto nitrocellulose membranes. Microsomal protein from nontransformed cells served as a control (lane 3). Expressed proteins were visualized by using monoclonal antibody 5 against Na+,K+-ATPase alpha  and the commercially available enhanced chemiluminescence Western blot analysis system. The figure demonstrates that NGH26 (lane 1) and alpha 3 subunits (lane 2) are expressed in yeast in comparable quantities. A similar protein at about 100 kDa is not detected in microsomes isolated from nontransformed yeast cells (lane 3).

The expression levels of the gastric proton pump beta  subunit (HKbeta ) were measured in yeast membrane preparations from cells expressing either NKalpha 3/HKbeta or NGH26/HKbeta using a monoclonal antibody raised against the gastric proton pump beta  subunit. The Western blot presented in Fig. 3A shows that the antibody recognizes two protein bands of about 40 and 43 kDa in membranes from cells expressing the NKalpha 3/HKbeta or NGH26/HKbeta heterodimers. Since corresponding bands are not detected in membranes from nontransformed cells, these bands at 40 and 43 kDa are gastric pump beta  subunits, possibly glycosylated to various extents. It is apparent from the figure that the abundance of the HKbeta subunit in membrane preparations from cells expressing the NKalpha 3/HKbeta complex is higher than in membranes from cells expressing the NGH26/HKbeta hybrid.



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Fig. 3.   Immunodetection of HKbeta subunits. A, microsomal proteins from untransformed yeast (lane 1), or yeast expressing either NGH26/HKbeta (lane 2) or NKalpha 3/HKbeta (lane 3) heterodimers were separated by SDS-polyacrylamide gel electrophoresis and then electrotransferred onto nitrocellulose membranes. Expressed proteins were visualized by using the monoclonal antibody 2/2E6 against HKbeta and the enhanced chemiluminescence Western blot analysis system. B, a total of 35 µg of microsomal protein was attached to the bottom of the wells of microtiter plates and was incubated first with the 2/2E6 antibody against HKbeta and then with an alkaline phosphatase-conjugated secondary antibody. After either 40 or 60 min of incubation with p-nitrophenyl phosphate, the p-nitrophenolate anion was determined by measuring the absorbance at 405 nm using a molar absorbance coefficient epsilon  = 18,500 liters/mol/cm. Microsomal protein from nontransformed cells served as a control, and values obtained with the control microsomes were subtracted from the rest.

Quantification of the HKbeta subunit expression levels in yeast microsomes from cells expressing either the NKalpha 3/HKbeta or the NGH26/HKbeta hybrid cannot easily be addressed by the Western blotting method. This was done instead by applying a variation of an antigen capture assay (42). In this assay the antigen (here HKbeta ) is affixed to the wells of a microtiter plate. This affixed antigen is then incubated with an antibody specific for the antigen (here antibody 2/2E6 against HKbeta ). Subsequently, an alkaline phosphatase-conjugated secondary antibody (here an anti-mouse IgG) is added to the well. With sufficient washes between additions, the antigen, the primary antibody, the secondary antibody, and alkaline phosphatase should be present in equal amounts. Thus, the amount of p-nitrophenyl phosphate hydrolyzed by the alkaline phosphatase is directly proportional to the number of HKbeta subunits present in each well, and, therefore, the amount of the p-nitrophenolate anion produced by the alkaline phosphatase can be used as measure of the relative abundance of HKbeta subunits.

Fig. 3B shows the results of this experiment. After either 40 or 60 min of incubation with substrate, the amount of the p-nitrophenolate anion formed in samples containing the HKbeta from cells expressing the NGH26/HKbeta heterodimers accounts for only 13-17% of the value obtained from the microsomes from cells expressing the NKalpha 3/HKbeta subunit combination.

Binding of [3H]Ouabain in the Presence of ATP, Na+, and Mg2+-- The sodium pump can be phosphorylated either by inorganic phosphate in the presence of Mg2+ (the conditions of the experiment in Fig. 1) or by ATP in the presence of Na+ and Mg2+ (44). In both cases, ouabain binds to the E2-P conformational state of the enzyme and forms a stable and easily measurable [E2*-P·ouabain] complex. By measuring the formation of the [E2*-P·ouabain] complex as a function of the ATP concentration, one can determine whether ATP is hydrolyzed to yield the phosphoenzyme, and, if so, it is possible from these experiments to obtain an EC50 value indicating the relative affinity of the enzyme for ATP (43, 45).

Fig. 4 shows that ATP promotes binding of [3H]ouabain to membranes containing the NKalpha 3/NKbeta 1 heterodimer with an EC50 of 3.9 ± 0.16 µM. Similarly, ATP promotes binding to the NKalpha 3/HKbeta heterodimer with an EC50 of 1.05 ± 0.16 µM. The Bmax values for [3H]ouabain binding to these two membrane preparations at saturating levels of ATP are nearly identical (0.76 pmol/mg). The EC50 for [3H]ouabain binding to yeast membranes containing the NGH26/HKbeta heterodimer in an ATP-dependent reaction is 1.13 ± 0.51 µM; the maximum amount of ouabain bound, however, ~0.13 pmol/mg, is 17% of the amount bound by either NKalpha 3/NKbeta 1 or NKalpha 3/HKbeta .



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Fig. 4.   Promotion of ouabain binding by ATP. Membranes isolated from yeast cells expressing NKalpha 3/NKbeta 1 (black-down-triangle ) or either of the chimeric proteins NKalpha 3/HKbeta () and NGH26/HKbeta (*) were incubated for 5 min at 30 °C in 10 mM Tris/HCl, pH 7.5, 50 nM [3H]ouabain, 5 mM MgCl2, 50 mM NaCl, and various concentrations of ATP (Tris form) as described under "Experimental Procedures." Afterward, bound [3H]ouabain was measured by scintillation counting.

Palytoxin-induced Potassium Efflux from Yeast Cells-- Yeast cells expressing the NKalpha 3/NKbeta 1, NKalpha 3/HKbeta , or NGH26/HKbeta subunits were incubated for 2 h with various concentrations of palytoxin. Thereafter, cells were removed by centrifugation and K+ in the supernatant was determined by flame photometry. As shown in Fig. 5, the interaction of palytoxin with the NKalpha 3/NKbeta 1, NKalpha 3/HKbeta , and NGH26/HKbeta complexes results in a loss of up to 80% of cytosolic K+ levels in yeast. The EC50 value for the palytoxin-induced K+ efflux from cells expressing the NKalpha 3/NKbeta 1 subunits is 136 nM. The corresponding value obtained with cells expressing the NKalpha 3/HKbeta subunits is approximately 2-fold higher (313 nM), and the EC50 obtained with cells expressing the NGH26/HKbeta subunits is about 6-fold higher (822 nM) than for NKalpha 3/NKbeta 1. Palytoxin has no effect on nontransformed yeast cells and only a small loss of K+ is observed from yeast cells that express the chimeric NGH26 subunit together with the rat sodium pump beta 1 subunit (data not shown).



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Fig. 5.   Palytoxin concentration dependence of K+ efflux. Yeast cells expressing NKalpha 3/NKbeta 1 () or either of the chimeric proteins NKalpha 3/HKbeta (black-down-triangle ) and NGH26/HKbeta (*) were incubated for 120 min with various concentrations of palytoxin as described under "Experimental Procedures." Cells were then centrifuged, and the K+ concentration in the supernatant determined by flame photometry. Percentage of total refers to [K+] calculated as a percentage of [K+] measured in samples treated with lithium dodecyl sulfate.

To estimate the initial rates of the palytoxin-induced efflux, cells expressing either NKalpha 3/NKbeta 1, NKalpha 3/HKbeta , or NGH26/HKbeta complexes were incubated at various concentrations of palytoxin (see "Experimental Procedures") and K+ was measured in the extracellular medium using a flow ionometer. During the period of recording, the initial rate of K+ efflux (Vin) at each palytoxin concentration was linear and increased with increasing palytoxin concentrations. The rate of efflux can be described by the formula Vin = kK+·N·Po where kK+ is the single channel conductance, N is the number of channels and is equal to the Bmax for ouabain binding, and Po is the probability that the channels are open. Po = k·An, where A is the concentration of palytoxin, the exponent n is the number of palytoxin molecules required to open the channel, and k is a constant of proportionality. A plot of log Vin versus log A showed a straight line with the slope near 1 for all subunit combinations, indicating that the channels are opened by the binding of a single palytoxin molecule. Substituting k' = kK+·k, the rate of efflux can be described by the equation Vin = k'·N·A. A plot of Vin versus A resulted in a straight line for each heterodimer with a slope of k'N. Values of k'N for a given alpha beta heterodimer were not significantly different at different palytoxin concentrations, and are reported in Table II as mean values for all palytoxin concentrations.

Inhibition of Palytoxin-induced Potassium Efflux by Ouabain and SCH 28080-- The palytoxin-induced K+ efflux from yeast cells expressing alpha 1/beta 1 subunits of the sodium pump has been shown to be completely inhibited by ouabain (37). Similarly, the palytoxin-induced K+ efflux from yeast cells expressing any of the subunit combinations NKalpha 3/NKbeta 1, NKalpha 3/HKbeta , or NGH26/HKbeta is also inhibited by ouabain (Fig. 6). At 400 nM palytoxin, ouabain inhibits the K+ efflux from cells expressing the NKalpha 3/NKbeta 1 heterodimer with IC50 = 26.5 ± 0.1 µM (Table II). Comparable results are obtained with cells expressing either NKalpha 3/HKbeta or NGH26/HKbeta heterodimers; ouabain inhibits the palytoxin-induced K+ efflux with IC50 values of 23.1 ± 3.4 µM and 27.6 ± 1.9 µM, respectively (Table II).



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Fig. 6.   Determination of IC50 values for inhibition of the palytoxin-induced K+ efflux by ouabain. Yeast cells expressing the NKalpha 3/NKbeta 1 () or either of the chimeric proteins NKalpha 3/HKbeta (black-down-triangle ) and NGH26/HKbeta (*) were incubated with 400 nM palytoxin and various concentrations of ouabain as described under "Experimental Procedures." Incubation was allowed to proceed for 120 min at 30 °C. [K+] was determined in the supernatant as described. The IC50 value is the negative of the intercept with the abscissa.

To investigate the effect of the gastric proton pump-specific inhibitor SCH 28080, yeast cells expressing NKalpha 3/NKbeta 1, NKalpha 3/HKbeta , or NGH26/HKbeta complexes were incubated at 400 nM palytoxin with various concentrations of SCH 28080. After an incubation period of 2 h, K+ in the supernatant was determined as described under "Experimental Procedures." As shown in Fig. 7A, only cells expressing the NGH26/HKbeta complexes are sensitive to SCH 28080. The palytoxin-induced K+ efflux from these cells is almost completely inhibited by 50 µM SCH 28080 with an IC50 of 14.3 ± 2.4 µM (Fig. 7B).



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Fig. 7.   Inhibition of the palytoxin-induced K+ efflux by SCH 28080. Yeast cells expressing NKalpha 3/NKbeta 1 () or either of the chimeric proteins NKalpha 3/HKbeta (black-down-triangle ) and NGH26/HKbeta (*) were incubated with 200 nM palytoxin and various concentrations of SCH 28080 as described under "Experimental Procedures." After incubation for 120 min at room temperature, cells were centrifuged and [K+] in the supernatant was determined by flame photometry (A). Under these conditions, SCH 28080 inhibits the palytoxin-induced K+ efflux with an IC50 of 14.3 ± 2.4 µM for NGH26/HKbeta (*) as shown in B.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Because of their importance as receptors for clinically relevant drugs, much effort has been put into identifying sequences of the proton and sodium pumps that participate in the recognition of omeprazole, SCH 28080, and ouabain and related cardiac steroids. Analysis of the properties of sodium pump mutants allowed for the identification of several sites involved in recognition of ouabain (27, 28). Comparatively little is known, however, about amino acids or peptides involved in the binding of omeprazole or SCH 28080 to the gastric proton pump. The omeprazole binding site contains Cys892 within the extracellular loop connecting the M7 and M8 membrane spans of the proton pump alpha  subunit (17). Since omeprazole binding to the proton pump can be inhibited by SCH 28080 (31), it is possible that the binding sites for both substances are identical or in close proximity to each other. Thus, the M7/M8 extracellular peptide of the gastric pump alpha  subunit might be part of the binding site for SCH 28080. This peptide corresponds to a sequence of the sodium pump alpha  subunit that has been shown to contain amino acids involved in the binding of ouabain (28). The corresponding region of the rat gastric proton pump alpha  subunit contains the peptide Gln905-Val930, which is involved in assembly with the gastric proton pump beta  subunit (13), much as the corresponding Asn886-Ala911 peptide of the sodium pump alpha  subunit is involved in assembly with the sodium pump beta  subunit (46).

To investigate a possible involvement of the M7/M8 extracellular peptide of the gastric proton pump alpha  subunit in SCH 28080 recognition, wild-type sodium pump alpha  subunits or alpha  subunit chimeras containing Gln905-Val930 of the gastric proton pump alpha  subunit were coexpressed with either sodium or proton pump beta  subunits in yeast (Table I) and were investigated with respect to their ability to recognize either the sodium pump-specific inhibitors ouabain and palytoxin or the gastric proton pump-specific inhibitor SCH 28080. 


                              
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Table I
Vector description and vector combinations used in the experiments

Ouabain Binding-- Microsomal membranes prepared from yeast cells expressing the various subunit combinations bind [3H]ouabain with high affinity (Fig. 1; Table II). The NGH26/HKbeta heterodimer was characterized by a reduced ouabain binding affinity (Kd = 21.1 ± 2.5 nM; Table II), and a lower maximum ouabain binding capacity (0.18 ± 0.01 pmol/mg) than the NKalpha 3/NKbeta complex. The ouabain binding capacity of the NGH26/HKbeta heterodimer is only 17% of the total binding obtained with the wild-type sodium pump (Fig. 1; Table II), and is similar to the expression level that has previously been reported for the NGH26/HKbeta complex (13). To investigate the reasons for the lower ouabain binding capacity, the expression levels of the NKalpha 3, NGH26, and HKbeta were measured in microsomal preparations by Western blots and antibody capture assays. Fig. 2 shows that the level of expression of the NGH26 subunit is approximately the same as the level of expression of the alpha 3 subunit. Fig. 3, however, indicates that HKbeta is present in microsomal membranes from yeast cells expressing the NGH26/HKbeta complex at lower levels than HKbeta is present in microsomes from yeast expressing NKalpha 3/HKbeta . Results of the antibody capture assay indicate that only about 13-17% of the amount of p-nitrophenolate obtained from NKalpha 3/HKbeta was formed by membranes containing the NGH26/HKbeta complex (Fig. 3B). Thus, it appears that the reduced expression level of HKbeta in membranes from cells expressing the NGH26/HKbeta heterodimer limits the number of NGH26/HKbeta complexes, and the low level of ouabain binding shown in Fig. 1 is a measure of the number of these complexes in the membranes. The limiting effect of HKbeta expression on the number of assembled pumps can be explained by the requirement of the expression system for two different plasmids to direct the synthesis of the alpha  and beta  subunits independently in the yeast cells. Transformation of yeast with the two plasmids results in cells with different copy numbers for the two plasmids.


                              
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Table II
Summary of results determined for yeast expressing various sodium/proton pump subunit combinations described in text
The EC50 values for palytoxin are from a single experiment. The EC50 values for ATP are the means ± S.D. of four measurements obtained with two membrane preparations. All other values represent the mean ± S.D. from three independent experiments.

Extraction of the yeast membranes with SDS is necessary to measure ouabain-sensitive ATP hydrolysis because the detergent inactivates endogenous yeast ATPases and enriches yeast membranes in the heterologously expressed Na+,K+-ATPase (40). The NGH26/HKbeta complex is easily denatured when yeast membranes containing this complex are incubated with SDS, however, and it was not possible to measure ouabain-inhibited ATPase activity in yeast membranes containing this chimera. Thus, to show that the NGH26/HKbeta complex binds ATP and is autophosphorylated by ATP, we measured binding of ouabain in the presence of Na+, Mg2+, and ATP. Under these conditions (see "Experimental Procedures"), ATP hydrolysis and autophosphorylation of the enzyme by ATP promotes binding of ouabain (44).

ATP promoted binding of [3H]ouabain by all alpha beta complexes examined. The maximum amount of ouabain bound by membrane preparations containing either NKalpha 3/NKbeta 1 or NKalpha 3/HKbeta was nearly the same, and was similar to the maximum amount of ouabain bound when the pumps are phosphorylated by inorganic phosphate (Fig. 1). The maximum amount of ouabain bound by the NGH26/HKbeta heterodimer was only about 17% of the amount bound by NKalpha 3/NKbeta 1 or NKalpha 3/HKbeta , however, consistent with the results obtained from the experiments shown in Figs. 1 and 3. The EC50 of the NGH26/HKbeta chimera for ATP (1.13 ± 0.51 µM) is also very similar to the EC50 of the wild-type Na+,K+-ATPase for the nucleotide in the same assay (Table II). These data confirm that the NGH26/HKbeta chimera is capable of forming a phosphoenzyme both from inorganic phosphate and from ATP, although they do not address the question whether the NGH26/HKbeta chimera is capable of completing the catalytic cycle.

Ouabain binding to yeast membranes containing the different pumps was measured at 0, 1, and 10 µM ATP in the presence of 25 µM SCH 28080. SCH28080 is a specific inhibitor of gastric H+,K+-ATPase and inhibits the binding of omeprazole to this enzyme. At this concentration, SCH 28080 had no effect on the binding of ouabain to membranes containing either NKalpha 3/NKbeta 1 or NKalpha 3/HKbeta hybrids (data not shown). This result is in agreement with data published previously about the effect of SCH 28080 on sodium pump activity (11, 47). 25 µM SCH 28080 caused a small (25%) reduction in ouabain binding to NGH26/HKbeta , but because of the uncertainties associated with the measurements this result was not statistically significant.

Palytoxin-induced Potassium Efflux from Yeast Cells-- Palytoxin has no effect on nontransformed yeast cells, and only a small loss of K+ is observed from yeast cells that express the chimeric NGH26 subunit together with the rat sodium pump beta 1 subunit (data not shown). This small effect on cells expressing NGH26/NKbeta 1 may be due to interaction of palytoxin with the very low number of functional pumps in the yeast membrane (13). Alternatively, the assembly of NGH26 and NKbeta 1 may affect the binding of palytoxin or the conduction of ions through the channel after palytoxin binds to the heterodimer. Because of the small magnitude of the K+ loss, however, investigation of the effects of palytoxin on this subunit combination was not continued any further.

When yeast cells expressing either the NKalpha 3/NKbeta 1, NKalpha 3/HKbeta , or NGH26/HKbeta subunits are exposed to various concentrations of palytoxin, they lose up to 80% of their intracellular K+ within 2 h (Fig. 5). At all concentrations of palytoxin, the K+ concentration inside the cell at the end of the incubation period represents a steady state between K+ efflux through the palytoxin-induced channel and K+ influx through the yeast TRK1 and TRK2 uptake pathways. At the end of the incubation period with saturating palytoxin, the steady-state levels of K+ in the cells expressing different alpha beta complexes were the same. The concentration of palytoxin required for half-maximal loss of K+ under these conditions, however, is different for each complex (Table II). The EC50 for the palytoxin-induced K+ efflux from cells expressing the NKalpha 3/NKbeta 1 subunits is 136 nM. The corresponding value obtained with cells expressing the NKalpha 3/HKbeta subunits is approximately 2-fold higher (313 nM), and the EC50 obtained with cells expressing the NGH26/HKbeta subunits is about 6-fold higher (822 nM) than for NKalpha 3/NKbeta 1. These differences in EC50 values may be explained in several ways. First, the affinity of the complexes containing HKbeta for palytoxin may be reduce compared with NKalpha 3/NKbeta 1 due to distortion of the palytoxin binding site by assembly of the alpha  subunits with HKbeta . Alternatively, the rate of K+ ion conduction through heterodimers containing different combinations of alpha beta subunits may be different. Finally, the different EC50 values may reflect the different numbers of functional complexes present in the cells expressing different heterodimers. Although it was not possible to test for reduced palytoxin binding affinity since radiolabeled palytoxin is not available, analysis of the initial rates of K+ efflux from the cells at various palytoxin concentrations shows that a difference in the number of channels is sufficient to explain the data. From the data presented in Table II it can be seen that dividing k'N by the number of channels (Bmax) yields nearly identical values of the apparent single-channel conductance k' for all of the alpha beta heterodimers. These values are 0.23 ± 0.04 for NKalpha 3/NKbeta 1, 0.17 ± 0.09 for NKalpha 3/HKbeta , and 0.27 ± 0.02 for NGH26/HKbeta . The similarity of these values indicates that the product of the single channel conductance (kK+) and the constant of proportionality (k) between palytoxin concentrations and the open channel probability is the same. Although values for each of these constants cannot be determined independently from these measurements, it is possible that the single-channel conductance and the open probability are similar for all of the heterodimers.

Inhibition of Palytoxin-induced Potassium Efflux by Ouabain and SCH28080-- The palytoxin-induced K+ efflux from yeast cells expressing any of the subunit combinations NKalpha 3/NKbeta 1, NKalpha 3/HKbeta , or NGH26/HKbeta is completely inhibited by ouabain (Fig. 6). At 400 nM palytoxin, ouabain inhibits the K+ efflux from cells expressing the different heterodimers with IC50 values of 23-28 µM (Table II). The small differences between the IC50 values for ouabain inhibition of palytoxin-induced K+ efflux in the different heterodimers is similar to the pattern of Kd values for ouabain binding to the heterodimers (13).

As expected from the known insensitivity of Na+,K+-ATPase to SCH 28080, there was <10% inhibition by SCH 28080 of K+ efflux induced by 400 nM palytoxin from yeast cells expressing either the NKalpha 3/NKbeta 1 or the NKalpha 3/HKbeta subunits (Fig. 7A). In contrast, the palytoxin-induced K+ efflux from yeast cells expressing the NGH26/HKbeta subunits is almost completely inhibited by 50 µM SCH28080. The IC50 value for the SCH 28080 inhibition of the palytoxin-induced K+ efflux is 14.3 ± 2.4 µM (Fig. 7B). This value should be compared with the IC50 values determined for the inhibition of proton transport into porcine gastric vesicles (0.5 µM) or for SCH 28080 inhibition of aminopyrine uptake in rabbit gastric glands (0.2 µM) (16). Thus, sensitivity to SCH 28080 can be conferred upon Na+,K+-ATPase by substitution of 26 amino acids from gastric H+,K+-ATPase (Gln905-Val930) for the homologous residues in the extracellular loop connecting transmembrane segments 7 and 8 of Na+,K+-ATPase alpha 3 subunit.

It is not known whether the extracellular loop connecting transmembrane segments 7 and 8 is directly involved in binding SCH28080 or whether the binding site is induced elsewhere in the protein as a result of chimera formation. If the extracellular loop is part of the binding site, it is likely that the binding site also consists of additional parts of the polypeptide. Using chimeric ATPases, Blostein et al. (29) concluded that the amino-terminal half of the gastric proton pump must be involved in SCH 28080 binding, and this is in good agreement with the results of Munson et al. (30), who labeled a 9.9-kDa tryptic peptide starting at Gln104 of the gastric proton pump alpha  subunit with a photoactivated derivative of SCH 28080. An amino-terminal localization of the SCH 28080 binding site was further supported by the observation that the rat gastric proton pump peptide Val115-Ile126 from the M1 membrane-spanning domain confers SCH 28080 sensitivity to the sodium pump (11). In that report, however, SCH 28080 did not inhibit palytoxin-induced K+ efflux from yeast cells expressing this chimera. The results of that investigation have recently been questioned by Asano et al. (48), however, who found that replacement of the Val115-Ile126 sequence of gastric H+,K+-ATPase with the corresponding region of Na+,K+-ATPase alpha  subunit did not abolish SCH 28080 sensitivity. A possible explanation for this apparent discrepancy is suggested by the possibility that SCH28080 binding is induced indirectly by chimera formation and that the different chimeras have different properties because of their different sequences. Thus, mutations within Val115-Ile126 do not necessarily have to preclude interactions of SCH 28080 with the protein, since the other points of attachment are possibly enough to stabilize binding. Asano et al. implicated Thr825 and Pro829 as possible determinants of the affinity of the enzyme for SCH 28080 (48), together with Glu822 in the M6 membrane-spanning domain of gastric H+,K+-ATPase (49).

The finding that the extracellular peptide Gln905-Val930 of the proton ATPase confers SCH 28080 sensitivity to Na+,K+-ATPase is consistent with previous findings demonstrating that SCH 28080 competes with omeprazole and that omeprazole reacts with Cys892 from the M7/M8 connecting loop (31, 50). A recent investigation suggested that the binding sites for omeprazole and SCH 28080 are not identical but do overlap (51). Taking our results into consideration, one can imagine that the interaction of SCH 28080 with the Gln905-Val930 peptide reduces the accessibility of Cys892 for omeprazole. Furthermore, conformational changes induced by either inhibitor binding or chimera formation may easily account for participation of other benzimidazole-reactive cysteines in SCH28080 binding (51). The close proximity between the Gln905-Val930 peptide and Cys892, the fact that both SCH 28080 and omeprazole inhibit H+ secretion catalyzed by the proton pump, and the demonstration that SCH 28080 inhibits the palytoxin-induced K+ efflux from cells expressing the NGH26/HKbeta 1 heterodimer implicates the M7/M8 loop of alpha  subunit in ion conduction by the proton pump. The homologous region of the sodium pump has also been suggested to be involved in ion binding, occlusion, or translocation (34, 45, 52). Further investigation of this possibility and the identification of additional peptides and amino acids within the Gln905-Val930 sequence that are important for interactions between the protein and the transported ions, will require additional experiments with new chimeras and mutants. The approach developed in this study may be useful, therefore, in elucidating the mechanisms that lead to SCH 28080 inhibition of H+ secretion by the gastric proton pump.


    ACKNOWLEDGEMENT

The gastric proton pump inhibitor SCH 28080 was a gift of Schering-Plough (Marburg an der Lahn, Germany).


    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grants Sche307/4-1 and Sche307/4-2 (to G. S.-B.) and by National Institute of Health Grant GM28673 (to R. A. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Keck School of Medicine, University of Southern California, 1333 San Pablo St., MMR 250, Los Angeles, CA 90033. Tel.: 323-442-1240; Fax: 323-442-2283; E-mail: rfarley@hsc.usc.edu.

Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M008784200


    ABBREVIATIONS

The abbreviations used are: Na+, K+-ATPase, sodium- and potassium-activated adenosine triphosphatase (EC 3.6.1.37); H+, K+-ATPase, proton-and potassium-activated adenosine triphosphatase (EC 3.6.1.36); SCH 28080, 8-benzyloxy-3-cyanomethyl-2-methyl-imidazo[1,2-a]pyridine; M1-M10, membrane-spanning domains 1-10; PBS-T, phosphate-buffered saline with Tween 20.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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