From the 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
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ABSTRACT |
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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 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 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 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 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
Yeast Cells Used for Expression--
The S. cerevisiae strain 30-4 (MAT- Detection of NK
HK Estimation of the Gastric Proton Pump
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 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 NK 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 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
Replacement of the sodium pump Expression of NK
The expression levels of the gastric proton pump
Quantification of the HK
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
HK 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 NK Palytoxin-induced Potassium Efflux from Yeast Cells--
Yeast
cells expressing the NK
To estimate the initial rates of the palytoxin-induced efflux, cells
expressing either NK Inhibition of Palytoxin-induced Potassium Efflux by Ouabain and SCH
28080--
The palytoxin-induced K+ efflux from yeast
cells expressing
To investigate the effect of the gastric proton pump-specific inhibitor
SCH 28080, yeast cells expressing NK 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 To investigate a possible involvement of the M7/M8 extracellular
peptide of the gastric proton pump 3 subunit with the peptide
Gln905-Val930 of the gastric proton pump
subunit substituted in place of the original
Asn886-Ala911 sequence was expressed together
with the gastric
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
3 and
1 subunits of the sodium pump or the
3 subunit of the sodium pump
together with the
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit, for their function, in addition to a
larger ATP-recognizing
subunit. The
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
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
subunits must be very similar, since the proton pump
subunit
was shown in numerous investigations to form functional complexes with
the
subunit of the sodium pump when these proteins were expressed
together (10-14).
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
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
subunit might also
participate in interactions of the gastric proton pump with SCH 28080.
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
subunit and a gastric proton pump
subunit? Does a
similar phenomenon occur when yeast cells express a chimeric sodium
pump
subunit containing the extracellular sequence
Gln905-Val930 from the M7/M8 loop of the
gastric proton pump
subunit? Finally, is any palytoxin-induced
K+ efflux observed sensitive to ouabain or SCH 28080?
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 subunit. This vector is denoted YEpr
3 (39). The same expression plasmid was also used for the insertion of the cDNA coding for a
chimera of the sodium pump
3 subunit that was made to contain Gln905-Val930 of the rat gastric proton pump
subunit
in place of the original Asn886-Ala911 (13).
This vector is named YEpNGH26. Vectors pG1T-r
1 and pG1T-HK
were
used for the expression of the rat sodium pump
1 subunit and the rat
gastric proton pump
subunit, respectively (39).
, trp1,
ura3, Vn2, GAL+) was used for
transformation with either of the yeast expression vectors YEpNGH26
(coding for the chimeric sodium pump
subunit) or YEpr
3 (coding
for the wild-type sodium pump
3 subunit of the rat) together with
either of the vectors pG1T-r
1 or pG1T-HK
coding for the
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).
3, NGH26, and HK
Subunits in a Western
Blot--
The conditions for electrophoresis and immunoblot detection
of
3 or NGH26 subunits have been described previously in detail (13). Briefly, 100 µg of microsomal protein isolated from yeast expressing either the NK
3/NK
or NGH26/HK
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
3 and NGH26 subunits were visualized by using monoclonal antibody 5 against sodium pump
subunits (1:400 dilution) and the
commercially available enhanced chemiluminescence kit (ECL) following
the protocol of the provider.
subunits in membrane preparations from cells expressing either
NK
3/HK
or NGH26/HK
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
subunit
(1:2000 dilution). All other conditions were the same as above.
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 HK
in membrane preparations
from yeast expressing either NK
3/HK
or NGH26/HK
. Microsomal
proteins from nontransformed cells served as a control.
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).
3/NK
1; 0.5, 2, or 5 µM palytoxin for cells expressing NK
3/HK
;
and 1, 2.5, or 5 µM palytoxin for cells expressing the
NGH26/HK
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.
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 and
1 subunits (NK
3/NK
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 NK
3/HK
subunits
(Kd of 6.4 ± 1.4 nM). The
Bmax for ouabain binding to the NK
3/HK
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
subunit (NGH26) together with the sodium pump
1 subunit (NK
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 NK 3/NK
1 (
) or either of the chimeric
proteins NK
3/HK
(
) and NGH26/HK
(*) 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.
1 by the proton pump
subunit
results in the formation of NGH26/HK
complexes capable of binding
ouabain (Fig. 1). The equilibrium dissociation constant Kd for ouabain binding to the NGH26/HK
heterodimer (21.1 ± 2.5 nM; Table II) is slightly
increased compared with complexes assembled with NK
3, indicating a
reduction in affinity. In addition, the maximum amount of ouabain bound
by NGH26/HK
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).
3, NGH26, and HK
Subunits in Yeast--
To
investigate the relative level of expression of the
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
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
3 or NGH26 subunit in microsomes isolated from nontransformed yeast cells.
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Fig. 2.
Immunodetection of
NK 3 and NGH26 subunits. Microsomal
proteins from yeast expressing either NGH26/HK
(lane
1) or NK
3/NK
(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
and the commercially
available enhanced chemiluminescence Western blot analysis system. The
figure demonstrates that NGH26 (lane 1) and
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).
subunit (HK
)
were measured in yeast membrane preparations from cells expressing either NK
3/HK
or NGH26/HK
using a monoclonal antibody raised against the gastric proton pump
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 NK
3/HK
or NGH26/HK
heterodimers. Since corresponding bands are not detected in membranes
from nontransformed cells, these bands at 40 and 43 kDa are gastric
pump
subunits, possibly glycosylated to various extents. It is
apparent from the figure that the abundance of the HK
subunit in
membrane preparations from cells expressing the NK
3/HK
complex is
higher than in membranes from cells expressing the NGH26/HK
hybrid.
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Fig. 3.
Immunodetection of HK
subunits. A, microsomal proteins from
untransformed yeast (lane 1), or yeast expressing either
NGH26/HK
(lane 2) or NK
3/HK
(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
HK
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 HK
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
= 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.
subunit expression levels in yeast
microsomes from cells expressing either the NK
3/HK
or the
NGH26/HK
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 HK
) 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 HK
). 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 HK
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 HK
subunits.
from cells expressing the NGH26/HK
heterodimers accounts for
only 13-17% of the value obtained from the microsomes from cells
expressing the NK
3/HK
subunit combination.
3/NK
1
heterodimer with an EC50 of 3.9 ± 0.16 µM. Similarly, ATP promotes binding to the NK
3/HK
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/HK
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 NK
3/NK
1 or
NK
3/HK
.
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Fig. 4.
Promotion of ouabain binding by ATP.
Membranes isolated from yeast cells expressing NK 3/NK
1 (
) or
either of the chimeric proteins NK
3/HK
(
) and NGH26/HK
(*)
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.
3/NK
1, NK
3/HK
, or NGH26/HK
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 NK
3/NK
1, NK
3/HK
, and NGH26/HK
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 NK
3/NK
1 subunits
is 136 nM. The corresponding value obtained with cells
expressing the NK
3/HK
subunits is approximately 2-fold higher
(313 nM), and the EC50 obtained with cells
expressing the NGH26/HK
subunits is about 6-fold higher (822 nM) than for NK
3/NK
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
1 subunit (data not shown).
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Fig. 5.
Palytoxin concentration dependence of
K+ efflux. Yeast cells expressing NK 3/NK
1 (
)
or either of the chimeric proteins NK
3/HK
(
) and NGH26/HK
(*) 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.
3/NK
1, NK
3/HK
, or NGH26/HK
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
heterodimer were not
significantly different at different palytoxin concentrations, and are
reported in Table II as mean values for all palytoxin concentrations.
1/
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 NK
3/NK
1, NK
3/HK
, or NGH26/HK
is also inhibited by ouabain (Fig. 6). At
400 nM palytoxin, ouabain inhibits the K+
efflux from cells expressing the NK
3/NK
1 heterodimer with
IC50 = 26.5 ± 0.1 µM (Table II).
Comparable results are obtained with cells expressing either
NK
3/HK
or NGH26/HK
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).
View larger version (14K):
[in a new window]
Fig. 6.
Determination of IC50 values for
inhibition of the palytoxin-induced K+ efflux by
ouabain. Yeast cells expressing the NK 3/NK
1 (
) or either
of the chimeric proteins NK
3/HK
(
) and NGH26/HK
(*) 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.
3/NK
1, NK
3/HK
, or
NGH26/HK
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/HK
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).
View larger version (17K):
[in a new window]
Fig. 7.
Inhibition of the palytoxin-induced
K+ efflux by SCH 28080. Yeast cells expressing
NK 3/NK
1 (
) or either of the chimeric proteins NK
3/HK
(
) and NGH26/HK
(*) 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/HK
(*) as shown in B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
subunit might be part of the binding site for SCH
28080. This peptide corresponds to a sequence of the sodium pump
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
subunit contains the peptide Gln905-Val930, which is involved in assembly
with the gastric proton pump
subunit (13), much as the
corresponding Asn886-Ala911 peptide of the
sodium pump
subunit is involved in assembly with the sodium pump
subunit (46).
subunit in SCH 28080 recognition, wild-type sodium pump
subunits or
subunit chimeras containing Gln905-Val930 of the gastric proton
pump
subunit were coexpressed with either sodium or proton pump
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.
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/HK 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 NK
3/NK
complex. The ouabain binding capacity of
the NGH26/HK
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/HK
complex (13). To investigate the reasons for the lower
ouabain binding capacity, the expression levels of the NK
3, NGH26,
and HK
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
3 subunit. Fig. 3, however, indicates that HK
is present in microsomal membranes from yeast cells expressing the NGH26/HK
complex at lower levels than HK
is present in microsomes from yeast
expressing NK
3/HK
. Results of the antibody capture assay indicate
that only about 13-17% of the amount of p-nitrophenolate obtained from NK
3/HK
was formed by membranes containing the NGH26/HK
complex (Fig. 3B). Thus, it appears that the
reduced expression level of HK
in membranes from cells expressing
the NGH26/HK
heterodimer limits the number of NGH26/HK
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
HK
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
and
subunits independently in the
yeast cells. Transformation of yeast with the two plasmids results in
cells with different copy numbers for the two plasmids.
|
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/HK 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/HK
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
complexes examined. The maximum amount of ouabain bound by membrane
preparations containing either NK
3/NK
1 or NK
3/HK
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/HK
heterodimer was only about 17% of the amount bound by NK
3/NK
1 or NK
3/HK
,
however, consistent with the results obtained from the experiments
shown in Figs. 1 and 3. The EC50 of the NGH26/HK
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/HK
chimera is
capable of forming a phosphoenzyme both from inorganic phosphate and
from ATP, although they do not address the question whether the
NGH26/HK
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
NK3/NK
1 or NK
3/HK
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/HK
, 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 1 subunit (data not shown). This small effect on cells expressing NGH26/NK
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 NK
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 NK3/NK
1, NK
3/HK
, or
NGH26/HK
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
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 NK
3/NK
1 subunits
is 136 nM. The corresponding value obtained with cells
expressing the NK
3/HK
subunits is approximately 2-fold higher
(313 nM), and the EC50 obtained with cells
expressing the NGH26/HK
subunits is about 6-fold higher (822 nM) than for NK
3/NK
1. These differences in EC50 values may be explained in several ways. First, the
affinity of the complexes containing HK
for palytoxin may be reduce
compared with NK
3/NK
1 due to distortion of the palytoxin binding
site by assembly of the
subunits with HK
. Alternatively, the
rate of K+ ion conduction through heterodimers containing
different combinations of
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
heterodimers. These values are
0.23 ± 0.04 for NK
3/NK
1, 0.17 ± 0.09 for
NK
3/HK
, and 0.27 ± 0.02 for NGH26/HK
. 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 NK3/NK
1,
NK
3/HK
, or NGH26/HK
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
NK3/NK
1 or the NK
3/HK
subunits (Fig. 7A). In
contrast, the palytoxin-induced K+ efflux from yeast cells
expressing the NGH26/HK
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
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 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
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/HK1
heterodimer implicates the M7/M8 loop of
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.
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