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INTRODUCTION |
The gastric H,K-ATPase produces the acidic environment of the
gastric mucosa through an electroneutral exchange of H+ for
K+ (1, 2). It is a member of a phosphoenzyme-forming ATPase family (P2)1 that includes
the Ca-ATPase, the Na,K-ATPase, and the Neurospora H+ pump (3). This family of transporters and the more
distantly related P1 and P3 ATPase families display important
functional variations in the specificity and stoichiometry of
transported ions, inhibitor sensitivities, cellular distribution, and
subunit organization.
The H,K-ATPase gene family within the P2 ion pump family is composed of
at least five
-subunit isomers and, as known to date, a single
-subunit. The ATPase is a heterodimer composed of a Mr 114,000 catalytic subunit and a smaller,
Mr 34,000 glycosylated
-subunit (4-6). The
primary structure of the gastric
-subunit isoform is highly
conserved among various mammalian species including rat (7), rabbit
(8), hog (9), and man (10), but sequence conservation deteriorates in
H,K-ATPase isoforms present in the urinary bladder (11), skin (12),
colon (13), and kidney (14) as well as in other P2-type pump isoforms
(15, 16).
The P2 family of ion pumps consists of polytopic membrane proteins
generally modeled with 8-10 membrane spanning regions. A variety of
evidence has yielded a consensus for the identity of the transmembrane
spanning domains M1-M4 within the N-terminal half of the ATPases
(17-21) but has not conclusively defined the C-terminal transmembrane
spanning domains (22-25).
Although the organization of the C-terminal membrane spanning domains
remains a focus of investigation, there is general consensus that
charged residues within these domains participate in
cation-dependent function. Cation binding to
membrane-embedded proteolytic fragments is dependent upon the integrity
of the ATPase from M5 to the C terminus in both the Na,K-ATPase and the
H,K-ATPase (17, 19, 26, 27). The disruption of the C-terminal sequence
at the cytoplasmic-M7 interface facilitates the thermal inactivation of
ATPase (28, 29), release of the putative M5-M6 hairpin to the
supernatant (30), and exposure of Cys-983 (31). Amino acid
substitutions within membrane spanning segments of M4, M5, M6, and M8
have identified conserved residues in the expressed Na,K-ATPase,
Ca-ATPase, and H,K-ATPase that are essential for ion-dependent transport or cation-dependent
catalytic activity (32-39).
Chemical modification studies utilizing carboxyl reagents such as DEAC
(40) and DCCD (41-45) have investigated the role of hydrophobic
carboxyl groups in several pumps within the P2 pump family. These
studies show that protection from inhibition by the transported cation
is a general characteristic of the inhibitor-dependent inactivation of these ATPases. Even though the mechanism of
inactivation is controversial, the cation specificity and concentration
dependence providing protection have frequently been interpreted as
evidence for the modification of residues within the cation binding
site. A microsequence has now been obtained for several reactive
residues in or near transmembrane spanning domains within M5, M7, and
M9 of the native H,K-ATPase (45) and Na,K-ATPase (44, 46). Mutational
studies of the modified residue and analysis of the expressed
Na,K-ATPase have provided evidence to confirm biochemical studies that
Glu-779 (NIPE), a DEAC-reactive site, is important for
cation-dependent activity (46-48) but contradicted a
similar biochemical study that Glu-959 (FEET), a
[14C]DCCD-reactive site (44, 47, 49), is important for
Na,K-ATPase activity.
Glu-857 (LVNE), a residue at the fourth putative
cytosolic-seventh transmembrane spanning region interface (C4-M7) of
the gastric H,K-ATPase has been identified as a
K+-protected, [14C]DCCD-reactive site (45).
To clarify whether DCCD-dependent inhibition is the result
of its modification of an essential residue within the cation binding
site of the H,K-ATPase or whether inhibition may result from a
secondary effect, such as cross-linkage or cation-dependent protection of a DCCD-reactive site at an alternative locus, a series of
site mutants were prepared at this position varying the size, charge,
and hydrophobicity of the side chain residue. The site mutants were
transiently expressed in HEK293 T cells, and the expressed H,K-ATPase
was analyzed to determine the effect of the modifications on the
steady-state kinetic constants describing K+-stimulated
activation or SCH 28080-dependent inhibition of the ATPase.
The results of this investigation demonstrate that Glu-857 is not
essential for K+-dependent ATPase activity but
do suggest that the C4-M7 domain is an important region involved in
the conformational sensitivity of the molecule.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
Plasmids containing the
-subunit
(GenBank accession number X64694) and
-subunit (GenBank accession
number M35544) of the rabbit (Oryctolagus cuniculus) gastric
H,K-ATPase in the vector pcDNA3 (Invitrogen) were a kind gift of
Dr. George Sachs (Wadsworth Veterans Affairs Medical Center, UCLA).
Bidirectional sequence analysis of the 5' and 3' regions verified the
integrity of the ORF in the end regions of each construct. The
-subunit plasmid, pcDNA3-
, is a 6778-bp construct with its
ORF flanked by original 5'- and 3'-untranslated regions (UTR) of 27 and
405 base pairs, respectively. The 5' UTR of the
-subunit is linked to the multiple cloning site of the pcDNA3 vector through a 16-bp sequence (AAT GGG GTA CCG AAT T), and the 3'-UTR is linked by an
11-bp sequence (TTC CTC GAA AT). The
-subunit, pcDNA3-
, is an 8784-bp construct with its ORF flanked by original 5'- and 3'-UTR of
12 and 203 base pairs, respectively. The 5'-end UTR of the
-subunit
is linked to the multiple cloning site of the pcDNA3 vector with a
21-bp linker sequence (GGT ACC CAA TTC CTG CAG CCC), and the
3'-UTR is linked with a 24-bp linker sequence (GAT CCA CTA GTT CTA GGG GGA TCC).
DNA Sequencing--
All site mutations were verified by DNA
sequencing performed by the W. M. Keck Foundation Biotechnology
Resource Laboratory at Yale University. Sequence analysis was carried
out on an Applied Biosystems 373A Stretch DNA Sequencer. The sequencing
reactions utilized fluorescently labeled dideoxynucleotides and
Taq FS DNA polymerase in a thermal cycling protocol. The
submitted samples, containing 750 ng of circularized plasmid DNA and
0.4 µM oligonucleotide sequence primer, yielded
approximately 600-bp sequences with >99% accuracy. The sequence
primers were constructed either as sense primers complementary to a
25-bp sequence preceding the M5 transmembrane region or as antisense
primers complementary to a 21-bp sequence immediately preceding the M8
transmembrane region.
Site-directed Mutagenesis--
Site-directed mutations were
introduced into pcDNA3-
via the Quick Change site-directed
mutagenesis kit (Stratagene) using the manufacture's instructions with
modifications. 125 ng of pcDNA3-
was extended in a thermal
cycler (Perkin-Elmer 2400) for 20 cycles (45 s at 95 °C, 1 min at
55 °C, 21 min at 68 °C) using 125 ng of both the mutant sense and
antisense primers indicated in Table I.
Following elongation, the parental strand was digested for a minimum of
1 h at 37 °C with 10 units of DpnI. Competent XL-1 Blue bacterial cells were heat shock-transformed with 1.3 µl of the
DpnI-treated extension reaction, plated on LB Amp medium
(0.1 mg of ampicillin/ml), and grown overnight at 37 °C. Single
colonies devoid of satellites were selected and expanded by growth at
37 °C for 12-24 h in 250 ml of LB Amp medium. The plasmid was
isolated using the Wizard Midi-prep kit (Promega). An aliquot of the
plasmid DNA was repurified on Qiagen Spin Mini-prep columns and
submitted for sequence analysis.
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Table I
Mutant primers for site mutants of Glu-857
Each mutation utilized a primer pair consisting of the sense primers
shown below and their identical antisense complements.
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Cell Culture and Transient Transfection--
HEK293 T cells were
cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine
serum, 2.0 mM glutamine, and 0.1 mg/ml penicillin and
streptomycin at 37 °C in 95% CO2, 5% O2
atmosphere. At confluency, the cells were released by trypsin
treatment, uniformly dispersed, and cultured overnight in 100-mm tissue
culture dishes. At approximately 60-80% confluency, the cells were
washed with serum-free Opti-MEM I supplemented with 0.9 mM
CaCl2 and co-transfected with 9 µg of pcDNA3-
and
18 µg of pcDNA3-
. The co-transfection procedure utilized 1.3 µl of LipofectAMINETM/µg of DNA. Following overnight incubation,
20 ml of the standard culture medium was added to the serum-free
culture medium, and the cells were grown for an additional 48 h.
The transiently co-transfected cells were harvested for membrane
preparation at 72 h post-transfection.
Membrane Preparation--
The transiently transfected cells were
washed once with PBS supplemented with 5 mM EDTA and were
harvested from the plate following a brief incubation in the same
medium. The cells were collected by 5 min of centrifugation at 2000 rpm
and resuspended in ice-cold homogenization buffer containing 10 mM Pipes, Tris, pH 7.4, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, and 10 units/ml bovine
lung aprotinin. Membranes were prepared from the cells by Dounce
homogenization utilizing a 7-ml vessel and tight fitting pestle.
Following a 200-stroke homogenization cycle, the membranes were diluted
to a final concentration of 165 mM sucrose with 0.5 M sucrose, homogenized a final 10 strokes, and layered on a
sucrose column containing 10 mM Pipes, Tris, pH 7.4, 1 mM EDTA, and 44% sucrose. Membrane fractions were resolved
by 1 h of centrifugation in a Beckman SW-28 rotor centrifuged at
22,000 rpm. The membrane fraction obtained from the 44% sucrose
interface was diluted 1:2 (v/v) with homogenization buffer and
collected in a Beckman Ti-50.2 rotor centrifuged at 34,000 rpm for 20 min. The pellet obtained by this procedure was resuspended in 10 mM Pipes, Tris, pH 7.4, and frozen at
80 °C until assay.
Protein Quantification and Western Analysis--
Total
membrane-bound protein was quantified using the Bio-Rad assay kit with
-globulin as standard. The quantitative Western analysis was
performed on the AMBIS image acquisition and analysis system utilizing
an XC-75/75CE CCD video camera module and AMBIS core (ver. 4)/ONE-Dscan
software. 35-55-µg aliquots of total membrane protein were resolved
by SDS-PAGE run under reducing conditions according to the method of
Laemmli (50) and transferred to nitrocellulose for Western analysis.
The gel was resolved by a two-step electrophoresis procedure utilizing
25 mA (constant current) for 20 min and 200 V (constant voltage) for
1 h. The SDS-PAGE-resolved protein was transferred to
nitrocellulose for 3 h at 150 mA (constant current). For Western
analysis the nitrocellulose membranes were blocked in 1% Carnation
instant non-fat dry milk and probed with anti-
mAb 2B-6 (1:10,000)
or anti-
mAb 12.18 (1:5,000). The probed membranes were incubated
with secondary anti-mouse horseradish peroxidase-conjugated antibody
and developed according to the Amersham protocol. The expressed protein
was visualized by a 15-s exposure to ECL Hyperfilm.
ATPase Analysis--
ATPase activity was measured in membrane
preparations of nontransfected (control) and transiently co-transfected
HEK293 T cells. Typically, 55 µg of membrane-bound protein was
assayed by the method of Yoda and Hokin (51) using 1 ml of ATPase
reaction buffer containing 2 mM ATP, 2 mM
MgCl2, 0.1 mM ouabain, 1 mM EGTA, 50 mM Tris-HCl, pH 7.4, and 1 µg of oligomycin ± 10 mM KCl. The assay was started by the addition of ATP,
incubated at 37 °C for 45 min and stopped with the addition of 1 ml
of 80% ammonium molybdate, 20% perchloric acid. The expressed
H,K-ATPase was differentiated from an indigenous, nonspecific
phosphatase by a characteristic SCH 28080-sensitive, KCl-stimulated
activity. The reported values for the K0.5 (KCl)
and Ki (SCH 28080) were calculated from the global
fit of n = 3 measurements from each of three separate transient co-transfection experiments. The steady-state kinetic constants were calculated from the nonlinear best fit to:
K0.5 = Vmax × [K+]/K0.5 + [K+] and
Y = 1/(1 + [SCH 28080]/Ki) + nsb (nonspecific).
Cell Line, Antibodies, and Materials--
HEK293 T cells were a
kind gift of Dr. Jamboor K. Vishwanatha. Anti-
mAb 2B-6 was a kind
gift from Dr. M. Maeda, and anti-
mAb 12.18 was a kind gift from Dr.
A. Smolka. The sense and antisense mutant and sequencing primers were
purchased from Midland Certified Reagents. LipofectAMINETM was
obtained from Life Technologies, Inc.
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RESULTS |
Transient Expression of Wild Type H,K-ATPase--
The H,K-ATPase
of mammalian species is organized as an
/
heterodimer. The
porcine
-subunit resolved by SDS-PAGE and probed with H,K-ATPase
-subunit antibody, mAb 12.18, is shown in Fig. 1, upper panel, lane 4. The
-subunit appears as a single band at Mr
94,000. The
-subunit probed with H,K-ATPase
antibody, mAb 2B-6,
is shown in lane 4 of the lower panel of Fig. 1.
Because of its extensive glycosylation the
-subunit exhibits a
mobility ranging from Mr 70,000 to 80,000. Membrane preparations from HK293 T cells transiently transfected with
the H,K-ATPase cDNA are shown in the remaining lanes of Fig. 1. As
shown in lanes 1 and 2, either the
- or
-subunit is expressed in the HK293 T cells. The cells transiently
transfected with the
-subunit express a subunit of comparable
mobility to the native H,K-ATPase. There is no evidence of smaller
protein fragments suggestive of premature termination of protein
synthesis or of enhanced proteolysis of the mature subunit. In contrast
to the native H,K-ATPase
-subunit, the expressed H,K-ATPase
-subunit is resolved into four bands ranging from Mr 54,000 to 77,000. This heterogeneity is most
likely because of the differential glycosylation of the
Mr 34,000 core protein of the
-subunit. As
expected, because of the individual subunit expression, cells
co-transfected with both
- and
-cDNA also co-express both
subunits. The co-expression of the
- and
-subunits does not
change the profile of
-subunit maturation products from that
observed with the expression of the
-subunit alone. The membrane
preparation from untransfected cells is shown in lane 5.
Neither gastric H,K-ATPase
- or
-subunits are expressed at detectable levels in the untransfected cells.

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Fig. 1.
Subunit expression of the H,K-ATPase in
HEK293 T cells. 55 µg of cell membrane protein per lane was
resolved by SDS-PAGE and transferred to nitrocellulose. Top
panel, lanes probed with H,K-ATPase -subunit antibody, mAb
12.18. Bottom panel, lanes probed with H,K-ATPase
-subunit antibody, mAb 2B-6. Lane 1, transfection with
H,K-ATPase -subunit; lane 2, transfection with H,K-ATPase
-subunit; lane 3, co-transfection with - and
-subunits; lane 4, 0.3 µg of the native H,K-ATPase;
lane 5, untransfected HEK293 T cells.
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A quantitative measurement of the
- and
-subunits co-expressed in
three separate membrane preparations of co-transfected HK293 T cells is
shown in Table II. The expressed
-subunit ranged from 3.8 to 7.0 µg with a mean of 5.2 ± 1.6 µg/mg of total membrane protein, whereas the
-subunit ranged from
3.7 to 8 µg with a mean of 5.5 ± 2.3 µg of expressed
protein/mg of total membrane protein. The ratio of
- to
-subunit
expressed in each membrane preparation ranged from 0.5 to 1.2 (w/w),
although the molar ratio of the total
-subunit population was always
expressed in excess of the
-subunit.
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Table II
Co-expression of - and -subunits in transiently transfected
HK293 T cells
HK293 T cells were co-transfected with plasmid DNA constructs
containing both and H,K-ATPase subunits in three separate
experiments. Cell membranes were prepared 72 h post-transfection
and probed for subunit expression using monoclonal antibodies to each
subunit.
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H,K-ATPase Activity in Transiently Transfected HK293 T
Cells--
To investigate the subunit requirement for H,K-ATPase
function, the
-subunit was transiently expressed in the presence or absence of the H,K-ATPase
-subunit. The H,K-ATPase activity
associated with
-subunit expression with or without the
-subunit
is summarized in Fig. 2. As indicated in
the first bar of each experimental cluster, all membrane
preparations of the HK293 T cells possess a nonspecific nucleotidase
activity in the presence of 0.1 mM ouabain and 1 µg of
oligomycin (per 55 µg of protein). This normalized activity, reported
as 1, ranged from 0.8 to 1.7 µmol/mg/h in four preparations. This
basal nucleotidase activity was stimulated approximately 10% by 10 mM KCl and inhibited by 55 µM SCH 28080. This activity profile was not changed with the transient transfection and expression of the
- or
-subunit alone. In contrast, the co-transfection and expression of both subunits significantly increased
the K+-stimulated ATPase. The mean normalized
K+-stimulated activity reported in Fig. 2 is approximately
50% above that of the untransfected controls. This normalized activity
represents an enhanced K+-stimulated ATPase ranging from
0.6 to 1.1 µmol/mg/h in each membrane preparation. As shown, this
K+-stimulated component is inhibited by 55 µM
SCH 28080.

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Fig. 2.
Subunit dependence of the functional
H,K-ATPase. ATPase activity in n = 3 replicates
was measured in the standard ATPase buffer containing 2 mM
MgCl2 ( ), 2 mM MgCl2 + 10 mM KCl ( ), or 2 mM MgCl2 + 10 mM KCl + 22 µM SCH 28080 ( ). The activity
profiles in each preparation were normalized (norm.) to the
activity measured in the presence of Mg+2 alone:
activitynorm = Actmeas/ActMg2+. The specific
activity in µmol/mg/h obtained in co-transfected cell membranes was:
Mg+2, 1.6 ± 0.4; Mg+2 + K+,
2.3 ± 0.7; Mg+2 + K+ + SCH 28080, 1.7 ± 0.04.
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The concentration dependence of the K+-stimulated
activation and the SCH 28080-dependent inhibition were
measured to provide a comparison of the steady-state kinetic properties
of the native gastric H,K-ATPase to that expressed in the membranes of
HEK293 T cells. The data from four separate co-transfected cell
populations were combined in Fig. 3. The
concentration dependence of the K+-stimulated activity is
shown in panel A, where the activity is saturable with a
K0.5 of 0.6 ± 0.1 mM KCl.
Panel B shows the concentration dependence of the
SCH 28080-inhibited component, where the Ki is
0.96 ± 0.3 µM. These steady-states kinetic
constants resemble those of the native gastric H,K-ATPase reported in
Table III.

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Fig. 3.
Activation and inhibitory kinetics of the
expressed H,K-ATPase. A, concentration dependence of the
K+-stimulated H,K-ATPase. The data report the normalized,
K+-stimulated component of H,K-ATPase activity from three
separate co-transfected cell preparations. The normalized activity is
reported as Vmeas/Vmax,
where Vmax is the activity measured in 10 mM KCl. The line is drawn to the best fit to
Y = Vmax × [K+]/K0.5 + [K+].
K0.5 = 0.6 ± 0.1 mM KCl.
B, concentration dependence of SCH 28080-inhibited
H,K-ATPase. The data report the SCH 28080-inhibited component of
H,K-ATPase ((Mg2+ + K+) (Mg2+ + K+ + SCH 28080)) activity from three separate
co-transfected cell preparations. The normalized activity is reported
as the fraction of K+-stimulated activity sensitive to
SCH 28080: activitynorm. = ( K+max SCH 28080)/ K+max. The line is drawn to
the best fit to Y = 1/(1+
(SCH 28080)/Ki) + nsb (nonspecific).
Ki = 1.0 ± 0.3 µM, nsb = 0.08.
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Table III
Kinetic constants for site mutants of Glu-857
Each constant was obtained from a data set composed of three separate
transfected cell membrane preparations with n = 3 replicates of each preparation. The analysis utilized the best fit to
the combined data set.
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Structure and Function of Glu-857--
To investigate the role of
size and charge at Glu-857, site mutants were prepared to reverse the
charge from negative to positive or vary the size of the uncharged side
chain at this position. Each of these site mutants was co-transfected
into HEK293 T cells with the H,K-ATPase
-subunit. The
-subunit
expressed for each site mutant is shown in Fig.
4. The Western analysis of the expressed protein confirms that all of the
-subunit site mutants are expressed at approximately equivalent levels. The small distortion in lane 2 is attributable to the difference in protein loading between the
native H,K-ATPase (0.3 µg in lane 1) and the expressed
H,K-ATPase (55 µg in lane 2) preparations.

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Fig. 4.
Expression of Glu-857 site
mutants. Lanes 2-7 contain 55 µg of cell membrane protein
produced by the transient co-transfection of H,K-ATPase - and
-subunits. All lanes are probed with H,K-ATPase subunit
antibody, mAb 12.18. Lane 1, 0.3 µg of native H,K-ATPase;
lane 2, E857R; lane 3, E857K; lane 4,
E857Q; lane 5, E857A; lane 6, E857L; lane
7, untransfected control.
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The steady-state activation and inhibitory constants of each site
mutant were derived from pooled data taken from three separate membrane
preparations of co-transfected cells. As shown in Table III, the
concentration dependence of both the
K+-dependent stimulation of the ATPase and the
SCH 28080-dependent inhibition of the co-transfected
HEK293 T (wild type) cell membranes and the native hog gastric
H,K-ATPase were comparable. In addition, the specific activity of the
H,K-ATPase expressed in each of the site mutants was similar to that
measured for the wild type activity. Thus, the site mutations at
Glu-857 do not influence the level of expression of the
-subunit
expression nor inhibit the specific activity of the H,K-ATPase activity.
In contrast to the specific activity of the H,K-ATPase, the
steady-state kinetic constants describing the
K+-dependent activation and the
SCH 28080-dependent inhibition of the ATPase were
sensitive to various site mutants. The role of the side chain charge
was evaluated either by eliminating the charge by substituting
glutamine for glutamate or reversing the charge by substituting
arginine or lysine for glutamate. The removal of the charge by the
conservative replacement of glutamine for glutamate had little effect
on the kinetic constants. In contrast, the reversal of the side chain
charge from negative to positive enhanced the sensitivity to
K+ but decreased the sensitivity to SCH 28080. As shown in
Table III, the introduction of arginine or lysine for glutamate reduced the K0.5 (KCl) from 0.6 ± 0.1 to 0.2 ± .05 and 0.2 ± .03 mM, respectively, while
increasing the Ki (SCH 28080) from 1.0 ± 0.3 to 6.5 ± 1.4 and 5.9 ± 1.5 mM, respectively.
The effect of changing the side chain size was intermediate to that of
reversing its charge, but the reciprocal changes noted in the
charge-reversed mutants also occurred with the introduction of the
bulky leucine side chain group at this position.
 |
DISCUSSION |
This report provides evidence of the functional expression of the
gastric H,K-ATPase in a mammalian HEK293 T cell line. The finding that
both the
- and
-subunits were required for expression of the
functional H,K-ATPase confirms several biochemical investigations suggesting that the H,K-ATPase is an essential heterodimer (4-6). It
is interesting that whereas the expression of the H,K-ATPase
-subunit was essential for H,K-ATPase function, the mature
-subunit, free of degradation products, was expressed in the absence
of the H,K-ATPase
-subunit. This steady-state observation is
somewhat unexpected because the co-translation of the
- and
-subunits are necessary to stabilize the Na,K-ATPase
-subunit
expressed in Xenopus laevis oocytes (52, 53). It
is possible that the indigenous Na,K-ATPase
-subunit present in the
HEK293 T cells could stabilize the H,K-ATPase
-subunit, although it
would then be inadequate to properly fold the functional ATPase. The
data also suggest that the role of the H,K-ATPase
-subunit in ATPase function may be fulfilled by the immature
-subunit because several glycosylation variants of the
-subunit were resolved by SDS-PAGE. Expression studies of the Na,K-ATPase in Xenopus oocytes
have shown that the glycosylated
-subunit is important in assembly efficiency but is not required for the functional maturation of the
catalytic Na,K-ATPase
-subunit (54). The importance of
-subunit
glycosylation for H,K-ATPase function is uncertain because it has been
shown that glycosylation inhibitors inactivate the H,K-ATPase expressed
in Sf-9 cells (55, 56). The present study does not eliminate the
possibility that a sufficient quantity of a complex glycosylated
species is present.
The H,K-ATPase activity of cell membranes is displayed within an
activity profile containing a nonspecific nucleotidase. The expressed
H,K-ATPase is differentiated from the nonspecific nucleotidase by an
enhanced K+-stimulated component of the ATPase that is
sensitive to SCH 28080, a competitive inhibitor of the gastric
H,K-ATPase. The K+-dependence of the expressed H,K-ATPase
is comparable with that of the native gastric H,K-ATPase with a
K0.5 (KCl) of 0.6 ± 0.1 mM and
0.5 ± 0.2 mM, respectively. Similarly, SCH 28080
inhibits the K+-stimulated ATPase of each with a
Ki (SCH 28080) = 1.0 ± 0.3 µM
and 0.7 ± 0.1 µM, respectively. Overall, the
specific activity of the H,K-ATPase normalized for the quantity of
expressed protein within the total membrane protein population is
comparable with that of the native H,K-ATPase with a specific activity
of 158 µmol/mg/h.
This structure-function study of Glu-857 was initiated because this
residue has been identified as a K+-protected,
[14C]DCCD-reactive site associated with the
DCCD-dependent inhibition of the native, gastric H,K-ATPase
(45). The data here show that various substitutions at this site do not
inhibit the expressed H,K-ATPase and are inconsistent with the
interpretation that Glu-857 is an essential hydrophobic residue. Thus,
the interpretation of the previous report (57) of
[14C]DCCD incorporation and inhibitory kinetics of the
H,K-ATPase is that DCCD-dependent inhibition is most likely
because of a secondary effect of carboxyl activation such as the
cross-linkage of the carboxyl group to a nearby residue or steric
hindrance of the inhibitor molecule inserted at this site. The former
explanation has been proposed to account for carbodiimide inactivation
of the Na,K-ATPase (57).
This investigation does show that Glu-857 is important for the
concentration dependence of cation-dependent activation and inhibitor-dependent inhibition kinetics. The reversal of
the side chain charge from negative to positive (Glu to Lys or Arg)
altered the kinetic profile by decreasing the
K0.5 for K+-dependent
activation and increasing the Ki for
SCH 28080-dependent inhibition of the fully active
H,K-ATPase. A partial explanation consistent with previous fluorescein
isothiocyanate quench studies of the native H,K-ATPase is that the
residue is involved in setting the conform-ational equilibrium,
Kc, between the E1 and E2 enzyme conformations. The apparent
dissociation constant, Kk', derived from the
K+ concentration dependence of steady-state measurements of
fluorescein isothiocyanate quench, was related to Kc
and the intrinsic K+ dissociation constant,
Kk, through the relationship Kk'
Kk/Kc +1. This model predicts that the K+-dependent concentration profile
will shift from 0.6 to 0.2 mM K+ in response to
a 3-fold increase in the conformational equilibrium of
Kc toward E2 (58). It is also
likely that the structural consequence of the introduced mutations is
more complex than a shift in the
E1/E2 conformational
equilibrium, because the reciprocal pattern of the concentrations
dependence for the activating and inhibiting ligands necessarily
implies that the reactivity of the catalytic intermediate,
E2P, is shifted to favor
K+ over SCH 28080. A further analysis of the functional
consequences of these site modifications, including peptide mapping of
ligand-stabilized proteolytic digests, the analysis of noncompetitive
inhibitor kinetics, and the analysis of partial reactions, is beyond
the scope of the present investigation.
The fourth cytoplasmic domain (C4) defined by the transmembrane
spanning domains M6-M7 is a stretch of 23 amino acids beginning at
Glu-835 (LAYE) and extending to Glu-857 (LVNE).
Both the N- and C-terminal stretches of the domain exhibit a
probability of
-helix formation by Chou-Fasman indices. Residues
Glu-835 (LAYE), Glu-838 (KAES), and Asp-840
(DIMH) within the predicted helix of the N-terminal stretch
of this domain are essential for the expression of the
SCH 28080-sensitive phosphoenzyme in Sf-9 cells (59). The present
investigation shows that Glu-857 in the second predicted helix of the
C-terminal stretch is not essential but is an effector of the
inhibitor-dependent and, to a lesser extent, the
ligand-dependent kinetics of ATPase activity. The C4 domain is well conserved in the Na,K-ATPase (74%) but is nonconserved in the
Ca-ATPase (1 in 23 residues). One could speculate from the kinetic
signature of ATPase function and the limitation of sequence
conservation to the heterodimeric isomers of the P2 ATPase family that
this domain is of special significance for the relay of information
between the
- and
-subunits. It seems likely that this domain is
one of a complex set of structural elements that contribute to the
conformational equilibrium of this ATPase.