(Received for publication, September 14, 1995; and in revised form, October 23, 1995)
From the
Gastric H,K
-ATPase was
functionally expressed in the human kidney HEK293 cell line. The
expressed enzyme catalyzed ouabain-resistant
K
-dependent ATP hydrolysis. The
K
-ATPase activity was inhibited by SCH 28080, a
specific inhibitor of gastric proton pump, in a dose-dependent manner.
By using this functional expression system in combination with
site-directed mutagenesis, we investigated effects of mutations in the
putative cation binding site and the catalytic center of the gastric
H
,K
-ATPase. In
Na
,K
-ATPase, the glutamic acid
residue in the 4th transmembrane segment is regarded as one of the
residues responsible for the K
-induced conformational
change (Kuntzweiler, T. A., Wallick, E. T., Johnson, C. L., and
Lingrel, J. B.(1995) J. Biol. Chem. 270, 2993-3000).
When the corresponding glutamic acid (Glu-345) of
H
,K
-ATPase was mutated to aspartic
acid, lysine, or valine, the SCH 28080-sensitive
K
-ATPase activity was abolished. However, when this
residue was replaced by glutamine, about 50% of the activity was
retained. This mutant showed a 10-fold lower affinity for K
(K
= 2.6 mM)
compared with the wild-type enzyme (K
= 0.24 mM). Thus, Glu-345 is important in
determining the K
affinity of
H
,K
-ATPase. When the aspartic acid
residue in the phosphorylation site was mutated to glutamic acid, this
mutant showed no SCH 28080-sensitive K
-ATPase
activity. Thus, amino acid replacement of the phosphorylation site is
not tolerated and a stringent structure appears to be required for
enzyme activity. When the lysine residue in the fluorescein
isothiocyanate binding site (part of ATP binding site) was mutated to
arginine, asparagine, or glutamic acid, the SCH 28080-sensitive
K
-ATPase activity was eliminated. However, the mutant
in which this residue was changed to glutamine had about 30% of the
activity, suggesting that amino acid replacement of this site is
tolerated to a certain extent.
H,K
-ATPase is the proton pump
responsible for gastric acid secretion(1, 2) . It
consists of
- and
-subunits. The
-subunit is the
catalytic subunit with a molecular mass of 114 kDa (3) and
contains the phosphorylation site, the ATP binding site, and the
binding sites for proton pump
inhibitors(4, 5, 6, 7) . The
-subunit is a glycoprotein with a molecular mass of 60-80
kDa (8) . One of the roles of the
-subunit is to stabilize
the
-subunit in the membrane. Although the cDNAs of both subunits
of many species were cloned, there have been no reports of
structure-function studies using site-directed mutagenesis because
there has been no effective functional expression system. Here we
report the functional expression of rabbit gastric
H
,K
-ATPase in human HEK293 cells.
When the cells were co-transfected with the cDNAs of the
- and
-subunits, ouabain-resistant K
-dependent ATPase
activity was observed. The activity was inhibited by SCH 28080 and
scopadulcic acid B, specific inhibitors of the gastric
H
,K
-ATPase(9, 10) .
By using this functional expression system, we investigated the role of
amino acid residues of the putative cation binding site and the
catalytic center.
H,K
-ATPase is a
member of the P-type ATPase family. Sarcoplasmic and endoplasmic
reticulum Ca
-ATPases and
Na
,K
-ATPase also belong to the same
family. They actively transport the ions coupled with the hydrolysis of
ATP. It has been considered that P-type ATPases have the common
structures in the catalytic center including the phosphorylation site
and the ATP binding site. On the other hand, their cation recognition
sites and transport pathways are hypothesized to be common to some
extent, but divergent depending on the species of transporting cations.
From the site-directed mutation and chemical labeling experiments,
Glu-327 in the
-subunit of
Na
,K
-ATPase (sheep
-1) has been
recognized as one of the pivotal residues for cation-induced
conformational changes or for K
occlusion (11, 12, 13) . The replacement of this
residue by glutamine partly reduced the affinity of the enzyme for
Na
and K
(14) . Glu-309 in
sarcoplasmic Ca
-ATPase (the counterpart of Glu-327 of
Na
,K
-ATPase) has been suggested to be
responsible for Ca
high affinity binding. The
replacement of this residue by glutamine completely eliminated the
Ca
transport activity and the Ca
sensitivity in the phosphorylation reaction(15) . Here we
mutated the corresponding residue (Glu-345) of the
H
,K
-ATPase
-subunit and compared
the property of the mutant with those of
Na
,K
-ATPase and
Ca
-ATPase.
The sequences around the
phosphorylation site and the FITC ()binding site are well
conserved in some of the P-type ATPases(16) . In
Na
,K
-ATPase and
Ca
-ATPase, amino acid replacement in the
phosphorylation site is not tolerated (17, 18) . In
Ca
-ATPase, amino acid replacement at the FITC binding
site is tolerated, and the structure is able to withstand basic amino
acids, but not a negatively charged amino acid(18) . In the
present paper, we also replaced Asp-387 of the phosphorylation site and
Lys-519 of the FITC binding site of the
H
,K
-ATPase
-subunit, measured
the enzyme activity of the mutants, and compared the effects of these
mutations with those of Na
,K
-ATPase
and Ca
-ATPase.
Protein was measured using the BCA Protein Assay Kit from Pierce with bovine serum albumin as a standard.
Figure 1:
Immunoblotting with Ab1024 directed
against the H,K
-ATPase
-subunit.
Thirty micrograms of HEK293 cell membrane fractions (lanes
1-4) and 0.5 and 1 µg of gastric vesicles (lanes 5 and 6) were applied to the gel. Lanes: 1, mock-transfected cells; 2, cells transfected with
the
-subunit cDNA; 3, the
-subunit cDNA; 4,
- and
-subunit cDNAs; 5, gastric vesicles (0.5
µg); 6, gastric vesicles (1
µg).
Figure 2:
Effects of SCH 28080 on the expressed
K-ATPase activity. The K
-ATPase
activity was measured as a function of the SCH 28080 concentrations
described under ``Experimental Procedures.'' SCH 28080 was
dissolved in ethanol. The final concentration of ethanol was below 1%.
The K
-ATPase activity was expressed as the percentage
of the control value measured in the absence of SCH 28080. The values
are mean ± S.E. for 3 observations. The control value is 1.05
± 0.09 µmol/mg/h (means ± S.E. n =
3).
Fig. 3shows the effect of K concentration on the expressed SCH 28080-sensitive
K
-ATPase activity. Low concentrations of K
(less than 3 mM) stimulated the ATPase activity, while
high concentrations of K
(more than 10 mM)
were inhibitory. The K
value for K
obtained from the least-squares curve-fitting in the range of the
low K
concentrations was 0.24 mM, which is in
agreement with the value obtained with gastric
vesicles(29, 30) .
Figure 3:
K dependence of the
expressed SCH 28080-sensitive K
-ATPase activity. The
K
-ATPase activity was measured as a function of the
K
concentrations.
, wild-type;
, mutant
E345Q. The values are mean ± S.E. for 3
observations.
Proton transport activity in the membrane fraction was measured using acridine orange fluorescence; however, no significant quenching of the fluorescence was observed.
Hereafter, we studied the significance of the putative functional
sites on the -subunit of
H
,K
-ATPase: 1) putative cation
binding site, 2) phosphorylation site, and 3) FITC binding site
(putative ATP binding site) by using the present functional expression
system.
Figure 4:
Immunoblotting with Ab1024 of the membrane
fraction of HEK cells transfected with the mutant -subunit and
wild-type
-subunit cDNAs. A, E345D (lane 1),
E345K (lane 2), E345Q (lane 3), E345V (lane
4), and wild-type (lane 5). B, D387E (lane
1), D387H (lane 2), D387N (lane 3), and
wild-type (lane 4). C, K519Q (lane 1), K519R (lane 2), K519E (lane 3), K519N (lane 4),
and wild-type (lane 5).
Gastric H,K
-ATPase belongs
to the family of P-type ATPases, which form phosphorylated
intermediates in their catalytic cycles and are inhibited by
vanadate(37) . Sarcoplasmic and endoplasmic reticulum
Ca
-ATPases and
Na
,K
-ATPase also belong to this
group, and these ATPases were cloned and expressed, and their
structure-function relationships have been studied extensively (14, 17,
18, 31, 33-35, 38-41). There are many reports describing
the cDNA cloning of the
- and
-subunits of the gastric
H
,K
-ATPase from rat, pig, human,
rabbit, and
dog(3, 19, 20, 42, 43, 44, 45, 46, 47) .
There have been few reports, however, of the functional expression of
the gastric H
,K
-ATPase. Recently,
gastric H
,K
-ATPase subunits were
expressed in renal proximal tubular epithelial cells
(LLC-PK
), but the enzyme functions were not
measured(48) . Very recently, Mathews et al.(49) reported the functional expression of the ATPase in Xenopus oocytes. The lack of an effective expression system
slowed the study of the structure-function relationships of the gastric
H
,K
-ATPase. Here we report the
functional expression of gastric
H
,K
-ATPase in HEK293 cells. The cDNAs
for the
- and
-subunits of
H
,K
-ATPase were introduced separately
to pcDNA3 vectors, and the cDNAs were transfected separately or
simultaneously into HEK293 cells with the calcium phosphate method.
When the
-subunit cDNA alone was transfected, the
-subunit
was not detected by immunoblotting, suggesting that there is no
endogenous H
,K
-ATPase in HEK293
cells. No significant SCH 28080-sensitive K
-ATPase was
detected in the membrane fraction of these cells. When the
-subunit cDNA was transfected without the
-subunit cDNA, a
slight band of the
-subunit was observed, but SCH 28080-sensitive
K
-ATPase activity was not detected. When the
-
and
-subunit cDNAs were co-transfected, the
-subunit was
clearly seen on immunoblot, and a significant and reproducible SCH
28080-sensitive K
-ATPase activity could be
demonstrated. Therefore, the
-subunit increases the expression of
the
-subunit and is essential for the functional expression of the
H
,K
-ATPase. As
Na
,K
-ATPase is a ubiquitous enzyme,
it is likely that endogenous
Na
,K
-ATPase
-subunit exists in
HEK293 cells. It would appear, however, that the
-subunit of the
H
,K
-ATPase does not assemble with the
-subunit of the Na
,K
-ATPase in a
functional form(50) , although the possibility that a very weak
ouabain-resistant K
-ATPase activity was manifested by
the
H
,K
-
/Na
,K
-
hybrid molecule cannot be excluded. So far, there has been no report
that indicates the functional assembly between
H
,K
-ATPase
-subunit and
Na
,K
-ATPase
-subunit. The
H
,K
-ATPase
-subunit seems to
discriminate the H
,K
-ATPase
-subunit from the Na
,K
-ATPase
-subunit. On the other hand, there are several reports that the
Na
,K
-ATPase
-subunit can
assemble with the H
,K
-ATPase
-subunit in Xenopus oocytes (51, 52) and
HeLa cells(53) . The hybrid molecule (Na/K-
and H/K-
)
showed Na/K pump current and Rb
uptake, although these
activities were much smaller than those in the authentic
Na
,K
-ATPase
/
complex(51) . In this case, the
H
,K
-ATPase
-subunit manages to
act as a surrogate for the
Na
,K
-ATPase
-subunit.
The
expressed K-ATPase activity described here did not
increase in the presence of gramicidin, which stimulates
K
-ATPase activity in gastric vesicles(54) .
This may be due to the leakiness of the HEK cell membrane to
K
and H
. In fact, when we measured
proton transport of the membrane fraction using acridine orange, the
fluorescence was not significantly quenched.
We mutated amino acid
residues involved in the putative cation binding (Glu-345), the
formation of the phosphorylated intermediate (Asp-387), and the ATP
binding (Lys-519). We expressed these mutants in our system and
compared the properties of the mutants with those of wild-type enzyme.
For the Glu-345 mutants, three in four mutants we prepared (E345D,
E345K, E345V) did not show the SCH 28080-sensitive
K-ATPase activity. The remaining mutant, E345Q,
retained 50% of the K
-ATPase activity of the wild-type
enzyme. In sarcoplasmic reticulum Ca
-ATPase, Glu-309
(the counterpart of Glu-345 of the
H
,K
-ATPase) is supposed to be one of
the amino acid residues constituting the Ca
high
affinity site, because the replacement of Glu-309 residue by glutamine
resulted in complete loss of Ca
transport activity
and phosphorylation from ATP, and because the phosphorylation of this
mutant with inorganic phosphate was observed even in the presence of
Ca
(15) . This residue is well conserved in
P-type ATPases, including
Na
,K
-ATPase, plasma membrane
Ca
-ATPase, yeast H
-ATPase, and
gastric H
,K
-ATPase. However, Glu-329
in rat kidney Na
,K
-ATPase
-subunit has been shown not to be essential for active transport
of Na
and K
, because the replacement
of this residue to glutamine retains the enzyme activity(14) .
Furthermore, mutations of Glu-327 (the counterpart of Glu-345 in
Na
,K
-ATPase
-subunit) to glutamine and leucine allow the enzyme to
retain function, whereas mutations to aspartic acid and alanine do not (31) . Our results presented here suggest that Glu-345 is not
absolutely essential for the ATPase function in gastric
H
,K
-ATPase as is the case with
Na
,K
-ATPase. Because Glu-345 can be
replaced by glutamine, the negative charge of the glutamic acid residue
in this site is not indispensable for the function of
H
,K
-ATPase. Rather, the bulkiness of
the side chain in this site appears to be important, because the
glutamic acid cannot be replaced by aspartic acid. These features are
also comparable to those of
Na
,K
-ATPase(31) . In the
present experiment, replacement of Glu-345 by glutamine reduced the
affinity for K
10-fold. Therefore, this residue
appears to be involved in determining the K
affinity.
The role of this glutamic acid residue in
H
,K
-ATPase is also comparable to that
in Na
,K
-ATPase, whose affinity for
Na
and K
was reduced by the
replacement of glutamic acid by glutamine(14, 31) .
The difference in the manner by which this glutamic acid residue
contributes to the functioning of
H
,K
-ATPase,
Na
,K
-ATPase, and
Ca
-ATPase might reflect the difference in the
structures of the ion sensors or the difference in the manner by which
K
participates in the reaction cycles. Both
H
,K
-ATPase and
Na
,K
-ATPase actively translocate
K
, while K
functions as an
accelerator from one side of the membrane in
Ca
-ATPase(55) . Recently, Kuntzweiler et
al.(12) studied the effects of cations on
[
H]ouabain binding and have shown that Glu-327 in
sheep Na
,K
-ATPase
-subunit stabilizes the K
-induced
conformation in the reaction cycle. Until now there has been no direct
evidence as to whether the K
recognition system is
common between H
,K
-ATPase and
Na
,K
-ATPase. The present results
suggest that the structure and mechanism for K
recognition is similar (or partly identical) between
Na
,K
-ATPase and
H
,K
-ATPase. However, there are
striking functional differences between
Na
,K
-ATPase and
H
,K
-ATPase; the former
electrogenically transports Na
and K
,
and the latter non-electrogenically transports H
and
K
.
As to the Asp-387 mutants, all the mutants
prepared (D387E, D387H, and D387N) were inactive, although they were
expressed in sufficient quantities. The experimental results on the
mutations to asparagine and histidine have shown that the existence of
phosphate acceptor moiety at this site is indispensable for the enzyme
function. Because the aspartic acid cannot be replaced by glutamic
acid, the bulkiness of the side chain in the phosphorylation site
appears to be very strict. These results are in agreement with the
results obtained with sarcoplasmic Ca-ATPase and
Na
,K
-ATPase(17, 18) .
The primary structure around the phosphorylation site is well conserved
in the P-type ATPases. The result presented here suggests the common
three-dimensional structure around the phosphorylation site among these
three P-type ion-transporting ATPases, and the requirement of a
stringent structure for their functions.
As to the Lys-519 mutants,
three in four mutants we prepared (K519E, K519N, and K519R) were
inactive. The K519Q mutant retained 30% of the
K-ATPase activity of the wild-type enzyme. In
sarcoplasmic Ca
-ATPase, mutation of the corresponding
lysine residue to arginine, glutamine, and glutamic acid led to
activities of 60%, 25%, and 5% of the activity of the wild-type enzyme,
respectively (18) , indicating that this site is tolerant of
amino acid replacement, although it cannot withstand a negative charge.
This is also the case with
H
,K
-ATPase. However, it was
surprising that the effect of mutation to a basic amino acid, arginine,
was quite different between Ca
-ATPase and
H
,K
-ATPase; the replacement did not
bring severe damage to Ca
-ATPase, whereas
H
,K
-ATPase could not withstand the
replacement. Although the amino acid sequence around the FITC binding
site is well conserved between
H
,K
-ATPase and
Ca
-ATPase(16) , some steric difference must
exist between the two FITC binding sites.