(Received for publication, March 14, 1997, and in revised form, April 15, 1997)
From the Molecular Genetics Research Center and the
¶ Faculty of Pharmaceutical Sciences, Toyama Medical and
Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan
A compound, SCH 28080 (2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile),
reversibly inhibits gastric and renal ouabain-insensitive
H+,K+-ATPase, but not colonic
ouabain-sensitive H+,K+-ATPase. By using the
functional expression system and site-directed mutagenesis, we analyzed
the putative binding sites of SCH 28080 in gastric
H+,K+-ATPase -subunit. It was previously
reported that the binding site of SCH 28080, which is a
K+-site inhibitor specific for gastric
H+,K+-ATPase, was in the first extracellular
loop between the first and second transmembrane segments of the
-subunit; Phe-126 and Asp-138 were putative binding sites. However,
we found that all the mutants in the first extracellular loop including
Phe-126 and Asp-138 retained H+,K+-ATPase
activity and sensitivity to SCH 28080. Therefore, amino acid residues
in the first extracellular loop are not directly involved in the SCH
28080 binding nor indispensable for the
H+,K+-ATPase activity. Here we propose a
candidate residue that is important for the binding with SCH 28080, Glu-822 in the sixth transmembrane segment. Mutations of Glu-822 to Asp
and Ala (mutants termed E822D and E822A, respectively) decreased the
ATPase activity to about 45% and 35% of the wild-type enzyme,
respectively, while the mutations to Gln and Leu abolished the
activity. Mutant E822A showed a significantly lower affinity for
K+ than the wild-type enzyme, indicating that Glu-822 is
involved in determining the affinity for K+. The
sensitivity of mutant E822D to SCH 28080 was 8 times lower than that of
the wild-type enzyme. The counterpart of Glu-822 in gastric
H+,K+-ATPase is Asp in
Na+,K+-ATPase and other colonic
ouabain-sensitive H+,K+-ATPase, which are
insensitive to SCH 28080. These results suggest that Glu-822 is one of
important sites that bind with SCH 28080.
H+,K+-ATPase is the proton pump
responsible for gastric acid secretion (1). It is the target molecule
for irreversible proton pump inhibitors such as omeprazole (2),
rabeprazole (E3810) (3), and SCH 280801
(4). SCH 28080 is a reversible inhibitor of gastric
H+,K+-ATPase, which competitively binds to the
luminal K+ high affinity site of the enzyme (5). This
inhibitor does not inhibit Na+,K+-ATPase,
therefore, discriminating the K+ site of
H+,K+-ATPase from that of
Na+,K+-ATPase. Furthermore, SCH 28080 inhibits
renal ouabain-insensitive H+,K+-ATPase (6), but
not colonic ouabain-sensitive H+,K+-ATPase (7,
8). The binding site of SCH 28080 in the gastric H+,K+-ATPase -subunit was reported to be in
the first extracellular loop between the M1 and M2 transmembrane
segments from the study in which a photo-affinity derivative of this
inhibitor was used (9). Furthermore, Phe-126 and Asp-138 (in the rabbit
-subunit) were proposed to be involved in the interaction with the
derivative of SCH 28080 based on a computer-generated model (9). The
corresponding loop between M1 and M2 transmembrane segments of
Na+,K+-ATPase is known to be important for
determining ouabain sensitivity (10). Here we mutated amino acid
residues in the first extracellular loop including Phe-126 and Asp-138,
studied the sensitivity of the mutant
H+,K+-ATPase to the inhibitor, and found that
all mutants studied here retained H+,K+-ATPase
activity and sensitivity to SCH 28080, suggesting that this loop is not
directly involved in SCH 28080 binding.
Several acidic and polar amino acid residues in the M5 and M6
transmembrane segments of Na+,K+-ATPase
-subunit and sarcoplasmic Ca2+-ATPase are involved in
the interaction with cations and are important for these ATPase
activities (11-18). Here we mutated one of the acidic amino acids in
the M6 transmembrane segment of rabbit
H+,K+-ATPase
-subunit, Glu-822, and studied
the properties of the mutants. When this residue was mutated to
aspartic acid and alanine, the mutants E822D and E822A partially
retained the ATPase activity. E822A mutant showed significantly lower
affinity for K+ and higher affinity for ATP. E822D mutant
showed significantly lower sensitivity to SCH 28080, although the
affinities for K+ and ATP were unchanged. We propose the
possibility that Glu-822 is involved in determining the affinity for
K+ and the binding with SCH 28080.
Materials
HEK-293 cells (human embryonic kidney cell line) were a kind gift from Dr. Jonathan Lytton (Brigham & Women's Hospital, Harvard Medical School, Boston, MA). pcDNA3 vector was obtained from Invitrogen Co. (San Diego, CA). MutanK kit was from Takara Shuzo Co. (Ohtsu, Japan). Vent DNA polymerase was obtained from New England Biolabs. Restriction enzymes and other DNA and RNA modifying enzymes were from Toyobo (Osaka, Japan), New England Biolabs, Life Technologies, Inc., or Pharmacia Biotech Inc. (Tokyo, Japan). SCH 28080 was obtained from Schering Co. (Bloomfield, NJ). All other reagents were of molecular biology grade or the highest grade of purity available.
cDNAs of - and
-Subunits of
H+,K+-ATPase
cDNAs of the - and
-subunits of
H+,K+-ATPase were prepared from rabbit gastric
mucosae as described elsewhere (19). The
- and
-subunit cDNAs
were digested with EcoRI and XhoI. The obtained
fragments were each ligated into pcDNA3 vector treated with
EcoRI and XhoI.
DNA Sequencing
DNA sequencing was done by the dideoxy chain termination method using an Autoread DNA sequencing kit and an ALF-II DNA sequencer (Pharmacia Biotech Inc.).
Site-directed Mutagenesis
Introduction of site-directed mutations in the M1-M2 domain of
the H+,K+-ATPase -subunit was carried out by
sequential polymerase chain reaction (PCR) steps (20), in which
appropriately mutated
-subunit cDNAs (segments between
EcoRI site (nucleotide
28) and BstEII site
(nucleotide 456)) were prepared. Two kinds of flanking sequence primers
were prepared, one is the 5
-flanking sense primer,
5
-CCGAATTCAAGGAGGGCAGCGCAGCGAG-3
(nucleotide
28 to
9,
EcoRI site is underlined), and the other is the 3
-flanking
antisense primer, 5
-GCCTCGAGCTCGGATCACCGTGGCTTGC-3
(nucleotides 534-553, XhoI site is underlined).
In addition, sense and antisense synthetic oligonucleotides, each 21 bases long containing one or two mutated bases near the center, were
designed (referred as the sense mutating primer and the antisense
mutating primer). In the first PCR amplification step, the
H+,K+-ATPase
-subunit cDNA subcloned in
pBluescript SK(
) (19) was used as a DNA template. Two fragments were
prepared in this step: one between the 5
-flanking sense primer and the
antisense mutating primer, and the other between the sense mutating
primer and the 3
-flanking antisense primer. Each amplified fragment
was purified by gel electrophoresis, combined and incubated with the
5
-flanking sense primer and the 3
-flanking antisense primer in the
second PCR amplification. The amplified fragment was purified by gel electrophoresis, subcloned in pBluescript SK(
), and sequenced. PCR
was routinely carried out in the presence of 300 µM each
dNTP, 6 µM primers, 10 mM KCl, 20 mM Tris-HCl, pH 8.8, 10 mM
(NH4)2SO4, 2 mM
MgSO4, 0.1% Triton X-100, 100 µg/ml bovine serum
albumin, and 2 units of Vent DNA polymerase for 25 cycles. After
sequencing, the amplified fragment in the second PCR was digested with
EcoRI and BstEII, and ligated back into the
relevant position of the wild-type construct of the
-subunit.
Site-directed mutagenesis in Glu-822 was carried out as described
elsewhere by using the MutanK kit (19, 21).
Cell Culture, Transfection, and Preparation of Membrane Fractions
Cell culture of HEK-293 was carried out as described previously
(19). - and
-subunit cDNA transfection was performed by the
calcium phosphate method with 10 µg of cesium chloride-purified DNA/10-cm dish. Cells were harvested 2 days after the DNA transfection. Membrane fractions of HEK cells were prepared as described previously (19). Briefly, cells in a 10-cm Petri dish were washed with phosphate-buffered saline, and incubated in 2 ml of low ionic salt
buffer (0.5 mM MgCl2, 10 mM
Tris-HCl, pH 7.4) at 0 °C for 10 min. After the addition of
phenylmethylsulfonyl fluoride (1 mM) and aprotinin (0.09 units/ml) the cells were homogenized in a Dounce homogenizer, and the
homogenate was diluted with an equal volume of a solution containing
500 mM sucrose and 10 mM Tris-HCl, pH 7.4. The
homogenized suspension was centrifuged at 800 × g for
10 min. The supernatant was centrifuged at 100,000 × g
for 90 min, and the pellet was suspended in a solution containing 250 mM sucrose and 5 mM Tris-HCl, pH 7.4.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblot
SDS-polyacrylamide gel electrophoresis was carried out as
described elsewhere (22). Membrane preparations (30 µg protein) were
incubated in a sample buffer containing 2% SDS, 2%
-mercaptoethanol, 10% glycerol, and 10 mM Tris-HCl, pH
6.8, at room temperature for 2 min and applied to the
SDS-polyacrylamide gel. Immunoblot was carried out as described
previously (19).
Antibody
Ab1024 was previously raised against the carboxyl-terminal
peptide (residues 1024-1034) of the
H+,K+-ATPase -subunit (PGSWWDQELYY)
(23).
Assay of H+,K+-ATPase Activity
H+,K+-ATPase activity was assayed by following two different methods depending on the purpose and conditions of the experiments.
Measurement of Decrease in the Amount of NADH Coupled with Regeneration of ATP from ADP ("Coupled-enzyme Method")H+,K+-ATPase activity was measured in 1.2 ml of a reaction mixture containing 50 µg of membrane protein, 3 mM MgCl2, 160 µM NADH, 0.8 mM phosphoenolpyruvate, 3 units/ml pyruvate kinase, 2.75 units/ml lactate dehydrogenase, 5 mM NaN3, 1 mM ouabain, 15 mM KCl, 40 mM Tris-HCl, pH 7.4, and various concentrations of ATP. The decrease in the amount of NADH was measured at 37 °C from the absorbance at 340 nm by a Beckman spectrophotometer as described elsewhere (24). The SCH 28080-sensitive K+-ATPase was calculated as the difference between the K+-ATPase activities in the presence and absence of 50 µM SCH 28080.
Measurement of Inorganic Phosphate Released from ATPH+,K+-ATPase activity was measured in 1 ml of a solution containing 50 µg of membrane protein, 3 mM MgCl2, 3 mM ATP, 5 mM NaN3, 2 mM ouabain, and 40 mM Tris-HCl, pH 7.4, in the presence and absence of 15 mM KCl. After incubation at 37 °C for 30 min, the inorganic phosphate released was measured as described elsewhere (25). The K+-ATPase activity was calculated as the difference between activities in the presence and absence of KCl.
These two methods did not give qualitative differences in results, but values measured by the former method were about 20% higher than corresponding values measured by the latter method. Routinely we measured the ATPase activity by both methods. We usually showed results measured by the latter method unless indicated. When the ATPase activity was measured as a function of ATP concentrations, the former method was employed to maintain constant ATP concentrations during the incubation period.
Protein was measured using the BCA protein assay kit from Pierce with bovine serum albumin as a standard.
SCH 28080 is a reversible inhibitor specific for the
gastric H+,K+-ATPase (4). Previously, it was
proposed that Phe-126 and Asp-138 were directly involved in the
interaction with SCH 28080 (9). Here we mutated these two residues into
several different amino acids (F126A, F126L, F126Y, D138A, D138E,
D138N, D138V and double mutants F126A/D138A and F126Y/D138N) and
studied the effects on the expression levels and the ATPase activity.
The expression levels of the mutant -subunits were almost identical
with that of the wild-type judging from the immunoblot pattern (Fig.
1). Table I shows the
K+-ATPase activities of the mutants and the wild-type
enzyme in the absence of SCH 28080, and the inhibition percentage in
the presence of 10 and 50 µM SCH 28080. Significant
K+-ATPase activity was detected in all these mutants
prepared here, although there were significant differences in the
activities between the wild-type enzyme and some mutants such as F126L
and F126A, i.e. F126L and F126A mutants showed about 40%
and 61% of the activity of the wild-type enzyme, respectively. SCH
28080 at 10 and 50 µM inhibited the K+-ATPase
activities of these mutants by 74-98% and by 77-100%, respectively.
Fig. 2 shows the effects of various concentrations of
SCH 28080 on the K+-ATPase activity for several mutants.
IC50 values are 2.1, 2.0, 3.3, 1.6, and 3.8 µM for wild-type and mutants F126Y, F126L, D138E, and
D138N, respectively. These results (Table I and Fig. 2) show that the
affinities of the mutants for SCH 28080 were not significantly different from that of the wild-type enzyme, indicating that Phe-126 and Asp-138 are not directly involved in the binding with SCH 28080.
|
Fig. 3 shows effects of K+ concentrations on
the SCH 28080-sensitive K+-ATPase activity of the mutants
(F126A, F126Y, D138A, and D138N). The K+ sensitivity of the
mutants at Phe-126 (F126A and F126Y) was not significantly different
from that of the wild-type enzyme. Km values for
K+ obtained from the least-squares curve fitting in the
range of the low K+ concentrations were 0.23 mM
for mutant F126A and 0.14 mM for mutant F126Y, which were
comparable with the values obtained for the wild-type
H+,K+-ATPase expressed in HEK-293 cells
(Km = 0.24 mM) (19) and the
H+,K+-ATPase in gastric vesicles
(Km = 0.2 ~ 0.4 mM) (26, 27). On
the other hand, mutations at Asp-138 (D138A and D138N) slightly
decreased the affinity for K+. Km values
were 0.94 mM for mutant D138A and 0.48 mM for
mutant D138N, which were 2-4-fold higher than that of the wild-type
enzyme.
Site-directed Mutations in Other Amino Acid Residues in the First Extracellular Loop Segment
Amino acid sequences in the M1 and M2
transmembrane segments are considerably well conserved between members
of H+,K+-ATPase family (gastric, colonic, and
urinary bladder) and Na+,K+-ATPase, whereas
amino acids in the loop segment between M1 and M2 are not conserved
(28-31) (Fig. 4). Here we prepared mutants E132A,
G133A, G133E, D134A, L135A, T136A, and T137A. K+-ATPase
activity was detected in all these mutants prepared, and the activities
of mutants D134A and L135A were significantly lower than that of the
wild-type as shown in Table I. SCH 28080 at 10 and 50 µM
inhibited the K+-ATPase activity of these mutants by
56-85% and 83-100%, respectively. The sensitivity of mutant D134A
to SCH 28080 was slightly lower than that of the wild-type enzyme;
however, the sensitivity of this mutant was still much higher than
those of Na+,K+-ATPase and ouabain-sensitive
colonic H+,K+-ATPases. Therefore, we conclude
that the first extracellular loop between the M1 and M2 segments is not
an exclusive (or major) determinant of SCH 28080 sensitivity nor
directly involved in the binding with SCH 28080.
In the following, we propose that Glu-822 in the M6 transmembrane segment is one of the candidates involved in the binding with SCH 28080.
Site-directed Mutations in the Putative Cation Binding Site in the M6 Transmembrane SegmentSeveral amino acid residues responsible
for the cation binding or indispensable for the enzyme activity are
present in the M5 and M6 transmembrane segments of the
Ca2+-ATPase and the Na+,K+-ATPase
-subunit (11-18). We mutated Glu-822 in the M6 transmembrane segment of the H+,K+-ATPase
-subunit, and
studied the role of this amino acid in the ATPase function. In
sarcoplasmic Ca2+-ATPase, the corresponding amino acid is
Asn-796, which is involved in the high affinity binding with
Ca2+ (17). The counterpart in rat
Na+,K+-ATPase
2-subunit, Asp-803, is
indispensable for the function, and mutations of this residue to Asn,
Glu, or Leu abolished the function of the
Na+,K+-ATPase (12). We mutated Glu-822 of the
H+,K+-ATPase
-subunit into Ala, Asp, Leu,
and Gln. The expression levels of the mutant
-subunits were almost
identical with that of the wild-type enzyme (Fig. 5).
Table II shows the H+,K+-ATPase
activity of these mutants. When this glutamic acid was mutated to
leucine (E822L) and glutamine (E822Q), the ATPase activity was
abolished in both cases. However, the aspartic acid mutant (E822D) and,
surprisingly, the alanine mutant (E822A) expressed in the present HEK
cells retained about 45% and 35% of the activity of the wild-type
enzyme, respectively. These results show that this residue is important
for the ATPase function, but the carboxyl residue of the side chain is
not indispensable. Fig. 6 shows the effects of
K+ on the ATPase activity of the mutants. The affinity of
mutant E822D for K+ was not significantly different from
that of the wild-type enzyme, whereas mutant E822A showed a
significantly lower affinity for K+. Km
values were 0.24, 0.32, and 5.5 mM for wild-type, E822D and
E822A mutants, respectively. Therefore, Glu-822 is involved in
determining the affinity for K+ in the
H+,K+-ATPase. Fig. 7 shows the
effects of ATP on the ATPase activity of the mutants. The affinity of
mutant E822D for ATP was not significantly different from that of the
wild-type enzyme, whereas mutant E822A showed a significantly higher
affinity for ATP. Therefore, the conformation of mutant E822A with a
lower affinity for K+ and higher affinity for ATP is
considered to be shifted to an E1 form compared with that
of the wild-type enzyme.
|
Sensitivity of Glu-822 Mutants to SCH 28080
Fig.
8 shows effects of SCH 28080 concentrations on the
K+-ATPase activity of mutants E822A and E822D. Mutant E822D
showed a significantly lower sensitivity to SCH 28080 than the
wild-type, whereas E822A showed a significantly higher affinity.
IC50 values are 2.1, 15, and 0.58 µM for the
wild-type, mutants E822D and E822A, respectively (i.e. these
mutations did not induce parallel changes in affinities for
K+ and SCH 28080). Taking the molecular shape and size
difference between K+ and SCH 28080 into consideration, it
is considered that these mutations induced some conformational changes,
resulting in complicated changes in the affinities. Therefore, Glu-822
is suggested to be involved in bindings with K+ and SCH
28080.
H+,K+-ATPase was originally found in
gastric mucosae. Recently new members of the
H+,K+-ATPase -subunit were found and cloned
from distal colons of rat (29) and guinea
pig,2 toad urinary bladder (30), and human
skin (32). The ouabain-sensitive K+-ATPase activity in the
membrane preparation from the guinea pig colon was insensitive to 100 µM SCH 28080 (7). The cDNAs of rat colonic
H+,K+-ATPase (8), urinary bladder
H+,K+-ATPase (30), and ATP1AL1 from human skin
library (33) were expressed in Xenopus levis oocytes. When
the
- and
-subunit cRNAs were co-injected (for colonic
H+,K+-ATPase and ATP1AL1, these
-subunit
cRNAs were co-injected with the
-subunit cRNA of rabbit gastric
H+,K+-ATPase), the expressed ATPases
transported rubidium inward, and proton outward. The
K+-ATPase activities of urinary bladder ATPase and ATP1AL1
were sensitive to both SCH 28080 and ouabain (30, 33). However, they
were much less sensitive to SCH 28080 than the gastric
H+,K+-ATPase, the Ki value
of the urinary bladder H+,K+-ATPase being 230 µM (30). The Rb+ transport by ATP1AL1 was
only partly inhibited by 500 µM SCH 28080 (33). The rat
colonic H+,K+-ATPase expressed in
Xenopus oocytes was sensitive to ouabain, but insensitive to
SCH 28080 (8), while the same ATPase expressed in Sf9 cells was
slightly inhibited by SCH 28080 (18% inhibition with 100 µM SCH 28080) and was insensitive to ouabain (34). The
amino acid sequences around the first extracellular loop are well
conserved in gastric H+,K+-ATPases obtained
from various species, i.e. rat (35), pig (36), human (37),
rabbit (28), dog (38), Xenopus, and mouse (39). In Fig. 4,
amino acid sequences around the first extracellular loop segment of the
-subunit are compared in the members of
H+,K+-ATPase and
Na+,K+-ATPase. The amino acid at the edge of
the M1 transmembrane segment is phenylalanine (Phe-126 in rabbit
gastric H+,K+-ATPase) and conserved in these
gastric H+,K+-ATPases and rat colonic
H+,K+-ATPase (29), whereas the corresponding
residue is tyrosine in the guinea pig colonic
H+,K+-ATPase, urinary bladder
H+,K+-ATPase (30), and sheep kidney
Na+,K+-ATPase (31), which are all insensitive
to SCH 28080. Therefore, we initially considered that mutation at
Phe-126 in the gastric H+,K+-ATPases would
cause some change in the sensitivity to SCH 28080, if SCH 28080 binds
with this loop as suggested previously (9). Surprisingly, when the
residue was replaced by tyrosine (F126Y) or alanine (F126A), the
K+-ATPase activity of the mutants still remained sensitive
to SCH 28080, and the sensitivity of these mutants to SCH 28080 was not significantly different from that of the wild-type enzyme. Because mutants F126A and F126Y are able to interact with SCH 28080, the phenyl
group in the side chain of Phe-126 is not supposed to be directly
involved in the interaction with the inhibitor, and the side chain of
the tyrosine residue does not interrupt the binding of SCH 28080. Therefore, the low sensitivity to SCH 28080 in the guinea pig colonic
H+,K+-ATPase and the urinary bladder
H+,K+-ATPase is not due to the amino acid
substitution to Tyr on this site (7, 30). The aspartic acid residue
(Asp-138 in rabbit gastric H+,K+-ATPase) at the
edge of the M2 transmembrane segment is also well conserved in gastric
H+,K+-ATPases from different species, but
divergent among Na+,K+-ATPase and colonic and
urinary H+,K+-ATPases. In the
Na+,K+-ATPase, the corresponding amino acid
residue is asparagine. In the colonic and urinary bladder
H+,K+-ATPases this residue is leucine and
arginine, respectively. When this residue of gastric
H+,K+-ATPase was replaced by asparagine
(D138N), alanine (D138A), glutamic acid (D138E), and valine (D138V),
the K+-ATPase activity remained sensitive to SCH 28080, and
the sensitivity of the mutants to SCH 28080 was not significantly
different from that of the wild-type enzyme. Therefore, Asp-138 is not
directly involved in the interaction with SCH 28080. To exclude the
possibility that the interaction between
H+,K+-ATPase and SCH 28080 remains unaffected
when only one of the two binding determinants (Phe-126 and Asp-138) is
mutated, we prepared double mutants in Phe-126 and Asp-138 (F126Y/D138N
and F126A/D138A). These double mutants are also active and have similar sensitivity to SCH 28080 compared with the wild-type enzyme. Our results altogether suggest that the putative SCH 28080 binding sites,
Phe-126 and Asp-138, are, at least, not major determinants of SCH 28080 sensitivity nor directly involved in binding with SCH 28080.
Next we introduced mutations in other residues in the first extracellular loop segment. Amino acid residues between Glu-132 and Thr-137 were replaced with alanine to study the role of the side chain in binding with SCH 28080 (mutants termed E132A, G133A, D134A, L135A, T136A, and T137A). In addition, Gly-133 was replaced with glutamic acid, a counterpart residue in sheep kidney Na+,K+-ATPase (mutant termed G133E). All these mutants prepared were active as H+,K+-ATPase, suggesting that the side chains of amino acids in this segment are not indispensable for the ATPase function. Sensitivity of the mutants to SCH 28080 was not significantly different from that of the wild-type enzyme. Therefore, it is concluded that every single amino acid residue in the first extracellular loop is not a major binding site of SCH 28080.
We found that Glu-822 is one of the amino acids that determine the
affinity for SCH 28080. When Glu-822 was mutated with Gln and Leu,
H+,K+-ATPase activity of the mutants was
completely abolished. For Ala and Asp mutants,
H+,K+-ATPase activity was partially preserved.
The affinities of mutant E822D for K+ and ATP were not
changed; however, those of mutant E822A were significantly lower for
K+ and higher for ATP than the corresponding affinities of
the wild-type enzyme. These results suggest that this site is important
for the function of H+,K+-ATPase, especially
for determining the affinity for K+, but that the carboxyl
moiety of Glu-822 is not indispensable for the ATPase function. Very
recently Swarts et al. (40) reported that Glu-820 of rat
gastric H+,K+-ATPase -subunit (counterpart
of Glu-822 of rabbit gastric H+,K+-ATPase) is
involved in K+ binding, and that E820Q mutant expressed in
Sf9 cells showed no K+-ATPase activity, which is in good
agreement with our findings. The corresponding residues in rabbit
sarcoplasmic Ca2+-ATPase and rat
Na+,K+-ATPase
2 subunit are Asn-796 and
Asp-803, respectively. Asn-796 in the Ca2+-ATPase is
involved in the high-affinity binding with Ca2+ (17).
Asp-803 in Na+,K+-ATPase is indispensable for
the ATPase function and unchangeable (12). Therefore, the roles of
Glu-822 and the corresponding amino acid residues in the ATPase
function are different among H+,K+-ATPase,
Ca2+-ATPase, and Na+,K+-ATPase.
Mutant E822D showed 8 times lower sensitivity to SCH 28080, whereas the
E822A showed a higher sensitivity than the wild-type enzyme. These
results suggest that Glu-822 is the site involved (directly or
indirectly) in binding with SCH 28080. It is noteworthy that the
corresponding amino acid residue of Glu-822 is an aspartic acid in
Na+,K+-ATPases and non-gastric type
H+,K+-ATPases that are insensitive to SCH 28080 (28-30). Furthermore, the sensitivity of mutant E822D to SCH 28080 is
lower than that of any mutant in the first extracellular loop segment,
but still higher than those of Na+,K+-ATPase
and non-gastric H+,K+-ATPases.
Our present results do not exclude the possibility that the binding sites of SCH 28080 are on the multiple domains in the H+,K+-ATPase. In fact, amino acid residues that are involved in determining the affinity for ouabain (or cardiac glycosides) in Na+,K+-ATPase are on the segments between M1 and M2 (10), M3 and M4 (41), M5 and M6 (42, 43), and M7 and M8 transmembrane segments (44). Blostein et al. (45) reported that the SCH 28080 binding site is in the N-terminal half of the H+,K+-ATPase from the study using chimeric ATPases between H+,K+-ATPase and Na+,K+-ATPase.
In conclusion, we have reported here that the amino acid residues in
the first extracellular loop of the
H+,K+-ATPase -subunit including the putative
SCH 28080 binding sites, Phe-126 and Asp-138, are in fact not
exclusively involved in the interaction with this inhibitor, and that
Glu-822 is involved in determining the affinity for SCH 28080.