(Received for publication, November 23, 1994; and in revised form, January 12, 1995)
From the
The effects of Na on gastric H,K-ATPase were
investigated using leaky and ion-tight H,K-ATPase vesicles.
Na
activated the total ATPase activity in the absence
of K
, reaching levels of 15% relative to those in the
presence of K
. The Na
activation,
which takes place at the luminal side of the membrane, depended on the
ATP concentration and the type of buffer used. The steady-state ATP
phosphorylation level, studied with leaky vesicles, was reduced by
Na
due to both activation of the dephosphorylation
reaction and a shift to E
in the E
E
equilibrium. By
studying this equilibrium in ion-tight H,K-ATPase vesicles, it was
found that Na
drives the enzyme via a cytosolic site
to the nonphosphorylating E
conformation. No
H
-like properties of cytosolic Na
could be detected. We therefore conclude that Na
behaves like K
rather than like H
in the H,K-ATPase reaction.
H,K-ATPase is an intrinsic membrane protein complex, which is
located in the secretory vesicles of the gastric parietal cell and is
able to generate a proton gradient of 10 across the
membrane in exchange for potassium. Na,K-ATPase, present in plasma
membranes of all mammalian cells, is responsible for the maintenance of
the intracellular levels of potassium and sodium and can generate a
Na
gradient of about 10
. The catalytic
-subunits of both ATPases have been cloned from several species
and have a molecular mass of 112-114 kDa(1, 2) .
The homology between these two ATPases is higher than with other
transport ATPases(3, 4) . Na,K-ATPase and H,K-ATPase
have additionally in common that they both contain a glycosylated
-subunit with a core mass of 33-35
kDa(5, 6) . Such
subunit is absent in other
related ATPases like Ca-ATPases from both sarcoplasmic reticulum and
plasma membrane(3) . Both Na,K-ATPase and H,K-ATPase belong to
the P-type ATPases as (i) the catalytic subunits can be phosphorylated
at an aspartyl residue, (ii) they are inhibited by submicromolar
vanadate concentrations, and (iii) two different enzyme conformations (E
and E
) can be
distinguished. The E
form has high-affinity cation
binding sites at the cytosolic side, whereas the E
form has high affinity cation binding sites at the luminal side
of the membrane. The Post-Albers scheme, which is based on the
alternate formation of these two conformations, is often used to
explain the reaction mechanisms of both Na,K-ATPase and H,K-ATPase. In
the overall H,K-ATPase reaction cycle(7, 8) , where
H
is transported from the cytosol to the lumen in
exchange for K
(steps 1-7, Fig. 1),
different partial reactions can be distinguished such as (i) the
steady-state ATP phosphorylation reaction (steps 2, 3, and 4), (ii) the
dephosphorylation reaction (steps 5 and 6), and (iii) the E
E
transition (steps 7
and 1).
Figure 1: The Post-Albers scheme for H,K-ATPase. The normal reaction cycle turns clockwise. In the text, opposite reactions are notated by negative signs.
Due to the common characteristics the ion specificities of
the two ion transporting enzymes have been studied intensively.
Proton-like effects of sodium on H,K-ATPase (a
``Na,K''-ATPase activity) (9) and sodium-like effects
of protons on Na,K-ATPase (a ``H,K''-ATPase activity) have
been claimed(10) , although the latter effects were not found
when ATP phosphorylation was studied(11) . In Na,K-ATPase,
Na shows, besides effects of its own,
K
-like properties in the absence of
K
. This Na,Na-ATPase or Na-ATPase activity is the
result of activation of both the ATP phosphorylation, and the
dephosphorylation reaction by Na
(12) . The
data regarding the effects of Na
on H,K-ATPase is
somewhat confusing. In some H,K-ATPase studies an identical activation
by Na
of the ATP hydrolysis reaction, a H,Na-ATPase
activity, has been observed(13) . Similar
K
-like effects of Na
have been found
on the rate of ATP
phosphorylation(13, 14, 15) , but the
dephosphorylation reaction has been claimed to be either
activated(16) , or insensitive toward
Na
(15) . Furthermore, two studies (9, 17) indicate that Na
behaves more
like H
and drives the enzyme to an E
conformation.
In preliminary experiments De Jong (18) observed that the K for ATP in the
phosphorylation reaction was considerably increased by
Na
. Such an effect of Na
cannot
easily be explained when Na
behaves like
H
. It could be explained, however, when Na
behaves as a K
analog. In that case the ion
activates the dephosphorylation reaction and drives the enzyme into the E
conformation.
With the use of ion-tight
H,K-ATPase vesicles, where no activation of the dephosphorylation
process by extravesicular cations can occur(19) , and by
comparing their properties with those of leaky vesicles, where such
activation does occur, the effects of Na on the total
ATPase reaction, the steady-state ATP phosphorylation level, the
dephosphorylation reaction, and the E
E
transition were investigated. The results show
that Na
displays K
-like actions under
those reaction conditions, thus activating the dephosphorylation
process at the luminal side of the membrane and driving the enzyme into
an E
conformation by interacting at the cytosolic
side.
Figure 2:
The effect of Na on the
steady-state ATP phosphorylation level and on the ATPase activity. A, the combined effect of Na
and ATP on the
steady-state ATP phosphorylation level. A leaky H,K-ATPase preparation
(0.012-200 µg/80 µl) was preincubated at 22 °C in the
presence 50 mM Tris acetate (pH 7.0), 0.1 mM MgCl
, 0.2 mM ouabain, and 12.5, 25, 62.5,
125, and 250 mM NaCl. After 20 min the steady-state ATP
phosphorylation level was determined by incubating for 5 s with
0.006-80 µM [
-
P]ATP (20
µl). The ATP phosphorylation level (nmol of E-P per mg of protein)
at the different [NaCl] and [ATP] present during
the phosphorylation period is plotted. B, comparison between
the effects of Na
on the steady-state ATP
phosphorylation level and the ATP hydrolysis rate. H,K-ATPase (0.01
mg/ml (
) and 0.125 mg/ml (
) was incubated at 37 °C in
the presence of 20 µM [
-
P]ATP,
0.12 mM MgCl
, 50 mM Tris acetate (pH 7.0)
and NaCl as indicated. After 3 (
) or 120 (
) s the
reactions were terminated and the steady-state ATP phosphorylation
level (
, nmol E-P per mg protein) or the ATPase activity
(
, µmol of ATP hydrolyzed per mg of protein/h) were
determined as described under ``Materials and
Methods.''
Figure 3:
The effect of KCl and NaCl on the
H,K-ATPase activity at varying concentrations of ATP. A leaky
H,K-ATPase vesicle preparation (1-400 µg/ml) was incubated
for 1-10 min at 37 °C in the presence of 30 mM Tris-HCl (pH 7.0), 5-5000 µM [-
P]MgATP, 0.1 mM MgCl
, 0.1 mM ouabain, and the KCl (A) or NaCl (B) concentrations as indicated.
Maximally 30% of the ATP was converted. Activity is given as µmol
of ATP hydrolyzed per mg of protein/h.
The activation of the ATPase activity by
Na depended, like the K
-activation,
on the ATP concentration (Fig. 3B). In the presence of
5 µM ATP we observed a K
for
Na
of 10-20 mM while in the presence of
5 mM ATP this value increased to about 100 mM. The
maximal Na
-ATPase activity reached levels of about
15-25 µmol of ATP hydrolyzed/mg of protein per h, which is
about 15% of the activity obtained in the presence of
K
.
Second, with the use of the K ionophores, valinomycin and nigericin, the K
activation of ATPase activity was studied in ion-tight vesicles.
In this type of H,K-ATPase vesicles the K
activation
site is located intravesicularly(20) . Fig. 4A shows that in these vesicles the basal (Mg-ATPase) activity was
very low and that activation by extravesicular (cytosolic) K
was not possible. In the presence of nigericin, a K
for H
exchanger, the K
activation profile was nearly identical to that of a leaky
H,K-ATPase preparation, in which the K
activation site
is freely accessible (Fig. 3A). In the presence of the
specific K
ionophore valinomycin, however, there was
only a slight activation, probably due to the ionophore-induced voltage
difference across the vesicle membrane(23) . The lack of
activation could partially be overcome by the extra addition of the
protonophore CCCP. When similar experiments (Fig. 4B)
were carried out in the presence of Na
, activation of
the ATP hydrolysis was only observed in the presence of nigericin,
which ionophore can also, but to a lesser extent, exchange
Na
for H
(23) . Valinomycin
either alone or in combination with CCCP could not induce a
Na
-activated P
production due to the
absolute selectivity of this ionophore for K
.
Figure 4:
The effect of ionophores on the ATPase
activity in ion-tight H,K-ATPase vesicles in the presence of varying
concentrations of either K or Na
.
Ion-tight H,K-ATPase vesicles (60 µg/ml) were preincubated at room
temperature in 20 mM Tris acetate (pH 7.0) and 250 mM sucrose with 100 µM of the ionophores: nigericin
(
), valinomycin (
), valinomycin plus CCCP (
), or
with 2% ethanol (
) as a control. After 6 min 10 µl of the
enzyme suspension was mixed with 80 µl of different KCl or NaCl
solutions and incubated for 2 min at 37 °C. The K
-
or Na
-dependent ATPase activity was determined by
incubating another 2 min with 10 µl of 50 µM [
-
P]ATP and 0.15 mM MgCl
(final concentrations) in 250 mM sucrose, 20 mM Tris acetate. The total ATPase activity,
including the basal Mg-ATPase activity is plotted as function of the
KCl (A) or NaCl (B) concentrations present during the
ATPase assay. Activity is given as µmol of ATP hydrolyzed per mg of
protein/h.
These
observations indicate that the activation of the ATP hydrolysis by
Na is not due to a contamination by
K
, but that Na
itself activates the
dephosphorylation process at the luminal (intravesicular) side of the
membrane.
Figure 5:
The effects of NaCl, KCl, and choline
chloride on the dephosphorylation reaction and the EE
transition. A, cation specificity of the dephosphorylation reaction. Leaky
H,K-ATPase vesicles (2 µg/50 µl) were incubated in the presence
of 1 µM [
-
P]ATP, 0.1 mM MgCl
, and 20 mM Tris acetate (pH 7.0). After
10 s, 0.45 ml of dephosphorylation mixture was added and incubated for
another 3 s as described under ``Materials and Methods.''
Final concentration of ATP was 20 µM. % E-P hydrolyzed is the percentage E-P hydrolyzed during 3 s incubation. B, cation specificity in the E
E
transition. Ion-tight H,K-ATPase vesicles (1
µg in 180 µl) were preincubated for 5 s at room temperature,
under iso-osmotic conditions with 20 mM Tris acetate (pH 7.0)
and different chlorides (XCl) at the concentrations as
indicated. The steady-state ATP phosphorylation was determined during a
3-s incubation with 50 nM [
-
P]ATP
and 0.1 mM MgCl
. The percentage of phosphoenzyme
is plotted as function of the cation concentration present during the
phosphorylation reaction (200 µl). The osmolarity was kept constant
with sucrose.
In ion-tight vesicles Na was unable to activate the dephosphorylation reaction. In the
presence of nigericin the dephosphorylation reaction was again
activated. This shows directly that the activating Na
site is located intravesicularly. Extravesicular (cytosolic)
Na
did not change the kinetics of the
K
-activated dephosphorylation reaction (studied with
valinomycin and CCCP, data not shown).
Helmich-De Jong et
al.(24) , showed that ATP inhibits the basal and the
K-activated dephosphorylation reaction. With the use
of ion-tight vesicles, in combination with valinomycin and CCCP it was
possible to show that the site of inhibition is located at the
extravesicular (cytosolic) side of the membrane. Parallel to
K
, the Na
-activated dephosphorylation
rate was also reduced in the presence of ATP (data not shown).
Figure 6:
The effect of Na on the E
E
transition at pH
8.0. Ion-tight H,K-ATPase vesicles (2 µg) were incubated at room
temperature, under iso-osmotic conditions (corrected with choline
chloride) in the presence of 50 mM Tris acetate (pH 8.0),
0-100 mM NaCl, 0.2, 2.0, and 20 µM [
-
P]MgATP, and 0.1 mM MgCl
. After 5 s the amount of phosphoenzyme (nmol of
P/mg of protein) was determined as described under ``Materials and
Methods'' and plotted as function of the NaCl concentration
present during the phosphorylation
reaction.
In this study data is presented which clearly shows that
Na ions behave like K
ions in the
H,K-ATPase reaction cycle. Na
activates the
dephosphorylation reaction (steps 5 and 6, Fig. 1) in a
preparation in which both cytosolic and luminal ion binding sites are
accessible. In intact H,K-ATPase vesicles no enhancement of this
process was observed, showing that the Na
-activation
site, like the K
-site(19, 25) , is
located intravesicularly (the luminal side). In ion-tight vesicles the
phosphorylation capacity was used as a measure for the relative amount
of the E
form of H,K-ATPase, as only the E
form can be phosphorylated and the
dephosphorylation reaction cannot be activated by the ligands
Na
or K
. It was found that
Na
, like K
(19) , reduces the
phosphorylation capacity of the enzyme, meaning that Na
shifts the equilibrium to the E
conformation
(reactions -1 and -7). Even at low H
concentrations no increase in the steady-state ATP
phosphorylation level, measured at suboptimal conditions, could be
observed, excluding H
-like properties of
Na
. The observation that cytosolic Na
has K
-like properties, driving the enzyme to the E
form, is in line with the inhibition of the ATP
phosphorylation rate (13, 14, 15) and the
reduction of the H
-transport rate (15, 25, 26) by this ion.
Variable effects
of Na, in the absence of K
, on the
overall H,K-ATPase activity (steps 1-7) have been
reported(13, 15) . These variations might be due to
differences in assay conditions, since we demonstrate that high
concentrations of ATP (Fig. 3B), Mg
,
and imidazole have marked effects on the Na
(and the
K
(19) ) affinity in the overall ATPase
reaction. Moreover, high [Na
] inhibits the
latter activity. In the overall ATPase experiments we were able to show
that Na
, like K
, at relative low
concentrations activated the hydrolysis of ATP, via the
dephosphorylation reaction (steps 5 and 6), and inhibited the ATPase
reaction at high concentrations by driving the E
E
equilibrium to the
right (steps -1 and -7). The combination of both effects
explains the increasing effect of Na
on the K
for ATP in the phosphorylation reaction (Fig. 1A).
In both Na,K-ATPase and H,K-ATPase,
K activates the dephosphorylation reaction. The role
of K
can be performed in both enzymes by Na
( (27) and (28) and this study), although the
affinity for Na
is much lower than that of
K
. In both enzymes K
also drives the
equilibrium E
E
to the E
form, whereas Na
(for
Na,K-ATPase) and H
(for H,K-ATPase) shifts the
equilibrium to the E
form. The present study shows
that with H,K-ATPase Na
can perform the latter role of
K
, but not that of H
. With
Na,K-ATPase there is no indication for an E
promoting effect of Na
in the absence of
K
. The ion specificity of Na
and
H
as E
promoters in Na,K-ATPase
and H,K-ATPase, respectively, is much more prominent. Neither an effect
of H
on the steady-state phosphorylation level of
Na,K-ATPase (11) nor of Na
on this parameter
of H,K-ATPase (this study) was observed.
The data seems to conflict
with studies by Rabon et al.(17) , who used a
fluorescein isothiocyanate-labeled H,K-ATPase preparation to test the
effects of Na. The fluorescence of this modified
enzyme, incapable of being phosphorylated by ATP, increased in the
presence of Na
. Although an antagonism between
H
and Na
was observed, the increase
in fluorescence was interpreted as an increase in the E
form of the enzyme, analogous to that with Na,K-ATPase. It has
not been proven, however, that an increase in fluorescence under these
circumstances actually means a shift of the E
E
equilibrium to the
right. Another interpretation is that the E
Na form
has a higher fluorescence than the E
K form.
A
comparable ``H-like'' effect of
Na
has also been observed by Polvani et
al.(9) , who measured an increased
Na uptake
in H,K-ATPase vesicles, under special conditions of low
[H
] (pH > 8.0),
[Na
] between 2 and 5 mM, and the
presence of intravesicular K
. The
Na
uptake and the ATP hydrolysis rates
were in a 1:1 ratio activated by luminal K
. Cytosolic
Na
failed to increase the hydrolytic rate of ATP, what
should have been the case if Na
has
H
-like properties. Therefore, it is most likely that
they measured H
- and K
-activated ATP
hydrolysis together with an exchange of cytosolic
Na
for luminal K
(normally the K
-K
exchange) and
not a Na,K-ATPase activity.
Our observation of an antagonism between
ATP and either K or Na
was not made
by Wallmark et al.(16) . The main difference between
their and our approach is that they used constant (2 mM)
Mg
and we always used 0.1 mM Mg
in excess of [ATP]. In our experience the latter
combination gives maximal activities. When we also used 2 mM Mg
we found the effect of ATP on the K
for K
activation to be
reduced, e.g. at 20 µM ATP the K
for K
doubled from 0.16 mM at 0.12
mM Mg
(our conditions) to 0.33 mM at 2 mM Mg
(conditions of Wallmark et al.(16) ). The maximal H,K-ATPase activity was not
affected. Hence, Mg
decreases the affinity of the
enzyme for K
and consequently the
K
/ATP antagonism.
Another fundamental question is:
if Na can substitute for K
as
activating cation for H,K-ATPase, why then is the maximal activity only
15% of that with K
? The most likely explanation is the
difference between the affinities at the luminal and the cytosolic
K
sites. Since Na
has a higher
affinity for the cytosolic site than for the luminal site (affinity
ratios of 88 versus 500), the inhibitory action on the ATP
hydrolysis overrules the stimulatory effect. We cannot exclude that an
additional reason for the lower activity of H,K-ATPase in the presence
of Na
is due to a decreased maximal rate of
dephosphorylation in the presence of the latter ion.
In the
Post-Albers model (Fig. 1) the activating K site (and/or Na
site) is located at the luminal
side of the membrane. Upon K
binding to the E
-P complex the enzyme changes to a conformation
with ``low'' affinity for K
at the cytosolic
side. Our findings indicate that the ligands (K
or
Na
) which are promoters of this ``on
reaction'' (steps 5 and 6) are, at the same time inhibitors of the
``off reaction'' (steps 7 and 1). Inhibitors of the on
reaction, like H
(15, 25) ,
ATP(24) , and tertiary amines(19) , are in parallel,
promoters of the off reaction.