From the
Modification of aspartic acid 369 in the sheep
The Na
Cardiac
glycosides (such as ouabain) specifically inhibit the
Na
In this work the
conserved aspartic acid residue (Asp
The mutated sheep
To maximize the accuracy of the calculated K
To
ensure that the Mg
In order to
unequivocally determine if the D369N protein is assembled into the
plasma membrane of the 3T3 cells, one must probe the extracellular
surface of the intact transfected cells either with a monoclonal
antibody or with a specific extracellular probe (i.e. ouabain). Due to the lack of an available monoclonal antibody that
binds to the extracellular surface of the sheep
Models for the
mechanism of Na
Many investigators have questioned
whether a phosphorylated intermediate form of the
Na
Proposed models for the inhibitory effects of Na
We hypothesize
that Mg
MgATP inhibited Mg
The different characteristics induced by MgATP interactions with the
wild type
1
Na
,K
-ATPase to asparagine results in
a membrane-associated form of
Na
,K
-ATPase that can bind
[
H]ouabain with high affinity in the presence of
Mg
alone (K
=
20.4 ± 2.6 nM). Ouabain binding to the D369N mutant is
not stimulated by inorganic phosphate, confirming that Asp
is both the catalytic phosphorylation site and the only P
interaction site which stimulates ouabain binding. Cation
inhibition of Mg
-stimulated ouabain binding to the
D369N mutant demonstrated that three Na
and two
K
ions inhibit [
H]ouabain
binding and suggests that this inhibition must occur via a
cation-sensitive conformational change which does not directly involve
dephosphorylation of the enzyme. In the presence of 10 mM Mg
, ATP stimulates ouabain binding to the wild
type protein, (AC
= 21.4 ± 2.7
µM) but inhibits the binding to the D369N mutant
(IC
= 2.52 ± 0.17 µM)
indicating that the mutation does not destroy the high affinity site
for MgATP but does change the nature of the protein conformation
normally induced by a
nucleotide-Na
,K
-ATPase interaction.
Increasing the Mg
from 1 to 10 mM did not
alter the AC
or IC
values for ATP and reveals
that the Mg
interaction which stimulates ouabain
binding in the absence of nucleotide involves a distinct divalent
cation site not associated with the binding of the magnesium-nucleotide
complex. Thus, altering the catalytic phosphorylation site of
Na
,K
-ATPase does not affect the
expression of the ouabain-sensitive protein in the membrane fraction of
NIH 3T3 cells and does not disrupt the binding of Na
,
K
, Mg
, ouabain, or ATP to the
enzyme. However, the D369N substitution does inhibit the formation of a
nucleotide-protein complex with high affinity for ouabain.
,K
-ATPase is a member of
a class of active cation transporters which form a characteristic
-aspartyl phosphate intermediate during the ATP hydrolysis/ion
translocation cycle. Amino acid replacement of this conserved aspartic
acid residue in the Na
,K
-ATPase and
in related active cation transporters results in complete loss of
cation transport (1-3). This loss of cation transport was
attributed to either loss of ATPase activity (1, 2) or
lack of mutant protein expression and/or stability in the system being
utilized(3) . In addition to loss of ATPase activity, this amino
acid replacement in the Na
,K
-ATPase
expressed in oocytes was reported to abolish cardiac glycoside binding
to the exterior surface of these cells(2) .
,K
-ATPase through a mechanism which
is not completely understood. The affinity of the
Na
,K
-ATPase for ouabain is closely
linked to enzyme cycling as demonstrated by the effects of enzyme
substrates on the protein-drug interaction. For example, Mg
and inorganic phosphate (P
) stimulate ouabain binding
in the absence of monovalent cations (4, 5) and
Mg
and ATP stimulate ouabain binding in the presence
of Na
(6, 7) . Mg
alone increases the affinity of the ATPase for ouabain
300-fold over Tris buffer alone(8) . Based on this
substrate-dependent affinity of the enzyme for ouabain, it has been
established that the interaction site for P
and ATP which
stimulates ouabain binding is the conserved catalytic phosphorylation
site(9, 10, 11, 12, 13) .
Moreover, although ATP and P
increase the enzyme's
affinity for ouabain an additional 10- and 20-fold, respectively, over
magnesium alone, Mg
appears to be the essential
element for high affinity ouabain binding.
) was replaced with
an uncharged amino acid, asparagine, in the ouabain-sensitive sheep
1 isoform of Na
,K
-ATPase. The
mutant protein was expressed in NIH 3T3 cells which contain a
relatively ouabain-insensitive endogenous isoform of
Na
,K
-ATPase. In this system,
[
H]ouabain binding can be utilized as a highly
specific probe for the exogenous protein, even though the mutant D369N
is inactive(14, 15, 16) . The purpose of this
study was to determine if D369N is expressed in NIH 3T3 cells in a form
which can bind ouabain and to decipher whether the amino acid
substitution alters the mechanism by which ligands
(Na
, K
, Mg
, ATP,
and ADP) regulate the binding of ouabain.
Materials
Companies from which reagents were
purchased were previously reported(15, 16, 17) .
The specific radioactivity of [H]ouabain was
determined by the method of Wallick and Schwartz(1988)(18) .
General Methods
Site-directed
mutagenesis(19) , establishment of stable 3T3 cell
lines(15, 16, 17, 20) , isolation of
crude plasma membranes(17, 21) , and immunological
detection (22) were all performed using methods previously
described.
[
All ouabain binding studies were conducted under the
following conditions unless otherwise indicated in the figure legends
or text: 5 mM MgClH]Ouabain Binding to Crude
Membranes
and 50 mM Tris-HCl (pH
7.4) in a final volume of 0.5 ml. The amount of protein used was
typically 50 µg of total protein/assay tube, depending on the
specific activity of the individual membrane preparation. The
affinities for ouabain of both the D369N and the wild type proteins
under phosphate-free conditions were determined using unlabeled ouabain
in a self-competition assay with [
H]ouabain (See
Equation 1, Ref. 16). The apparent K
for
wild type sheep
1 was calculated to be
50 nM while
the D369N protein had a K
for ouabain of
20 nM. For the competition curves with unlabeled ouabain,
eight concentrations of unlabeled ouabain (including zero) were
examined in triplicate (Fig. 3). Ligand stimulation or inhibition
of [
H]ouabain binding was characterized using at
least 11 concentrations (including zero) of each ligand in duplicate.
To optimize the determinations of the different ATPase-ligand
interactions, the concentration of [
H]ouabain
utilized was approximately equal to the ouabain K
values calculated for each protein. Aliquots of each
reaction mixture (50 µl) were taken with each experiment to
determine the exact concentration of [
H]ouabain
utilized. The samples were filtered, and radioactivity analyzed as
described previously(16) . Data describing the competition of
[
H]ouabain with unlabeled ouabain was fit to the
self-competition model as described previously(16) .
P
, Mg
, Na
,
K
, ATP, and ADP data were fit to a four parameter
logistic function for either stimulation of binding:
{[(B
- B
)/(1 +
(AC
/x)
)] + B
} or inhibition of binding:
{[(B
- B
)/(1 + (x/IC
)
)] + B
}. B
and B
represent the maximum and minimum amounts of
bound [
H]ouabain, respectively. n represents the approximate number of ligands responsible for the
inhibition or activation of [
H]ouabain binding, a
factor which is similar to a Hill coefficient. The AC
or
IC
value is the concentration of ligand which produces 50%
of the activation or inhibition, respectively. x is the
concentration of inhibiting or stimulating ligand (i.e. P
, Mg
, Na
,
K
, or nucleotide). All data analysis was done using
the KaleidaGraph program by Abelbeck Software.
Figure 3:
Ouabain competition curves.
[H]Ouabain binding was measured in the presence
of various concentrations of unlabeled ouabain. The assay contained 5
mM MgCl
and 50 mM Tris-Cl (pH 7.4). The
symbols represent the mean of triplicate determinations and are coded
as follows: wild type (
) and D369N (
). The error was
calculated for each point and is shown unless it is smaller than the
symbol size. The data were fit to a simple self-competition model with
three adjustable parameters: K (dissociation constant for
ouabain), ET (total number of ouabain-binding sites), and NS
(proportionality constant for nonspecific binding) (see
``Experimental Procedures''). The fitted parameters
calculated for the wild type: (K = 49.6 ± 5.7
nM; ET = 0.244 ± 0.010 nM; NS =
0.000324 ± 0.000011) and for the D369N mutant: (K = 27.2 ± 2.0 nM; ET = 0.252 ±
0.016 nM; NS = 0.000244 ±
0.000012).
Expression of the D369N Mutant
Initially, these
experiments were designed to directly address whether a mutant form of
Na,K
-ATPase in which the catalytic
phosphorylation site was altered could be stably expressed and could
bind ouabain with high affinity. To this end, the sequence encoding the
amino acid replacement D369N was introduced into two cDNAs: one which
encodes a ouabain-resistant form of
Na
,K
-ATPase (sheep
1(RD))
(
)and a second which encodes a ouabain-sensitive form
of Na
,K
-ATPase (sheep
1). The
mutant sheep
1(RD) cDNA was transfected into HeLa cells and grown
in the presence of 1 µM ouabain. In this expression
system, only the exogenous ouabain-resistant
Na
,K
-ATPase can support cell
viability. Thus, if a mutation impairs the functional activity of the
exogenous Na
,K
-ATPase, no transfected
HeLa cells will survive the ouabain selection. No ouabain resistant
colonies were observed for the D369N mutant (data not shown) and thus
confirmed that this amino acid replacement disrupts ATPase activity
presumably by inhibiting the formation of the
-aspartyl-phosphorylated intermediate as previously suggested by
Ohtsubo et al.(2) .
1 cDNA
which encodes the D369N amino acid substitution in a ouabain-sensitive
form of Na
,K
-ATPase was cotransfected
into 3T3 cells with a plasmid encoding a neomycin resistance protein.
The transfected 3T3 cells were grown in the presence of 400 µg/ml
G418 (a neomycin analog), and G418-resistant colonies were isolated.
This expression system does not require that the exogenous
Na
,K
-ATPase be functionally capable
of supporting cell viability. Immunological detection was utilized to
screen the isolated cell lines for expression of sheep
1 protein.
Three clonal cell lines which expressed the sheep
1 proteins were
expanded, and crude membranes were prepared using NaI treatment. Twenty
µg of total protein from these membrane preparations (as determined
by a Lowry assay) were separated on a 7.5% SDS-polyacrylamide gel
electrophoresis and electroblotted onto nitrocellulose. The M7-PB-E8
monoclonal antibody grown against sheep kidney
Na
,K
-ATPase (23) was used to
probe the exogenously expressed sheep
1 protein without
cross-reacting with the endogenous mouse protein from 3T3 cells (Fig. 1). A single band of protein migrating at a molecular mass
of approximately 110 kDa was detected by the monoclonal antibody in the
stable transfectants but not in untransfected 3T3 cells. Several
membrane preparations of two clonal cell lines transfected with either
the D369N cDNA or the wild type cDNA showed relatively high expression
levels of sheep
1 protein and were utilized in the ouabain binding
experiments.
Figure 1:
Western analysis of
sheep 1 cDNA transfectants. Twenty µg of total protein
isolated from the neomycin-resistant NIH 3T3 cell lines were probed
with the monoclonal antibody, M7-PB-E8, which specifically reacts with
the exogenous sheep
1 protein and does not cross-react with the
endogenous mouse
1 protein of NIH 3T3 cells. This autoradiograph
of an immunoblot shows the content of sheep
1 protein in membrane
preparations from one wild type clonal line and two D369N clonal lines.
The arrows indicate the positions of the prestained molecular
weight markers (Bio-Rad), phosphorylase b (
106,000 Da) and
-galactosidase (
116,500 Da) which were used as a reference to
identify the Na
,K
-ATPase (
110,000
Da).
Interaction of Inorganic Phosphate with the Expressed
Proteins
To test for additional phosphate interaction sites
which might stimulate ouabain binding, [H]ouabain
binding to both the expressed wild type sheep
1 and the D369N
mutant protein was measured at 23 concentrations of inorganic phosphate
ranging from 0 to 30 mM. A portion of the curve defining this
P
effect is displayed in Fig. 2. No stimulation of
[
H]ouabain binding was observed for the mutant
D369N for P
concentrations up to 30 mM. P
stimulation of the wild type sheep
1 displayed a
half-maximum stimulation (AC
) of 0.12 ± 0.02 mM P
, which is identical to the value previously
reported(15) . These data are consistent with this aspartic acid
at position 369 in the sheep
1 isoform being the only interaction
site for inorganic phosphate which stimulates ouabain binding as well
as being the site of
-aspartyl phosphorylation during the
enzymatic cycle(24) .
Figure 2:
Inorganic
phosphate stimulation of [H]ouabain binding.
[
H]Ouabain binding was measured in the absence
and presence of various concentrations of inorganic phosphate as
displayed on the x axis. The symbols represent the mean of
triplicate determinations, and the calculated standard error is shown
unless it was less than the symbol size. All assay solutions contained
5 mM MgCl
, 50 mM Tris-Cl (pH 7.4), and 20
nM [
H]ouabain and were incubated for 6 h
at 37 °C. The data characterizing the expressed wild type sheep
1 are shown by the open squares (
) while the data
for the D369N mutant are symbolized by closed circles (
). The wild type data were fit to the four parameter
logistic function for activation (see ``Experimental
Procedures''). The fitted parameters for the wild type curve were: B
= 0.402 ± 0.012 nM; B
= 0.147 ± 0.001 nM;
AC
= 0.127 ± 0.017 mM; and n = 0.91 ± 0.05.
Mg
In the presence of
Mg Stimulated
[
H]Ouabain Binding
alone, both the wild type sheep
1 protein and
the D369N mutant protein were able to bind
[
H]ouabain present in nanomolar concentrations.
This is demonstrated clearly at 0 mM P
in Fig. 2, keeping in mind that nonspecific binding (NS) represents
a value which is less than 10% of the total amount bound (wild type, NS
= 0.0126 ± 0.0006 nM and D369N, NS =
0.0049 ± 0.0002 nM as determined from unlabeled ouabain
self-competition data). This observation suggested that the affinity of
both proteins for ouabain is relatively high in the presence of
Mg
alone and that [
H]ouabain
binding could be utilized as a probe for these expressed proteins. In
order to determine the K
values
characterizing these protein-drug interactions, competition curves were
carried out in the presence of 5 mM MgCl
and 50
mM Tris-Cl using [
H]ouabain and
unlabeled ouabain. Fig. 3shows a comparison of the competition
curves for one membrane preparation of D369N and of the wild type
protein. The mean K
values obtained for
three different preparations of each protein are presented in . In the presence of 5 mM Mg
,
the affinity of the wild type sheep
1 protein for ouabain is
34-fold lower than the affinity in the presence of P
and
Mg
(K
= 1.53
± 0.08 nM, 5 mM P
, and 5 mM Mg
(15) ). This increase in apparent K
for ouabain caused by the absence of
P
is similar to the 23-fold increase previously calculated
from rate constants observed for purified sheep
Na
,K
-ATPase(8) . In the
presence of Mg
alone, the mutant D369N displayed a
2.5-fold higher affinity for ouabain compared to the wild type protein.
The high affinity of D369N for ouabain is evidence that the ouabain
interaction site on the mutant (presumably involving the extracellular
surface of the protein) has not been markedly altered compared to the
wild type protein.
values for all ligands, all competition
curves characterizing the wild type protein were done in the presence
of approximately 60 nM [
H]ouabain, and
those describing the D369N mutant were done in the presence of
approximately 20 nM [
H]ouabain. The
observed nonspecific binding is primarily due to
[
H]ouabain binding to the glass fiber filters and
is essentially independent of the enzyme concentration employed. The
maximum level of [
H]ouabain bound to the
membranes is dependent on the expression levels of the exogenous
proteins and differs slightly between membrane preparations.
site(s) on these proteins were
saturated at 5 mM Mg
, activation of
[
H]ouabain binding to the
Na
,K
-ATPase was examined with respect
to Mg
concentrations (data not shown). In previous
studies, an apparent AC
value for Mg
stimulation of ouabain binding to the wild type protein was
determined to be 0.20 ± 0.03 mM in the presence of 5
mM P
and 50 mM Tris (pH 7.4)(15) .
In the absence of P
, the apparent AC
value for
Mg
stimulation of ouabain binding to the wild type
Na
,K
-ATPase is increased 3.6-fold to
0.71 mM (). The AC
value for
Mg
stimulation of ouabain binding in the absence of
P
to the D369N mutant (0.12 mM) is 5.9-fold lower
than that of the wild type. The Mg
AC
values and n values shown in are averages
of three experiments done in duplicate on three separate membrane
preparations of two clones for each protein. Although the cause for the
alterations in the Mg
AC
values are not
completely understood, it appears that 5 mM Mg
provides an excess of divalent cation such that all detectable
Mg
activation sites are occupied in the absence of
additional ligands. Therefore, the changes in the amount of
[
H]ouabain bound at various inhibitor
concentrations (i.e. unlabeled ouabain, Na
or
K
, in the upcoming experiments) reflect protein
inhibitor (ligand) interactions and are not a direct result of
unoccupied Mg
sites on the protein. It is interesting
to note that the number of Mg
interactions (n) which stimulate ouabain binding to the two proteins were
calculated to be greater than one and may be an indication that at
least two Mg
activation sites are located on the
Na
,K
-ATPase.
K
Ouabain binding
stimulated by either Mg Inhibition of
[
H]Ouabain Binding
and P
or by
Mg
, Na
, and ATP is antagonized by
the addition of K
to the equilibrium medium (4, 6).
Presumably, K
induces a conformational change in the
Na
,K
-ATPase such that the resulting
form of the protein has a lower affinity for ouabain. As demonstrated
in Fig. 4, K
also inhibits ouabain binding in
the presence of Mg
alone. The average apparent
IC
and the pseudo-Hill coefficient (n) for
K
determined from four separate membrane preparations
of each cell line are presented in . The D369N mutant
demonstrates a 3-fold lower AC
value for K
compared to the wild type protein under P
-free
conditions. Thus, it appears that the two K
inhibition
sites previously observed in the presence of Mg
and
P
(14, 15, 16) can be probed on the
wild type sheep
1 protein in the presence of Mg
alone, and these K
sites remain intact in the
D369N mutant form of the sheep
1 protein.
Figure 4:
K inhibition of
[
H]ouabain binding. The amount of
[
H]ouabain bound was measured in the presence of
various concentrations of KCl, 5 mM MgCl
, and 50
mM Tris-Cl (pH 7.4). The symbols represent the mean of
duplicate determinations. The error bars display the range of the
duplicates and are not shown if smaller than the symbol size. The
symbol representation is the same as in Fig. 3. These data were fit to
a four parameter logistic function. The fitted parameters for the wild
type were: B
= 0.331 ± 0.006
nM; B
= 0.034 ± 0.001
nM; IC
= 1.41 ± 0.06 mM;
and n = 2.00 ± 0.08. The calculated parameters
for D369N were: B
= 0.217 ± 0.007
nM; B
= 0.0088 ± 0.0001
nM; IC
= 0.445 ± 0.022 mM;
and n = 1.75 ± 0.05.
Na
Unlike
K Inhibition of
[
H]Ouabain Binding
, Na
stimulates ouabain binding in
the presence of Mg
and ATP and antagonizes ouabain
binding in the presence of Mg
and P
(4-7). In the presence of Mg
alone,
Na
induces a conformational change in the wild type
Na
,K
-ATPase and the D369N mutant
protein which reduces the affinities of the proteins for ouabain. In Fig. 5, examples of the inhibitory effects of Na
on Mg
-stimulated
[
H]ouabain binding are displayed. These
inhibition data were fit to a four parameter logistic function (see
``Experimental Procedures''). The average IC
and n values for Na
of at least three trials on
two clones are presented in .
Figure 5:
Na inhibition of ouabain
binding. The amount of [
H]ouabain bound in the
presence of various concentrations of NaCl was measured in the presence
of 5 mM MgCl
and 50 mM Tris-Cl (pH 7.4).
The symbols represent the mean of duplicate determinations. The error
bars represent the range of the duplicates and are not shown if smaller
than the symbol size. The symbol representation is as follows:
(
) wild type sheep
1 and (
) D369N. These data were
fit to a four parameter logistic function. The calculated parameters
for the wild type were: B
= 0.161
± 0.003 nM; B
= 0.0204
± 0.0004 nM; IC
= 8.04 ±
0.36 mM; and n = 1.83 ± 0.07 and for
D369N were: B
= 0.195 ± 0.003
nM; B
= 0.0080 ± 0.0002
nM; IC
= 13.0 ± 0.3 mM;
and n = 2.31 ± 0.06.
MgATP and MgADP Effects on
[
In the absence of
NaH]Ouabain Binding
, MgATP was shown to stimulate ouabain binding to
purified Na
,K
-ATPase by increasing
the on-rate for ouabain and in turn decreasing the K
for the drug (8). We have utilized this stimulatory effect
to determine if the MgATP site has remained intact in the D369N mutant.
To avoid the inhibitory effects of Na
on
Mg
-stimulated [
H]ouabain
binding and to limit the amount of ATP hydrolysis due to ATPase
turnover in the wild type protein, no Na
was added to
these equilibrium studies. As shown in Fig. 6A, addition
of MgATP stimulated ouabain binding in the wild type protein both at 1
mM Mg
and 10 mM Mg
. Average AC
values for three
preparations at both concentrations of Mg
are shown
in . As shown in Fig. 6B, MgATP inhibits the
Mg
-stimulated ouabain binding to the D369N mutant in
the presence of either 1 or 10 mM Mg
.
Average IC
values are summarized in for three
membrane preparations of the mutant D369N. This MgATP induced effect
demonstrates that a high affinity ATP-binding site exists on the D369N
mutant protein and suggests that the cytoplasmic loop in which the
amino acid replacement is located resembles the wild type protein and
specifically binds ATP with high affinity.
Figure 6:
Nucleotide effects on
[H]ouabain binding.
[
H]Ouabain binding was measured in the presence
of various concentrations of ATP or ADP. The assays contained
approximately 20 nM [
H]ouabain, 50
mM Tris-Cl (pH 7.4), and either 1 mM MgCl
(closed symbols) or 10 mM MgCl
(open symbols). These assays were incubated for 3 h at
37 °C. Panel A shows the ATP activation of
[
H]ouabain binding to the wild type protein.
These data were fit with a four parameter logistic function for
activation, and the calculated constants were as follows: at 1 mM MgCl
, B
= 0.380 ±
0.014 nM; B
= 0.050 ±
0.001 nM; AC
= 19.2 ± 1.4
µM; and n = 0.896 ± 0.045 and at 10
mM MgCl
, B
= 0.349
± 0.010 nM; B
= 0.082
± 0.002 nM; AC
= 15.1 ± 1.4
µM; and n = 1.10 ± 0.08. Panel
B displays the inhibitory effect of increasing amounts of ATP on
[
H]ouabain binding to the D369N mutant. These
data were fit to a four parameter logistic function for inhibition (see
``Experimental Procedures''), and the calculated constants
were: at 1 mM MgCl
, B
= 0.069 ± 0.002 nM; B
= 0.011 ± 0.001 nM; IC
= 2.03 ± 0.11 µM; and n = 1.38 ± 0.09 and at 10 mM MgCl
, B
= 0.065 ±
0.002 nM; B
= 0.014 ±
0.001 nM; IC
= 2.21 ± 0.11
µM; and n = 1.64 ± 0.13. Panel
C displays the stimulatory effect of ADP on ouabain binding to the
wild type protein (
) and the inhibitory effect of ADP on
ouabain association with the D369N protein (
) at 10 mM MgCl
and 50 mM Tris-Cl (pH 7.4). The wild
type data were fit with the logistic function for activation, and the
calculated constants are: B
= 0.410
± 0.011 nM; B
= 0.177
± 0.005 nM; AC
= 7.54 ± 0.54
µM; and n = 1.47 ± 0.17. The D369N
data were fit to the four parameter logistic function for inactivation
with the calculated values being: B
=
0.134 ± 0.004 nM; B
=
0.036 ± 0.001 nM; IC
= 3.52
± 0.34 µM; and n = 1.30 ±
0.11.
To determine if the
MgATP-induced stimulation of ouabain binding in the wild type protein
is a result of nucleotide binding or a result of enzyme
phosphorylation, MgADP was used to mimic simple nucleotide binding in
the absence of Na. As shown in Fig. 6C,
like MgATP, MgADP stimulates ouabain binding in the wild type protein
and inhibits ouabain binding in the D369N mutant. Average AC
and IC
values for three experiments using two clonal
cell lines for each protein are shown in . It is apparent
that the nucleotide-induced conformational change normally observed as
an increase in the affinity of the
Na
,K
-ATPase for ouabain cannot occur
if the aspartic acid residue at position 369 is altered to an
asparagine.
Expression and Structure of the D369N
Mutant
Previous mutagenesis studies involving this conserved
aspartic acid residue in other P-type ATPases have demonstrated the
loss of cation transport and phosphorylation upon amino acid
replacement(2, 3, 25) . In the sarcoplasmic
reticulum Ca-ATPase, this mutant protein (D351N) was
transiently expressed in COS-1 cells and was characterized as impaired
with respect to Ca
transport and ATP-stimulated
formation of a phosphorylated enzyme intermediate(25) . When a
similar amino acid substitution was introduced in the yeast
H
-ATPase, the protein processing of the mutant was
thought to be interrupted such that no protein was detected in the
secretory vesicles of the yeast expression system(3) . Unlike
the yeast H
-ATPase, but similar to the SR
Ca
-ATPase,
Na
,K
-ATPase in which the essential
aspartyl residue has been replaced can be expressed in 3T3 cells as
shown here and in oocytes as demonstrated by Ohtsubo et
al.(2) . Both the immunological analysis and the presence
of a ouabain-sensitive protein in the isolated membrane fraction of
transfected 3T3 cells is direct evidence for the expression of the
mutant protein. The interaction between the D369N mutant and ouabain
suggests that the extracellular surface of the mutant resembles the
wild type protein while the MgATP-D369N interaction shows the
structural integrity of the cytoplasmic domain of the mutant. These
data are consistent with the D369N mutant being expressed in a membrane
of the 3T3 cells with an overall protein structure which is similar to
the wild type sheep
1
Na
,K
-ATPase.
1 protein, whole
cell ouabain binding was utilized to examine if the mutant was located
in the plasma membrane. No [
H]ouabain binding was
detected when intact 3T3 cells expressing the D369N mutant were
incubated with the radioligand (data not presented). Ouabain binding to
intact 3T3 cells is regulated by the presence of intracellular MgATP
and subsequent intracellular Na
binding. Both of these
substrates, MgATP and Na
, when added independently ( Fig. 5and Fig. 6B) or together (data not
presented) were shown to inhibit Mg
-stimulated
ouabain binding in the studies employing membrane fragments from 3T3
cells expressing the D369N mutant. Thus, due to the presence of these
intracellular components in the intact 3T3 cells, it was not surprising
that ouabain did not bind to the extracellular surface of whole cells
expressing the D369N mutant. Whether or not the D369N mutant is present
in the plasma membrane of 3T3 cells or oocytes(2) , our ouabain
binding data on isolated membrane preparations does explain why ouabain
interactions with whole cells expressing the D369N mutant may not have
been observed.
Ligand Sites of the
Na
This work suggests
that phosphate interaction at the catalytic phosphorylation site,
aspartic acid 369 in the sheep ,K
-ATPase
1 isoform, induces a conformational
change in the Na
,K
-ATPase which
increases the affinity of the enzyme for ouabain. Thus, catalytic
ATPase activity and ouabain binding are linked through this
phosphorylation site which supports the use of
[
H]ouabain binding as a method for probing the
functional sites of Na
,K
-ATPase. In
addition, this double role for aspartic acid 369 contradicts the theory
that phosphate interactions at a second site on
Na
,K
-ATPase increase the affinity of
the enzyme for ouabain(26, 27) .
,K
-ATPase have been
proposed in which the chemical moieties associated with position 369 (i.e. aspartyl group or covalently bound phosphate) act as
binding sites for the transported Na
and K
ions, directly linking the formation of the phosphorylated
intermediate with cation transport(28, 29) . From the
data presented here, three Na
and two K
ions interact with the
Na
,K
-ATPase when both of these
moieties are removed either by site-directed mutagenesis or simply by
eliminating P
from the equilibrium medium. Thus, assuming
that the cation sites which inhibit ouabain binding are also the cation
transport sites, it does not appear that either of these chemical
groups are directly associated with the binding of the monovalent
cations being transported.
,K
-ATPase is necessary for ouabain
binding(30, 31, 32, 33) . Conclusions
from these investigations have been met with some skepticism due to the
possibility that contaminating inorganic phosphate promotes
phosphoenzyme formation. No phosphoenzyme was present in the D369N
studies presented here due not only to the absence of added inorganic
phosphate but also due to the alteration of the catalytic
phosphorylation site. Thus, our data unequivocally demonstrate that a
phosphorylated Asp
site is not required for ouabain
binding to the Na
,K
-ATPase.
and K
on the ouabain binding properties of
Na
,K
-ATPase often postulate cation
stabilization and destabilization of the phosphorylated intermediate
(10, 13). Although our results do not discount these effects of
monovalent cations on phosphoenzyme formation, our studies suggest that
this is not the only mechanism by which Na
and
K
affect the ouabain binding properties of the ATPase.
The cation inhibition experiments involving the D369N mutant ( Fig. 4and 5) had no phosphoenzyme present due to the absence of
added P
and substitution of the catalytic phosphorylation
site. Thus, the Na
inhibition and K
inhibition of ouabain binding to the D369N protein are due to
cation-induced conformational changes in the protein and cannot
directly reflect the cation effects on the phosphoenzyme intermediate.
Mg
The importance of
Mg and MgATP Interactions with the
Na
,K
-ATPase
in active cation transport by
Na
,K
-ATPase was recognized in the
original characterization of this protein by Skou(34) .
Subsequent to these studies, both kinetic analysis and physical
chemical studies have suggested that Mg
interacts
with the Na
,K
-ATPase at two distinct
sites. The first site is observed in the presence of nucleotide and is
thought to be a site related to the binding of MgATP. This site
demonstrates a K
for Mg
of 5 µM as measured by ATP-ADP exchange in the
presence of 125 mM Na
, 5 mM ATP, and
1.25 mM [
C]ADP(35) . The second
Mg
site is a separate divalent cation site. This site
displays a K
for Mg
of
1-3 mM and is observed in the absence of nucleotide and
Na
in para-nitrophenylphosphate hydrolysis,
Mn
competition, and Rb
release
experiments (28, 35, 36). Mg
binding to the distinct
divalent cation site is thought to induce a conformation in
Na
,K
-ATPase characterized by a high
affinity for P
and K
and a lower affinity
for Na
(36) . In addition, the affinity of this
distinct divalent cation site for Mg
is increased
10-fold in the presence of P
(36) .
interaction with the low affinity site on the
wild type Na
,K
-ATPase induces a
protein conformation with a high affinity for ouabain. This hypothesis
is based on three characteristics of the Mg
-wild type
interaction. First, the AC
value calculated for
Mg
stimulation of ouabain binding was 0.71
mM, similar to the K
value which
characterizes this low affinity
site(28, 35, 36) . Second, this AC
value was decreased approximately 3.5-fold in the presence of
P
, consistent with the effect of P
on this low
affinity site observed with a
Rb-release
technique(36) . Third, ouabain binding to the wild type protein
was stimulated further upon formation of a E-MgATP complex (Fig. 6A). The absence of a Mg
-induced
shift in this MgATP stimulation of ouabain binding demonstrates that
the two Mg
interactions are not directly competitive
on the wild type Na
,K
-ATPase. These
data support the theory that the stimulation of ouabain binding
observed in the presence of Mg
alone is promoted by
Mg
interacting at a divalent cation site distinct
from the MgATP site.
-induced
ouabain binding in the D369N mutant suggesting that the
D369N
MgATP complex has a reduced affinity for ouabain. The
IC
value for MgATP was approximately 2 µM indicative of a high affinity ATP site in this mutant form of the
Na
,K
-ATPase. Thus, although
Mg
can interact with the D369N mutant to form an
enzyme complex with a high affinity for ouabain, neither MgATP nor
P
can induce a conformation in the D369N mutant which
displays a higher affinity for ouabain. Similar to the wild type
protein, the absence of a Mg
-induced shift in the
MgATP inhibition curve implies that the Mg
site which
stimulates ouabain binding and the MgATP site are kinetically distinct.
In addition, the dramatically different effects of Mg
and MgATP association with the D369N protein suggest that the
binding sites for Mg
and MgATP are mechanistically
distinct. Similar to the effects of MgATP, MgADP stimulated ouabain
binding in the wild type protein and inhibited binding to D369N (Fig. 6C). The similarity between the effects of MgATP
and MgADP suggests that the difference in nucleotide-induced
conformational changes between the two enzymes is not a direct result
of the inability of D369N to form a phosphorylated intermediate but may
be a direct result of magnesium-nucleotide binding to the protein.
Mg
complex and the
D369N
Mg
complex suggest that the charged
character of this aspartic acid is required to induce an enzyme complex
with high affinity for ouabain in the presence of nucleotide.
Therefore, we suggest that D369N is inhibited both by its inability to
form a
-aspartyl-phosphorylated enzyme intermediate and by its
inability to undergo the normal conformational change induced by the
binding of a magnesium-nucleotide complex. However, the tight
association of MgATP to D369N demonstrates that the nucleotide-binding
site of Na
,K
-ATPase is not destroyed
by the amino acid substitution. In future structural investigations,
the mutant D369N may serve as a stable model of the
Na
,K
-ATPase in which the
ligand-binding sites can be probed without the effects of varying
turnover numbers altering calculated affinity constants.
Table: Apparent
binding constants for protein-ligand interactions
1(RD), sheep
1 isoform modified to a ouabain-resistant
protein with the following amino acid substitutions: Q111R and N122D
(37); Na
,K
-ATPase, sodium and
potassium-activated adenosine triphosphatase; G418, geneticin, a
neomycin analog.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.