(Received for publication, October 5, 1994; and in revised form, December 1, 1994)
From the
By combining the tools of site-directed mutagenesis and
[H]ouabain binding, the functional role of
glutamic acid 327 in the fourth transmembrane domain of the sheep
1 isoform of Na
,K
-ATPase was
examined with respect to its interactions with ouabain,
Na
, K
, Mg
, and
inorganic phosphate. Using site-directed mutagenesis, this glutamic
acid was substituted with alanine, aspartic acid, glutamine, and
leucine. The mutant proteins were constructed in a sheep
1 protein
background such that [
H]ouabain binding could be
utilized as a highly specific probe of the exogenous protein expressed
in NIH 3T3 cells. Na
competition of
[
H]ouabain binding to the mutant forms of
Na
,K
-ATPase revealed only slight
alterations in their affinities for Na
and in their
abilities to undergo Na
-induced conformational changes
which inhibit ouabain binding. In contrast, K
competition of [
H]ouabain binding to all
four mutant forms of Na
,K
-ATPase
displayed severely altered interactions between these proteins and
K
. Interestingly, [
H]ouabain
binding to the mutant E327Q was not inhibited by the presence of
K
. This mutant was previously reported to be
functionally able to support cation transport with a 5-fold reduced K
for K
-dependent ATPase
activity (Jewell-Motz, E. A., and Lingrel, J. B.(1993) Biochemistry 32, 13523-13530; Vilsen, B.(1993)
Biochemistry 32,
13340-13349). Thus, it appears that this glutamic acid in the
fourth transmembrane domain may be important for stabilizing a
K
-induced conformation within the catalytic cycle of
Na
,K
-ATPase that is not rate-limiting
in the overall ATPase cycle but that displays a greatly reduced
affinity for ouabain.
The Na,K
-ATPase (
)is an integral membrane protein found in nearly all
eukaryotic cells that utilizes energy from the hydrolysis of
intracellular ATP to transport three sodium ions out of the cell and
two potassium ions into the cell (1, 2, 3) .
The enzyme is composed of an
subunit containing 8-10
transmembrane domains and a
subunit which has one transmembrane
segment. The catalytic cycle of
Na
,K
-ATPase involves an acid-stable
phosphorylated form of the protein, a characteristic shared by other
active cation transporters such as the sarcoplasmic reticulum
Ca
-ATPase, the plasma membrane
Ca
-ATPase,
H
,K
-ATPase, and
Mg
-ATPase(4, 5, 6, 7) .
Unlike other ATPases, Na
,K
-ATPase is
inhibited by cardiac glycosides such as ouabain.
In recent years,
structure-function studies of
Na,K
-ATPase have focused on
identifying the amino acids involved in binding and translocating
Na
and K
ions. Both chemical
modification studies and site-directed mutagenesis studies have
targeted anionic amino acid residues located in the transmembrane
domains of the
subunit. These residues contain negatively charged
side chains which are thought to neutralize the cations during
transport through the hydrophobic lipid bilayer. Six oxygen-containing
residues in the transmembrane domains of the sarcoplasmic reticulum
Ca
-ATPase have been implicated as essential amino
acids for Ca
transport using site-directed
mutagenesis(8) . Three of these residues contain carboxylic
acid side chains. These negatively charged residues are conserved in
the Na
,K
-ATPase and include
Glu
, Glu
, and Asp
in the
sheep
1 isoform.
N,N`-dicyclohexylcarbodiimide (DCCD) labels
Na,K
-ATPase in a K
protectable manner and blocks cation occlusion upon
modification(9) . Hence, DCCD is thought to bind to a residue
essential for potassium occlusion. By combining DCCD labeling and
limited proteolytic digestion, two sites of DCCD modification have been
located within the transmembrane domains (10) . Glutamic acid
953 in the COOH-terminal half of the sheep
1 protein has been
identified as one site of modification and glutamic acid 327 is
hypothesized to be another site of modification. Site-directed
mutagenesis studies, involving Glu
and a neighboring
glutamic acid residue, Glu
, have demonstrated that
altering the charged side chains at these positions does not
dramatically change the cation-dependence properties of ATP hydrolysis
by Na
,K
-ATPase(11) .
Therefore, it is possible that the residue labeled by DCCD in the
NH
-terminal half of the enzyme, Glu
, is the
carboxyl group important for cation occlusion.
Glutamic acid 327 is
located in the fourth transmembrane domain of the
Na,K
-ATPase and is highly conserved
throughout the amino acid sequences of related
ATPases(4, 5, 6, 7) . Previously,
site-directed mutagenesis was employed to change Glu
to
Gln, Leu, Ala, and Asp(12, 13, 14) . These
mutagenesis studies utilized similar expression schemes which take
advantage of species-specific variations in ouabain sensitivity to
examine the effects of the mutation on cell viability and on the
cation-dependent ATPase activity. These schemes involve introducing the
mutation into a cDNA which encodes a ouabain-resistant isoform of
Na
,K
-ATPase (K
= 1
10
M; i.e. rat
1, sheep
1(RD) (
)or rat
2*(
)), expressing this mutant in a cell line containing
a ouabain-sensitive endogenous form of the ATPase (K
= 1
10
M; COS-1
or HeLa) and selecting for cells expressing the ouabain-resistant
protein by growing the cells in the presence of ouabain (0.2, 1, or 5
µM). An active
Na
,K
-ATPase is essential for the
survival of mammalian cells due to its role in the establishment and
maintenance of the electrochemical gradient across the cell membrane.
By growing these transfected cells in the presence of ouabain, the
endogenous Na
,K
-ATPase is inhibited,
and only the exogenous ATPase can produce the essential cation
gradient. Only if the transfected cDNA encodes for an active enzyme
will cells be viable in ouabain. The mutant forms of
Na
,K
-ATPase, E327Q and E327L, were
able to transport cations, produce an electrochemical gradient, and
maintain cell viability. The mutants E327D and E327A were unable to
support cell viability in the presence of ouabain. The
cation-dependence of Na
,K
-ATPase
activity was examined in the presence of 1
10
M ouabain for mutant proteins E327L and E327Q. The
mutant E327L demonstrated a 4-fold higher K
for
Na
and a 2-fold higher K
for
K
than the wild type protein. The mutant E327Q
displayed a 2-fold higher K
for Na
and a 3-6-fold higher K
for
K
than the wild type ATPase. From these small changes
in the K
values, it was concluded that the
charge of this glutamic acid side chain was not essential for ATPase
activity. However, based on the cell viability results, the length of
the side chain was suggested to be important sterically in the
enzymatic cycle. Although these original site-directed mutagenesis
studies revealed some important facts about the glutamic acid at
position 327, the mechanistic step (i.e. cation binding or
conformational changes) that the substitutions E327D and E327A disrupt
was not identified, since these mutant enzymes were inactive and could
not be examined.
In order to study both inactive and active mutant
enzymes, we have examined the cation-enzyme interactions of
Na,K
-ATPase by observing the effects
of Na
and K
on ouabain binding to the
enzyme. The affinity of the
Na
,K
-ATPase for cardiac glycosides is
closely linked to enzyme cycling. Two assay environments which are
known to induce high affinity ouabain binding to the enzyme are
Mg
, ATP, and Na
or Mg
and P
(15, 16) . Both of these
conditions promote formation of a phosphorylated form of the enzyme (E2-P) which is cation-sensitive. Thus, the role specific
amino acids play in binding cations during the catalytic cycle of
Na
,K
-ATPase may be probed by studying
the effects of cations on [
H]ouabain binding to
mutant forms of the Na
,K
-ATPase.
The crude
plasma membrane preparations from NIH 3T3 cell lines were analyzed by
Western immunoblotting. The proteins contained in the membrane
preparations were resolved on a 10% SDS-polyacrylamide gel,
electrophoretically transferred onto nitrocellulose, and stained with
the monoclonal antibody M7-PB-E9 and an antimouse horseradish
peroxidase-conjugated secondary antibody. M7-PB-E9 was raised against
lamb kidney Na,K
-ATPase and is
subunit specific such that it distinguishes the transfected sheep
1 isoform from the endogenous mouse
1
subunit(23, 24) . M7-PB-E9 was a generous gift from
the laboratory of Dr. W. J. Ball (University of Cincinnati).
Figure 2:
Ouabain competition curves.
[H]Ouabain binding was measured in the absence
and presence of various concentrations of unlabeled ouabain as shown on
the x axis. The symbols represent the mean of triplicate
determinations. The error was calculated for each point and is shown
unless it is smaller than the symbol size. Assay conditions were as
described under ``Experimental Procedures,'' except for E327D
which was assayed in the presence of 15 mM Tris-P
and 10 mM MgCl
. Symbol representation is as
follows:
, wild type sheep
1;
, E327Q;
,
E327D;
, E327L; and
, E327A. The data were fit to a
simple self-competition model:
where [I] is the
concentration of unlabeled ouabain, E is
the amount of enzyme, [O] is the concentration of
[
H]ouabain, and NS is the
proportionality constant for nonspecific binding. Three adjustable
parameters were calculated for each curve and include K
(dissociation constant), E
and NS. The fitted parameters
were calculated: wild type sheep
1 (K
= 1.51 ± 0.18 nM; E
= 0.106 ± 0.003
nM; NS = 0.000343 ± 0.000012); E327Q (K
= 4.06 ± 0.35
nM; E
= 0.130 ±
0.004 nM; NS = 0.000322 ± 0.0000096); E327D (K
= 12.5 ± 0.8
nM; E
= 0.268 ±
0.009 nM; NS = 0.000379 ± 0.0000146); E327L (K
= 2.87 ± 0.31
nM; E
= 0.142 ±
0.004 nM; NS = 0.000470 ± 0.0000143); E327A (K
= 1.67 ± 0.13
nM; E
= 0.110 ±
0.004 nM; NS = 0.000365 ±
0.000012).
Figure 3:
Na competition curves.
[
H]Ouabain binding was measured for isolated
membranes in the absence and presence of various concentrations of NaCl
as shown on the x axis. The symbols represent the mean of
duplicate determinations. The error bars represent the range of the
duplicate determinations and are not shown if smaller than the symbol
size. The assay conditions were as described under ``Experimental
Procedures,'' except for E327D which was assayed in the presence
of 15 mM Tris-P
and 10 mM MgCl
. The symbol representation is as follows:
, wild type sheep
1;
, E327Q;
, E327D;
, E327L; and
, E327A. The curves displaying the data of
E327Q and the wild type were fit to a competitive model involving three
Na
sites:
where K is the dissociation constant for ouabain obtained from the
ouabain competition experiments (Fig. 2) and [O] is
the concentration of [
H]ouabain. E327D, E327L,
and E327A were fit to a similar competitive equation involving two
Na
sites such that the fourth term in the denominator
of this equation was eliminated. The two adjustable parameters
calculated were: K
, (Na
inhibition constant) and E
(amount
of ouabain-binding sites). The fitted values obtained were: wild type
sheep
1 (K
= 15.7 ±
0.1 mM; E
= 0.241
± 0.003 nM); E327Q (K
= 20.8 ± 0.3 mM; E
= 0.167 ± 0.003 nM); E327D (K
= 7.51 ± 0.16
mM; E
= 0.359 ±
0.008 nM); E327L (K
=
19.2 ± 0.4 mM; E
=
0.106 ± 0.002 nM); and E327A (K
= 24.2 ± 0.4 mM; E
= 0.289 ± 0.003
nM).
Figure 4:
K-Induced effects on
ouabain binding. [
H]Ouabain binding was measured
in NaI-treated membranes in the presence of various concentrations of
KCl as shown on the x axis. The symbols represent the mean of
duplicate determinations. The error bars represent the range
of the duplicate determinations and are not shown if smaller than the
symbol size. The assay conditions were (5 mM P
and
5 mM Mg
) as described under
``Experimental Procedures.'' The assay conditions for the inset data were the same with the exception that the
Mg
concentration was 10 mM and the
Tris-P
concentration was 15 mM. The symbol
representation is as follows:
, wild type sheep
1;
,
E327Q;
, E327D;
, E327L; and
, E327A. The amount
of bound [
H]ouabain (B) as a function of
K
concentration was calculated with the following
equation:
where E is the total enzyme concentration,
[O] is the concentration of [
H]ouabain,
is the interaction factor of inhibition, and NS is the
proportionality constant for nonspecific binding. Fitted parameters for
the inhibition phase were: wild type sheep
1 (at 5 mM P
and 5 mM Mg
) (K
= 1.02 ± 0.03
mM; E
= 0.115 ±
0.002 nM;
= 10.1 ± 0.2); E327A (at 5
mM P
and 5 mM Mg
) (K
= 0.879 ± 0.009
mM; E
= 0.117 ±
0.003 nM;
= 2.76 ± 0.11); E327L (at 5
mM P
and 5 mM Mg
) (K
= 1.18 ± 0.07
mM; E
= 0.139 ±
0.003 nM;
= 3.35 ± 0.11); wild type sheep
1 (at 15 mM P
and 10 mM Mg
) (K
=
1.17 ± 0.06 mM; E
= 0.112 ± 0.002 nM;
=
12.9 ± 0.3). Fitted parameters for the activation phases: E327A (K
= 84 ± 28 mM;
= 1.40 ± 0.31); E327D (at 5 mM P
and 5 mM Mg
) (AC
=
2.71 ± 0.54 mM); E327D (at 15 mM P
and 10 mM Mg
) (AC
= 2.23 ± 0.51 mM). K
values for E327A were calculated by fitting both the
inhibition and the activation phase of these data to the following
equation:
where is the
interaction factor of activation. Data describing the KCl effects on
E327Q were not fit to any activation or inhibition models, and a smooth
curve was drawn through the data.
The mutations were constructed in a sheep 1 cDNA which
encodes a ouabain-sensitive enzyme, and these cDNAs were transfected
into NIH 3T3 cells containing an endogenous protein with a low affinity
for ouabain. The exogenous proteins bind ouabain with a 1000-fold
higher affinity than the endogenous enzyme. Thus, at the low
concentrations of [
H]ouabain utilized in these
experiments, there is no interference from the binding of
[
H]ouabain to the endogenous
Na
,K
-ATPase(21) . Since the
exogenous protein is ouabain sensitive, no selectable function is
conferred to the cells upon expression of these cDNAs; therefore, an
alternative to ouabain selection was required. The mutated cDNAs were
cotransfected with a gene which codes for a neomycin resistance protein
and selected in 400 µg/ml of G418. cDNAs encoding all four
substitutions (E327A, E327D, E327Q, and E327L) were transfected into
NIH 3T3 cells, and clonal cell lines were established for each mutant.
Western analysis of crude membrane preparations from these cell lines
indicated that the sheep
1 proteins (wild type and mutants) were
being expressed. To establish that the transfected cDNAs were
integrated into the cell genome, genomic DNA was isolated from each
clonal cell line. Using sheep
1-specific primers, PCR was employed
to amplify a region of the DNA which encodes amino acids 230-512
of the exogenous DNA. The DNA fragment from each cell line was
sequenced and revealed a single altered codon which encoded the desired
amino acid at position 327. No additional mutations were detected in
the DNA fragment surrounding the mutated codon.
[H]Ouabain binding to intact 3T3 cells was
performed to establish that the mutant sheep
1 enzymes were
located in the plasma membrane. Fig. 1presents the amount of
[
H]ouabain (60 nM) bound/mg of total
protein for untransfected NIH 3T3 cells and for two clonal cell lines
of each mutant and wild type sheep
1
Na
,K
-ATPase. The nonspecific binding
(NS) of [
H]ouabain (60 nM) to each cell
line was measured in the presence of 1 µM unlabeled
ouabain. From these data, one can see that the cell lines which express
sheep
1 proteins (mutant or wild type) bind 5-10-fold more
[
H]ouabain/mg of total protein than the
untransfected 3T3 cells and at least 4-fold more than nonspecific
binding. The presence of [
H]ouabain binding in
the mutant cell lines reveals that at least some of the translated
mutant sheep
1 protein is located in the plasma membranes of these
clonal cell lines. Moreover, the [
H]ouabain
binding to intact cells indicates that the mutant proteins are
assembled with an extracellular protein conformation sufficiently
similar to the native sheep
1 protein to permit ouabain binding.
The variation in maximum binding levels for each mutant cell line may
be due to different expression levels, secondary to variable
insertional sites of the transfected cDNAs in the genomic DNA. It is
interesting to note that E327L has the lowest expression level in 3T3
cells as measured by [
H]ouabain binding to whole
cells, yet it is translated at high enough levels in HeLa cells to
support viability. Thus, it does not appear that the lower expression
levels of the mutant sheep
1 proteins are the result of the amino
acid substitution interrupting the protein processing steps of
Na
,K
-ATPase.
Figure 1:
[H]Ouabain
binding to intact NIH 3T3 cells. The amount of
[
H]ouabain/mg of total protein is displayed for
the clonal 3T3 cells expressing wild type sheep
1 protein (WT), untransfected NIH 3T3 cells (3T3), and clonal
3T3 lines expressing E327A, E327Q, E327D, and E327L. The maximum values
represent the amount of 60 nM [
H]ouabain
bound to intact cells in the presence of
K
/Ca
-free buffer with respect to the
total protein within these cells. Nonspecific binding (NS) to
the intact 3T3 cells was also measured in identical conditions with the
addition of 1 µM unlabeled ouabain. WT-NS, A-NS, Q-NS, D-NS, and L-NS display
the nonspecific binding observed for the clonal cell lines expressing
wild type sheep
1 Na
,K
-ATPase,
E327A, E327Q, E327D, and E327L, respectively. The values were obtained
by averaging the amounts bound from three separate experiments
performed in triplicate for two clonal cell lines of each
mutant.
The
effects of increasing ionic strength on
[H]ouabain binding were examined in a series of
equilibrium binding assays with increasing concentrations of choline
chloride. Similar to previous studies(26) , choline chloride
concentrations above 150 mM inhibited
[
H]ouabain binding to both mutant and wild type
Na
,K
-ATPase membrane preparations
(data not presented). IC
values obtained from fitting
these data to a simple Hill equation were approximately 195-210
mM for both the wild type and the mutant forms of the ATPase.
Therefore, the effects of Na
and K
observed at cation concentrations
100 mM can only be
explained through specific interactions of these monovalent cations
with the enzyme and do not reflect inhibition due to elevated ionic
strength.
Previously, a partially
competitive model for K effects on
[
H]ouabain binding to wild type sheep
1
protein was used to describe the incomplete inhibition of ouabain
binding at saturating K
concentrations(26) .
The data obtained for the wild type sheep
1 enzyme and for the
K
inhibition phase of E327L and E327A fit this model
(see Fig. 4legend). The Hill coefficients and the K
values characterizing the K
inhibition of ouabain binding to E327L and E327A are presented in Table 1. The potassium K
values are
approximately 1 mM for E327L and E327A, similar to the K
value for the wild type
Na
,K
-ATPase. Unlike the wild type and
E327L proteins, higher concentrations of K
(
10
mM) stimulate [
H]ouabain binding to the
mutant E327A protein. This increase in [
H]ouabain
binding is characterized by a K
value of 84
± 28 mM and a Hill coefficient of 0.97 ± 0.35.
Generally, it appears that both E327A and E327L bind low concentrations
of K
in a manner similar to the wild type (similar K
values) and undergo a K
-induced
conformational change which reduces their affinity for
[
H]ouabain.
In contrast to the results with
E327A and E327L, K did not inhibit
[
H]ouabain binding to the E327Q and E327D
mutants. The effect of K
on E327Q was not fit to any
model. The data describing K
interactions with E327D
demonstrated an increase in [
H]ouabain binding
with increasing concentrations of K
and were fit to a
simple Hill equation yielding a Hill coefficient of 1.9 ± 0.3
and an AC
of 3.14 ± 0.19 mM. Overall, it
appears that either the ability of E327D and E327Q to bind K
has been altered or that these mutants do not undergo the
K
-induced conformational change which normally reduces
the affinity of the enzymes for [
H]ouabain.
Apparent AC values for
P
stimulation of [
H]ouabain binding
in the presence of 5 mM Mg
are also
presented in Table 1. These values range from 0.08 to 1.25
mM. Cation competition data presented in Fig. 3and Fig. 4were obtained in 5 mM P
. Thus, all
the inorganic phosphate site(s) which stimulate ouabain binding were
occupied for the mutant proteins E327A, E327L, and E327Q in the absence
of monovalent cations. The E327D mutant demonstrated the largest change
in its AC
value for P
which was approximately
10-fold higher than the wild type
Na
,K
-ATPase. The ouabain,
Na
, and K
competition curves were
therefore repeated in the presence of 15 mM P
and
10 mM Mg
for the E327D mutant enzyme, and
these calculated kinetic constants are reported in Table 1. The
K
competition constants were not significantly changed
by the higher P
and Mg
concentrations for
either the E327D or wild type proteins (see inset of Fig. 4). This is consistent with the concept that the inhibition
of K
is a direct effect and not an indirect effect due
to a change in the degree of saturation by Mg
and/or
P
. Additional K
and Na
inhibition curves at various Mg
and P
concentrations must be done to fully understand the interaction
of all these ions (monovalent or divalent cations or P
)
with the Na
,K
-ATPase. Since all
Mg
and P
activation sites were saturated
in the absence of monovalent cations under these equilibrium condition,
we conclude that the inhibition and activation of
[
H]ouabain binding observed in Fig. 2Fig. 3Fig. 4were due to ouabain,
Na
, and K
, respectively.
The exact role that glutamic acid 327 plays in the sheep
1 Na
,K
-ATPase is unknown. ATPase
activity studies characterizing proteins containing amino acid
substitutions at this site have been limited by the nonfunctional
character of some of the mutants. In this work, the cation-protein
interactions of four mutant proteins, E327Q, E327D, E327L, and E327A,
were examined using Na
and K
inhibition of [
H]ouabain binding. This
radiolabeling technique can in principle detect any mutation in the
Na
,K
-ATPase which alters either the
number of cations associated with the enzyme or which alters the
stability of an intermediate within the
Na
,K
-ATPase pathway which is normally
sensitive to cation binding.
In addition to altered Hill
coefficients, the K values for Na
inhibition of ouabain binding were increased nearly 2-fold for
E327L, E327A, and E327Q. Previously, Na
interactions
with E327Q and E327L were investigated using cation-dependent ATPase
assays in the presence of ATP, Mg
, and saturating
concentrations of K
(13, 14) . The K
values for the Na
dependence
of ATPase activity for E327Q and E327L were reported to be 2-fold
higher than that of the wild type ATPase. This similar increase in K
values and K
values for
Na
is consistent with the concept that the
Na
sites which activate the ATPase activity may be
identical to the Na
sites which inhibit ouabain
binding. It appears that substitution of the carboxyl side chain of
Glu
disrupts Na
binding to the enzyme by
either reducing the number of Na
inhibition sites
(E327D, E327L, and E327A) or by increasing the inhibition constant of
Na
(E327Q, E327L, and E327A). No correlation exists
between the inability of E327A and E327D to support cell viability and
their ability to interact with Na
as measured by
inhibition of ouabain binding.
The K inhibition profile for
E327L is similar to that observed for wild type
Na
,K
-ATPase. The inhibition constant
for K
is approximately 2-fold higher than the K
value for K
inhibition of the
wild type and is similar to the K
value
determined for this mutant by K
-dependent ATPase
measurements (K
= 1.24 ± 0.05
mM, K
= 1.25 ± 0.13
mM)(13) . When the K
inhibition data
for E327L was fit to the partially competitive equation (see legend of Fig. 4), the interaction factor (
) was determined to be
3-fold lower than the
for wild type. This change in the
interaction factor is evident in the K
inhibition
profile of E327L as a higher level of [
H]ouabain
is bound at saturating K
concentrations. This is
consistent with the concept that the K
-complexed
mutant enzyme has a higher affinity for ouabain than does the
K
-complexed wild type
Na
,K
-ATPase. Thus, upon the
association of two K
ions with E327L, the mutant
undergoes only a portion of the conformational changes normally induced
by K
and retains an extracellular surface to which
ouabain can bind more readily than to the wild type. E327L with two
K
ions bound also retains sufficient structural
integrity in the membrane such that this mutant can support cell
viability, possibly because other ligand-enzyme interactions compensate
for the defect in the K
-induced conformational change.
For example, E327L exhibits a 5-fold decrease in the K
constant for ATP. (
)
[H]Ouabain binding to E327Q is
not inhibited by K
. Previously, it was reported that
E327Q demonstrated a 3-6-fold increase in its K
value for K
-stimulated ATPase activity compared
to the wild type protein(13, 14) , suggesting that the
K
-binding sites of this protein are intact but have
altered affinities for K
. Moreover, the mutant E327Q
supports viability of HeLa cells and transports Rb
from the extracellular to the cytoplasmic surface of the membrane
as measured with
Rb
uptake
experiments(36) . Thus, at the highest concentrations of
K
used in these [
H]ouabain
binding studies, one would predict that K
is bound to
the E327Q mutant but that the protein conformation induced by
K
binding contains an extracellular surface that can
associate with ouabain. There appears to be a greater defect in the
K
-induced conformational change in the E327Q mutant
enzyme than in the E327L mutant. Again, it is possible that the 10-fold
decrease in the K
value for ATP observed for
E327Q (14) compensates for the inability of the mutant to
undergo K
-induced conformational changes observed in
the presence of Mg
and P
. This increase
in ATP affinity along with the normal Hill coefficient for
Na
may enable E327Q to pump cations with enough
efficiency to support cell life.
Similar to E327Q in the
Na,K
-ATPase, the analogous amino acid
substitution (E309Q) was previously constructed and characterized in
the sarcoplamic reticulum Ca
-ATPase (8, 37, 38, 39) . This mutant
protein, E309Q, reacts with P
and Mg
to
form a phosphorylated protein in the presence of Ca
(a ligand which normally inhibits this phosphorylation). In
addition, this phosphorylated intermediate was shown to be extremely
stable with a low rate of dephosphorylation. The
[
H]ouabain binding studies done on E327Q of
Na
,K
-ATPase may indicate a possible
inability of K
to induce a conformational change
necessary for dephosphorylation. Thus, it appears that the
phosphorylated forms of these mutant proteins (E327Q or E309Q) in the
Na
,K
-ATPase and the
Ca
-ATPase formed in the presence of Mg
and P
are resistant to conformational changes
normally induced by the binding of cation ligands.
Unlike E327Q and
E327L, it is not known if E327D and E327A have intact K sites since these mutants could not support HeLa cell viability
and do not have measurable ATPase activity. Hence, direct correlation
of the K
effects on [
H]ouabain
binding to the transport cycle of these mutants is not possible. As
discussed with respect to E327Q, mutations of E327 inhibit
K
-induced conformational changes even in the cases
where K
sites are known to be intact. K
interactions with E327A as probed by
[
H]ouabain binding demonstrate a normal
inhibition constant at low K
concentrations (
10
mM). The stimulation of ouabain binding to E327A at higher
concentrations of K
suggests a possible third
K
interaction and major alterations in the
Na
,K
-ATPase conformation resulting
from this amino acid replacement. E327D does not demonstrate any ligand
affinities similar to the wild type sheep
1 enzyme
(Na
, K
, P
,
Mg
, and ouabain affinities are all altered). Hence,
the structural integrity of this E327D mutant of
Na
,K
-ATPase must be questioned. Data
which indicate that portions of the enzyme conformations of E327D and
E327A may resemble wild type
Na
,K
-ATPase include
[
H]ouabain binding to whole 3T3 cells and
profiles of ouabain competition and Na
competition.
Previously, it had been observed that the hydrolytic activity of
Na
,K
-ATPase is more sensitive to
alteration of protein structure (as induced by chemical modification)
than is ouabain binding to the enzyme(40) . Hence, we
hypothesize that the amino acid substitutions E327A and E327D have
changed the structure of the protein such that the hydrolytic activity
is severely impaired; however, the mutants retain enough native
structure that [
H]ouabain can still bind to the
proteins. Generally, we can conclude that the K
interactions with E327D and E327A as probed by
Mg
-P
supported
[
H]ouabain binding are different from the
K
interactions with the wild type
Na
,K
-ATPase; however, the cause of
the different K
-induced effects (absence of
K
-binding sites, unstable K
-induced
intermediates, or additional K
-binding sites) is not
fully understood.
The lowest ouabain affinity observed for a
K-complexed enzyme was associated with the wild type
protein in which the glutamic acid at position 327 is most likely
responsible for stabilizing a K
-induced conformation.
This charged residue may stabilize this conformation by forming an
internal salt bridge in which the charged side chain of Glu
would be neutralized in the K
-induced
intermediate by a neighboring amino acid. This may explain the
K
protectable characteristic of DCCD modification of
this residue(10) . In contrast to these chemical modification
studies, we do not believe that the charged side chain of Glu
is involved in ligating the K
ions being
transported since E327L and E327Q are able to transport
K
. However, it is significant that both chemical
modification studies and these site-directed mutagenesis studies
identified Glu
in the fourth transmembrane domain of
Na
,K
-ATPase as being essential for
K
interactions with the protein.