(Received for publication, April 17, 1995; and in revised form, July 17, 1995)
From the
The functional role of serine 775, predicted to be located in
the fifth transmembrane segment of the subunit of the Na,K-ATPase
(YTLTSNIPE), was studied using site-directed mutagenesis, expression,
and kinetic analysis. Substitutions S775A, S775C, and S775Y were
introduced into an ouabain-resistant
1 sheep isoform and expressed
in HeLa cells. cDNAs carrying substitutions S775C and S775A produced
ouabain-resistant colonies only when extracellular K
was increased from 5.4 mM to 10 or 20 mM,
respectively. No ouabain-resistant colonies were obtained for
substitutions S775Y at any tested K
concentration.
Kinetic characterization of S775C and S775A substituted enzymes showed
expression levels higher than control enzyme, reduced V
and turnover, and normal phosphorylation and
high affinity ATP binding. Dephosphorylation experiments indicated that
S775A substituted enzyme is insensitive to ADP but readily
dephosphorylated by K
. The K
K values for the activation of the Na,K-ATPase were markedly
altered, with S775C displaying a 13-fold increase and S775A exhibiting
a 31-fold increase. These large changes in the Na,K-ATPase affinity for
K
are consistent with the participation of this amino
acid in binding K
during the translocation of this
cation. Substitutions of Ser
did not change Na
affinity, indicating that this residue is likely not involved in
Na
binding and occlusion.
These data show that the
electronegative oxygen and the small side chain of Ser are required for efficient enzyme function. Moreover, these
results suggest Ser
plays a distinct role in K
transport and not in Na
interactions, revealing
a possible mechanism for the enzymatic differentiation of these cations
by the Na,K-ATPase.
The Na,K-ATPase uses the energy produced by ATP hydrolysis to
transport Na and K
ions across the
plasma membrane of eukaryotic
cells(1, 2, 3) . This enzyme belongs to a
class of ion pumps, termed P-type ATPases, that has a common catalytic
mechanism in which an aspartyl phosphate intermediate is transiently
formed(1, 4, 5, 6) . In addition to
the Na,K-ATPase, this group includes the gastric H,K-ATPase, the
sarco(endo)plasmic reticulum Ca-ATPase, and the plasma membrane
Ca-ATPase, among others.
The hydrolytic and transport cycles of the
Na,K-ATPase have been described in
detail(1, 2, 3) . However, because little is
known about the structure of the enzyme, the molecular events that take
place in the protein during the binding of Na,
K
, and ATP, the hydrolysis of ATP, the transfer of
energy to the cation sites, and finally the transport of ions, are
largely unknown.
The Na,K-ATPase consists of two major subunits,
(M
= 112,000) and
(M
= 35,000 for the protein
component)(1, 2, 7) . Studies using
hydropathy analysis, epitope localization, selective proteolytic
cleavage, and labeling of putative transmembrane segments with
hydrophobic probes suggest a topological arrangement of the
subunit involving 10 transmembrane segments (3, 8, see Fig. 3in (2) ). However, the assignment of particular sequences to some
transmembrane segments is still questioned; for instance, the exact
identity of the fifth and sixth transmembrane
segments(8, 9, 10) .
Figure 3:
Na dependence of
Na,K-ATPase activity of RD and Ser
-substituted enzymes.
The Na,K-ATPase activity was determined as indicated under
``Experimental Procedures.'' K
concentration
was kept at 20 mM, while Na
concentration was
varied. The values are the mean of results obtained with membrane
preparations from four independent clones, each one measured in
duplicate. Standard errors were lower than 5% and error bars are not
plotted for simplicity. The Na,K-ATPase activities corresponding to
100% were similar to the V
values presented in Table 1. The Na
K (mM) and
Hill coefficients were as follows: RD enzyme 7.06 ± 0.43, n = 2.36 (
); S775C 5.56 ± 0.53, n = 1.95 (
); S775A 5.98 ± 1.01, n = 1.65 (
).
A major objective of
our studies is to identify the amino acids that constitute the cation
binding and occlusion sites of this enzyme. It is accepted that these
sites reside in the transmembrane region of the
subunit(2, 8, 10, 11, 12) .
It has been shown that after removing large cytoplasmic portions of the
protein by protease treatment, the remaining transmembrane fragments
can still occlude K
(13, 14) . It is
reasonable to expect that carboxyl residues located within the membrane
could be part of the occlusion cage neutralizing the cation charges.
This concept is supported by findings in the Ca-ATPase from
sarcoplasmic reticulum, where intramembrane carboxyl residues seem to
play an important role in Ca
binding and transport (15, 16) . In the Na,K-ATPase there are probably seven
carboxyl residues within the membrane (Glu
,
Glu
, Asp
, Asp
,
Asp
, Glu
, and Asp
) (
)and they have been the target of chemical modification and
site-directed
mutagenesis(17, 18, 19, 20, 21, 22, 23, 24, 25, 26) .
Chemical modification studies using 4-(diazomethyl)-7-diethylamino)
coumarin (DEAC) (
)identified Glu
as involved
in cation binding(17, 18) . Although transfectants of
ouabain-resistant enzyme containing both E779L and E779D are unable to
confer ouabain resistance to ouabain-sensitive HeLa cells, the
substitutions E779A and E779Q yield functional enzymes, indicating that
neither the carboxyl group nor the electronegative atom are
``essential'' for enzyme
function(19, 20, 21, 22) . However,
it is possible that an oxygen atom or a hydrogen bonding capability is
required in this position for stabilizing the E
P(Na) form
of the enzyme(21) . The other carboxyl residues located in the
membrane have also been mutated with mixed
results(19, 20, 21, 22, 23, 24, 25, 26) .
Van Huysse et al. (23, 25) demonstrated that
carboxyl residues Glu
and Glu
are not
required for enzyme function. Likewise substitutions of Asp
did not lead to major alterations in the cation
affinity(19) . Similar to Glu
, substitutions of
Glu
and Asp
display mixed characteristics
with either severely impaired activity (E327A, E327D, D808L, D808Q) or
slightly altered kinetic parameters (E327L, E327Q, D808E, (
)D808A
)(19, 21, 24, 26) .
Furthermore, the functional studies that have been performed with some
of these mutants do not indicate a clear pattern to support a
particular role for these residues or to provide a convincing
structural model (based on structure-function relationships). At the
present, only Asp
appears to be important for enzyme
function, since no functional substitution has been found for this
residue
(19, 21) . Thus, in spite of
considerable work, there is no clear understanding of the ``cation
binding cage,'' although it seems probable that other residues
(possibly non-carboxyl) must be necessary for cation binding,
occlusion, and transport.
By analogy with other cation binding
proteins, it is likely that the cations are coordinated by six or eight
oxygen-containing residues, water, or even backbone carbonyl oxygen,
when they are bound or occluded in the
Na,K-ATPase(27, 28, 29) . Considering this,
we have begun testing the putative involvement of other (non-carboxyl)
oxygen-containing residues in cation binding and occlusion.
Particularly, we have targeted those located in the putative fifth
transmembrane segment
(YTLTSNIPEITPFLIFIIANI
). Previously, we
have observed that substitutions to alanine of several of these
residues yield apparently nonfunctional enzymes(20) .
Furthermore, the structural connection of this transmembrane segment
with the large cytoplasmic loop (where ATP binds and is hydrolyzed)
suggests that this segment might play a key role in cation binding and
also in the structural transitions required to transport Na
and K
through the plasma membrane.
We report
here the functional effects of substitutions of Ser (YTLTSNIPE). Our findings indicate that, while this serine is not
involved in Na
interactions with the enzyme, it is
very likely part of the K
-binding site. The
participation of this serine in the interaction of one cation but not
the other provides an initial structural basis for cation selectivity
and contributes to the understanding of the transport mechanism of the
Na,K-ATPase.
In experiments in which the
effect of extracellular K on cell survival was
examined, DMEM (that contains 5.4 mM KCl) was supplemented
with KCl to reach either 10 or 20 mM KCl. The cells were
adapted to the higher K
concentrations 2 days prior to
transfection and maintained at the desired K
concentration during the selection with ouabain. Controls with
choline-Cl instead of KCl were performed. Mock transfections were made
to determine if the inclusion of KCl had any effect on the selection
with ouabain. Colonies obtained after selection in high K
were consistently maintained in the KCl concentration in which
the cells were selected.
A mutagenesis and expression strategy previously developed in
our laboratory(7, 19, 20, 22, 23, 30) was
employed to study the role of Ser. This system uses a
cDNA encoding the sheep
1 subunit that has been made ouabain
insensitive by the introduction of substitutions Q111R and N122D (RD
enzyme). This cDNA is specifically mutated and is transfected into HeLa
cells which express a human
1 isoform with high affinity for the
inhibitor. The transfected cells are grown in the presence of 1
µM ouabain. This concentration of ouabain leads to the
death of the untransfected cells as well as those transfected with
cDNAs carrying substitutions that alter Na,K-ATPase function to the
extent that the expressed enzyme cannot support cell viability under
the cell culture conditions. In previous studies, only cells carrying
mutated pumps with relatively mild effects in overall Na,K-ATPase
activity have survived the selective pressure of ouabain, and the
stable expression has allowed the study of the enzymatic
characteristics of these enzymes.
Substitutions of serine at
position 775 were designed to remove the hydroxyl group S775A, to make
the most conservative alteration changing the hydroxyl to a sulfhydryl,
S775C, or to change the size while retaining the oxygen atom S775Y. In
initial studies we observed that none of the cells transfected with
cDNAs coding for substitutions at Ser were able to
produce colonies after selection in regular DMEM which contain 5.4
mM KCl (Fig. 1). Under similar conditions HeLa cells
transfected with control RD
cDNA yielded a large number of
ouabain-resistant colonies. It was apparent that
subunits
substituted at Ser
were unable to produce functional
enzymes, including the conservative substitution to cysteine. It was
possible that these substitutions disrupted the cation binding site of
the Na,K-ATPase, supporting our hypothesis that Ser
was
involved in the coordination of cations. Based on this assumption, if
these substitutions diminish the cation (Na
or
K
) affinities, then by increasing the cation
concentrations it could be possible to ``saturate'' the
enzyme allowing it to function at (or near) physiological levels,
allowing the survival of transfected cells. Experiments were designed
to determine if increasing K
in the culture media
would allow the cells to survive. In these experiments, substitutions
S775C and S775A that were unable to sustain HeLa cell growth in regular
DMEM (5.4 mM K
) produced numerous
ouabain-resistant colonies when K
was increased to 10
and 20 mM, respectively (Fig. 1). No colonies were
detected in plates transfected with mutant S775Y. While K
does not affect the total ouabain binding capacity, it does lower
the ouabain binding rate(40) , and therefore high K
concentrations may alter the ouabain selection procedure such
that untransfected HeLa cells can survive in the presence of the
inhibitor. Mock transfections were performed with no DNA and ouabain
selection in high K
. No colonies were detected,
indicating that the higher K
concentrations did not
affect the ouabain selection method. Likewise, colonies were not
observed in controls using choline-Cl instead of KCl. These results
indicate that although enzymes with the substitutions S775A and S775C
were severely impaired, they could function enough to support cell
growth under facilitating conditions. Furthermore, these findings
predicted that the interaction between K
and the
enzyme was affected by substitutions at Ser
.
Figure 1:
Number of
ouabain-resistant colonies resulting from the expression of RD control
and substituted enzymes, after selection in growth media containing
different K concentrations. Transfections and
selections were performed as indicated under ``Experimental
Procedures.'' Selection was stopped after 2 weeks in cells
transfected with RD control cDNA and after 4 weeks in the case of cells
transfected with mutated cDNA. Colonies were stained with Methylene
Blue and counted. The values are the mean ± S.E. of six
independent experiments.
In site-directed mutagenesis
studies, the expression system and the characteristics of a particular
protein can yield distinct expression levels for different
substitutions. These differences in expression levels are relevant when
parameters such as specific activity are necessary to understand
functional alterations. We have estimated the level of expression of
the two mutants S775A and S775C and the control RD enzyme by
quantifying immunostained dot-blots of serial dilutions of each
membrane preparation. A purified sheep enzyme was used as a standard. Table 1shows the level of each of these enzymes in our membrane
preparations. It was determined that the mutated Na,K-ATPases S775A and
S775C were expressed in significantly larger quantities than the
control RD protein. However, reduced V were
found in the S775A- and S775C-substituted enzymes when their activities
were compared to the RD control (Na,K-ATPase activities measured at
saturating concentrations of all ligands and expressed per milligram of
heterologous enzyme). These results imply that the substitutions of
Ser
produce a major alteration in the enzymatic activity.
Keeping in mind that purified sheep enzyme has been used as a standard,
the activity of the control RD enzyme (calculated per milligram of
heterologous enzyme) (Table 1) is lower than those usually
obtained for purified Na,K-ATPase. It is known that a significant pool
of unassembled (likely nonfunctional)
subunits is normally
present in cells(41, 42) . These subunits are probably
present in our preparations and may be detected in our immunoblot
determinations, lowering the calculated activity values (expressed per
milligram of heterologous enzyme). Other factors including
overestimation of the expression level due to the immunostaining method
or partial inactivation by NaI during membrane preparations may have
also decreased the calculated activity. In any case, our quantification
demonstrates that the substituted enzymes are expressed in larger
quantities than the control RD enzyme.
To determine the amount of
functional enzyme, the phosphorylation levels were measured. Under the
phosphorylation conditions used, the EE
P equilibrium is
largely displaced toward the phosphoenzyme form, thus the level of E
P (or E
P) is nearly equal to
the total amount of functional enzyme. Similar phosphorylation levels
(calculated per milligram of heterologously expressed protein) were
detected in the three enzymes (Table 1), indicating that the
fraction of functional
subunit was comparable for the three
proteins and that the activity values (per milligram of heterologous
enzyme) of S775C and S775A enzymes are lower than that of RD enzyme.
This last point is more evident when the turnover number, which is
independent of the protein concentration, is calculated. As expected,
the turnover numbers of the substituted enzymes were significantly
lower than that of RD control enzyme (Table 1) showing again the
effects of these replacements in the overall enzyme activity.
The
turnover value of the control RD enzyme is in good agreement with
others reported for purified Na,K-ATPase(43, 44) ;
however, it is much lower than those described for heterologously
expressed sodium pump (21, 22, 24, 45) . This discrepancy
is most likely based in the different procedure used to determine the
phosphoenzyme level. Previous studies have separated the phosphorylated
Na,K-ATPase from other phosphoproteins in the preparations using acidic
SDS-polyacrylamide gel electrophoresis gels. A common problem of these
gels is that not all the protein (after being precipitated with
trichloroacetic acid and partially resuspended with SDS) enters into
the gels. This eventually leads to an underestimation of the
phosphoenzyme and, consequently, an overestimation of the turnover
number. In place of the acidic gels, we directly measure the
phosphorylated Na,K-ATPase by subtracting the level of phosphorylated
protein formed in the presence of K (absence of
Na
) from the phosphoenzyme formed in the presence of
Na
(no K
). Controls using gels to
separate the different phosphorylated proteins showed that the only
K
- or Na
-sensitive phosphoprotein in
our preparations is the Na,K-ATPase(22) . Control experiments
were also performed using membranes from untransfected HeLa cells where
the human isoform is not phosphorylated if it is preincubated in the
presence of 0.01 mM ouabain. After preincubation with ouabain
the phosphoprotein was insensitive to K
and its level
identical to that obtained for membranes from transfected cells at
saturating K
(no Na
) (not shown). The
background levels in the presence of K
were never
higher than 50% of the phosphoprotein produced in the presence of
Na
.
The alterations described in Table 1indicate that Ser is an important residue
required for normal enzyme function. On the other hand, the data in Table 1also point out that the cells are compensating for the
lower activity of the S775A and S775C substitutions by producing large
quantities of these enzymes to reach the amount of Na,K-ATPase activity
(and consequent ionic homeostasis) required for cell survival. In this
way, no difference between the mutants and the control enzymes is
observed when comparing the total Na,K-ATPase activities (expressed per
milligram of total protein in the membrane preparation) at the
K
concentrations at which the cells were selected (RD:
12.4 ± 2.4 µmol/h/mg (5.4 mM K
);
S775C: 13.9 ± 2.5 µmol/h/mg (10 mM K
); S775A 10.8 ± 2.2 µmol/h/mg (20
mM K
)). Furthermore, if human Na,K-ATPase
from untransfected HeLa cells is isolated and measured under similar
conditions, the activity is comparable to the one produced by the
expressed proteins (13.5 ± 3.4 µmol/h/mg (5.4 mM K
)). This demonstrates that in this expression
system the activity of the heterologous protein must almost completely
replace the endogenous Na,K-ATPase activity to maintain cell viability.
Figure 2:
K dependence of
Na,K-ATPase activity of RD and Ser
-substituted enzymes.
The Na,K-ATPase activity was determined as indicated under
``Experimental Procedures.'' Na
concentration was kept at 30 mM (A) or 100
mM (B), while K
concentration was
varied. The values are the mean ± S.E. of results obtained with
membrane preparations from four independent clones, each one measured
in duplicate. The Na,K-ATPase activities corresponding to 100% were
similar to the V
values presented in Table 1. The K
K (mM) and Hill
coefficients were as follows: A, RD enzyme 0.43 ± 0.06, n = 1.56 (
); S775C 5.82 ± 0.30, n = 2.27 (
); S775A 13.32 ± 1.75, n = 1.61 (
). B, RD enzyme 0.99 ± 0.05, n = 1.59 (
); S775A 24.57 ± 3.69, n = 1.07 (
).
The curves shown in Fig. 2A were
performed at 30 mM NaCl which saturates the intracellular
Na sites (E
cation sites). It is
known that Na
can compete with K
for
the extracellular cation binding sites (E
cation
sites)(46, 47) . We determined if this competitive
effect was present in S775A, assuming initially that if K
binding to the external sites has been significantly affected by
the replacements at Ser
, the interaction of Na
at those sites would be largely removed (i.e. it would
not compete). However, it was observed that 100 mM NaCl was
able to displace to the right both curves, and the K
K is higher in both RD and S775A enzymes (compared to
that at 30 mM Na
) such that the relative
increase (near 30-fold) is maintained (Fig. 2B).
Previously, amino acid substitutions in the Na,K-ATPase have, to a
smaller or larger extent, affected the binding of both Na and
K
(19, 21, 22, 23, 24, 25, 26) .
Considering the simplest mechanism where the same ``sites''
that are involved in transporting Na
outward bind and
transport K
inward, it was expected that a
substitution affecting K
interactions would also alter
Na
binding. Fig. 3shows the dependence of the
Na,K-ATPase on Na
concentration. The K for
Na
activation of the Na,K-ATPase activity was not
affected by the substitutions of Ser
. Since external
K
sites of S775A are not saturated at 20 mM KCl, the Na
activation for this substitution was
also measured at 100 mM KCl; again under this condition the
substitution had no effect on Na
activation.
Na
K (mM) were: RD enzyme 16.50
± 2.81; S775A 21.22 ± 4.72 (curves not shown).
A
possible explanation for this unique result was that an altered
affinity for Na was masked by the turnover condition
at which the Na
-enzyme interactions were examined. To
test this alternative, the Na
K for the
activation of the phosphorylation by ATP was determined for the RD
control and the S775A-substituted enzymes (Fig. 4). Again no
alteration in the Na
activation of phosphorylation by
ATP was observed. These data strongly suggest that Ser
does not participate in the coordination of Na
when it interacts with the enzyme.
Figure 4:
Na dependence of
phosphorylation by ATP of RD- and S775A-substituted enzymes. The
sodium-activated ATP phosphorylation was measured at different sodium
concentrations as indicated under ``Experimental
Procedures.'' The values are the mean of results obtained with
membrane preparation from four independent clones, each one measured in
duplicate. Standard errors were between 5-10%, and error bars are
not plotted for simplicity. The maximum phosphorylation levels
corresponding to 100% were similar to the phosphorylation values
presented in Table 1. The Na
K and Hill
coefficients were as follows: RD enzyme 0.88 ± 0.24 mM, n = 0.95 (
); S775A 0.91 ± 0.14 mM, n = 1.46 (
).
Figure 5:
Phosphorylation by ATP of RD- and S775A-
substituted enzymes. The sodium-activated ATP phosphorylation was
measured at different ATP concentrations as indicated under
``Experimental Procedures.'' The values are the mean of
results obtained with membrane preparations from four independent
clones, each one measured in duplicate. Standard errors were between
5-10%, and error bars are not plotted for simplicity. The maximum
phosphorylation levels corresponding to 100% were similar to the
phosphorylation values presented in Table 1. The ATP K and Hill coefficients were as follows:
RD enzyme 0.58 ± 0.05 µM, n = 1.16
(
); S775A 0.48 ± 0.16 µM, n =
1.02 (
).
Figure 6:
ATP dependence of Na,K-ATPase activity of
RD- and Ser-substituted enzymes. The Na,K-ATPase activity
was determined as indicated under ``Experimental
Procedures.'' The ATP concentration was varied as indicated. The
values are the mean ± S.E. of results obtained with membrane
preparations from four independent clones, each one measured in
duplicate. The Na,K-ATPase activities corresponding to 100% were
similar to the V
values presented in Table 1. The ATP K (mM) and Hill coefficients
were as follows: RD enzyme 0.244 ± 0.056, n =
1.50 (
); S775C 0.088 ± 0.006, n = 0.98
(
); S775A 0.027 ± 0.005, n = 1.01
(
).
To understand
how these cells survive with a severely impaired Na,K-ATPase, it is
necessary to examine the expression levels in combination with the
activity of these pumps. The cells appear to compensate for the low
activity of the substituted enzymes by overproducing them to a level
which can support ionic homeostasis compatible with cell growth. Even
though our experiments were not designed to study the underlying
induction mechanism, it is interesting to consider our findings in
relation to other studies. It has been known for some time that the
extracellular K level influences both the Na,K-ATPase
expression and activity(53, 54, 55) . The
diminution of extracellular K
leads to slower pump
activity and disruption of the transmembrane ionic gradients. In this
case the cells increase the number of Na,K-ATPases in the membrane as a
compensatory mechanism to the lower activity. A similar situation is
observed with substitutions at Ser
, where the
extracellular K
(almost under the K) and the
slower turnover leads to low Na,K-ATPase activity and, in turn, to a
large production of enzyme.
It is clear that the substitutions at Ser did
not produce long range conformational effects that affect, for
instance, the binding of ATP with high affinity. However, these enzymes
have reduced turnover numbers indicating that one of the partial
reactions of their catalytic cycle is slower compared to the RD control
enzyme. Our determinations do not allow us to describe the affected
partial reaction, although neither the dephosphorylation step
(K
can readily dephosphorylate E
P) nor the E
E
transition (increase in the apparent affinity
for ATP at the low affinity nucleotide-binding site) appear to be
slower in the substituted enzymes.
A decrease in the affinity for
K could be due to either a specific alteration of the
binding site or to a tendency of the enzyme to remain in an E
conformation with low affinity for
K
. The Ser
-substituted enzymes do not
have any apparent preference to remain in the E
conformation. This is evident from the unaltered Na
K measured under turnover conditions. A stabilization of E
would decrease this K, as observed for
the L332A mutant (57) . It could be argued that in our case
this alteration could be masked by a real increase in the Na
K (observable under non-turnover conditions). We did not
see such a change when the Na
activation of the ATP
phosphorylation was analyzed. Furthermore, if the mutated enzymes had a
tendency to remain in E
, then a nonspecific
reduction in the interaction of any cation with E
should have been observed. On the contrary, the competition of
Na
with K
for the extracellular (E
) cation binding site seems largely unaltered.
However, the binding of ATP with low affinity is affected, and this
could indicate a displacement in the equilibrium between conformational
intermediates. ATP binds with low affinity to E(K)
(K
occluded). This binding accelerates the deocclusion
of K
and the E
E
conformational
transition(1, 48, 49, 50) . Other
substitutions, such as E779A, E779Q, and
E327Q(21, 22, 24) , also produce increases in
the affinity of the low affinity nucleotide binding site. However, the
changes in cation affinity observed in those enzymes (2-5-fold)
are minor compared to the change in K
K of
S775C and S775A enzymes (10-30-fold). Therefore, the changes in
the low affinity ATP binding of S775C and S775A enzymes cannot justify
the large increments observed in K
K. On the
other hand, since we observed that the K
enzyme
interaction (probably binding and occlusion) is affected by the
substitution of Ser
, then this increase in the affinity
of ATP might be more likely associated to an instability of E
(K) (that would accelerate deocclusion and
increase the apparent ATP affinity) than to a tendency of the
substituted Na,K-ATPase to go to an E
form.
Considering that a slow EP dephosphorylation
rate or a displacement of E
P
E
P toward E
P would lead to a
reduced affinity for K
, we also analyzed the status of
the phosphorylated forms of the S775A enzyme. Our results indicate a
displacement of the equilibrium E
P
E
P toward the E
P form (i.e. S775A is insensitive to ADP). This conformational effect
of the substitution also affects the K
-induced
dephosphorylation of the E
P form, which is
apparently faster (compared to control RD enzyme) at saturating
K
. The consequence, if any, of this conformational
effect on the K
affinity should be to increase the
affinity for the cation. On the contrary we detected a decrease in
affinity; thus, the conformational effect is perhaps masking an even
bigger alteration at the cation site.
Another distinctive
feature of the mutations of Ser is the correlation
between the structural modification and the kinetic effect observed.
The most disrupting substitution is the replacement S775Y in which the
introduction of the bulky phenol ring yields largely inactive enzyme
(taking into account that no ouabain-resistant colonies were obtained
for this substitution). Mutation S775A, where the hydroxyl group is
removed, shows large functional alterations, while S775C yields an
intermediate phenotype between S775A and RD enzyme. These data strongly
suggest that the enzyme requires a hydroxyl group as well as a
relatively small side chain at this position for efficient cation
transport.
Ser is conserved in all the sequenced
Na,K-ATPases, and it is also conserved in the sarcoplasmic reticulum
Ca-ATPase(7, 58) . The equivalent of Ser
in the sarcoplasmic reticulum Ca-ATPase (S767) has been subjected
to site-directed mutagenesis studies, and while no major alteration in
Ca
affinity was observed, the mutated enzyme
exhibited low activity(16) . It is interesting that Ser
(YTLTSNIPE) is replaced by a lysine (YTLTK
NIPE) in
the H,K-ATPase(59) . This structural difference in these two
K
-transporting enzymes is probably based in a
different stoichiometry for K
transport or in the
different counter ion being transported. The H,K-ATPase stoichiometry
has not been satisfactorily determined. Previous studies indicate that
one K
is transported in each cycle of the
H,K-ATPase(60) ; thus, requiring fewer coordinating residues
than the Na,K-ATPase. It is also possible that the H,K-ATPase uses the
amino group of this lysine to coordinate protons being transported as
hydronium ions (61) to form a proton relay (62) or to
provide the proper environment for carboxyl group
deprotonation(63) , as it has been proposed for other
proton-transporting proteins.
The role of Ser in
cation binding fits with the significance attributed to the fifth
transmembrane segment, which connects with the cytoplasmic loop
containing the phosphorylation site (18) . Thus, Ser
is likely to perceive conformational changes produced by ATP
hydrolysis or translate changes in the binding of K
affecting the phosphoenzyme intermediate. Furthermore, its
relative position within the membrane, i.e. the cytoplasmic
half, agrees with the proposed model for the cation binding path as the
ion well where the occlusion of cations takes place in the cytoplasmic
half of the membrane(64, 65) .
The kinetic
mechanism of the Na,K-ATPase indicates that Na and
K
are transported alternately through the membrane.
The simplest model would suggest that the same sites that bind
Na
from the intracellular milieu are then accessible
to bind K
from the extracellular media. Even if both
cations share some coordinating groups, others must be different to
accommodate the stoichiometry of the transport and the different
coordination requirements. Thus, some structural differences are
necessary to confer selectivity. Keeping in mind the functional
differences, it is interesting to consider the crystal structures of
dialkylglycine decarboxylase as an example of a single
``site'' using different residues to coordinate Na
or K
. This enzyme binds both cations at the same
``pocket''; however, a serine that coordinates K
is displaced when Na
binds the enzyme, while an
extra water molecule takes up the sixth coordinating
position(28) . Taking into account that substitutions of
Ser
affect to a large extent K
but not
Na
binding, it is tempting to hypothesize that this
serine confers the ion selectivity at the extracellular facing cation
sites.
In summary, the functional effects of minimal structural
alterations produced by substitutions of Ser in the
subunit of the Na,K-ATPase have been studied. Our findings indicate
that this is an important amino acid which is required for the binding
of K
with high affinity. Thus, it is likely that
Ser
participates in the coordination of K
when the cation is bound, occluded, or transported. Furthermore,
the Na,K-ATPase probably requires the hydroxyl group of a small amino
acid to function properly. On the other hand, Ser
does
not participate in the coordination of Na
, and its
removal seems not to have any effect in the binding of this cation.
These results are the first report of a large change in the enzyme
affinity for a cation. Furthermore, the critical role of Ser
in the binding of only one of the two transported cations is the
first evidence that suggests a possible mechanism for the
differentiation of Na
and K
by the
Na,K-ATPase.