(Received for publication, July 28, 1995; and in revised form, November 20, 1995)
From the
The intramembrane Glu residue of the Na,K-ATPase
subunit has been postulated to have a role in the binding and/or
occlusion of cations. To ascertain the role of Glu
, the
residue was substituted with an aspartate, alanine, or lysine residue
and the mutant Na,K-ATPases were coexpressed with the native
1
subunit in Sf9 insect cells using the baculovirus expression system.
All
mutants are able to efficiently assemble with the
1
subunit and produce catalytically competent Na,K-ATPase molecules with
hydrolytic activities comparable to that of the wild-type enzyme.
Analysis of the kinetic properties of the mutated enzymes showed a
decrease in apparent affinity for K
compared to
wild-type Na,K-ATPase, with the lysine and alanine substitutions
displaying the greatest reduction. All Na,K-ATPase mutants demonstrated
a significant increase in apparent affinity for ATP compared to
wild-type Na,K-ATPase, while the sensitivity to the cardiotonic
inhibitor, ouabain, was unchanged. The dependence on
Na
, however, differs among the mutant enzymes with
both the Glu
Asp and Glu
Ala
mutants displaying a decrease in the apparent affinity for the cation,
while the Glu
Lys mutant exhibits a modest
increase. Furthermore, in the absence of K
, the
Glu
Ala mutant displays a
Na
-ATPase activity and a cellular Na
influx suggesting that Na
is substituting for
K
at the extracellular binding sites. The observation
that trypsin digestion of the Glu
Ala mutant in
Na
medium produces a K
-stabilized
tryptic fragment also intimates a decreased capacity of the mutant to
discriminate between Na
and K
at the
extracellular loading sites. All together, these data implicate
Glu
of the Na,K-ATPase
subunit as an important
coordinate of cation selectivity and activation, although the modest
effect of Glu
Lys substitution seemingly precludes
direct involvement of the residue in the cation binding process. In
addition, the fifth membrane segment is proposed to represent an
important communicative link between the extramembraneous ATP binding
domain and the cation transport regions of the Na,K-ATPase.
The Na,K-ATPase, or sodium pump, is a membrane-spanning,
heterodimeric protein that uses the energy from the hydrolysis of ATP
to maintain the low intracellular sodium concentration and high
intracellular potassium concentration common to most animal cells.
During enzyme turnover, three intracellular sodium ions and two
extracellular potassium ions are countertransported across the plasma
membrane for each molecule of ATP hydrolyzed. The ion-motive enzyme
consists of a 110-kDa catalytic subunit and a smaller
noncovalently associated
subunit. The catalytic
subunit
contains the binding sites for ATP and the cardiotonic inhibitor,
ouabain; it is phosphorylated by ATP, and undergoes the
cation-dependent E
E
conformational
transitions associated with cation transport. The exact function of the
glycosylated
subunit remains unknown, although, it has been
implicated in the K
-binding process of the enzyme
(Lutsenko and Kaplan, 1993).
Understanding the molecular events
associated with the Na,K-ATPase catalytic cycle requires a knowledge of
the individual amino acids that coordinate the binding of the enzyme
ligands. While data from site-directed mutagenesis and chemical
modification studies have implicated specific residues within the
subunit in ATP binding and ouabain sensitivity (for review, see Lingrel
and Kuntzweiler(1994)), comparatively little is known concerning the
intramembrane amino acids that comprise the monovalent cation binding
and occlusion sites. It is generally believed that movement of sodium
and potassium through the low dielectric medium of the plasma membrane
involves the neutralization and stabilization of the positively charged
ions through coordination with carboxyl-bearing residues within or near
the membrane. The use of hydrophobic, carboxyl-selective reagents to
chemically modify and inactivate cation binding of the Na,K-ATPase has
proven useful in identifying intramembrane residues in the
subunit that may be involved in cation transport function (Goldshleger et al., 1992; Argüello and Kaplan, 1994).
Recently, a fluorescent, carboxyl-reactive derivative of
diazomethane, 4-(diazomethyl)-7-(diethylamino)coumarin
(DEAC)(), has been shown to inactivate cation occlusion of
the Na,K-ATPase in a K
and Na
preventable manner by covalently labeling Glu
, a
residue in the fifth transmembrane segment
(Argüello and Kaplan, 1994). DEAC inhibition
appears to be selective for the cation binding site as the modified
enzyme is still able to undergo the E
E
conformational transitions and displays normal levels of ATP/ADP
binding. The importance of this residue in Na,K-ATPase function is
further evidenced by the findings that (i) substitution of Glu
with a leucine or aspartate residue inhibits
Na,K-ATPase-dependent cell growth in transfected HeLa cells
(Jewell-Motz and Lingrel, 1993; Feng and Lingrel, 1995), (ii)
replacement of the corresponding glutamate residue, Glu
,
in the sarco(endo)plasmic reticulum Ca
-ATPase blocks
Ca
-dependent functions of the enzyme (Clarke et
al., 1989), and (iii) the glutamate residue is highly conserved
among members of the P-type family of ATPases
(Argüello and Kaplan, 1994). More recently,
expression of Glu
mutations in transfected mammalian
cells have shown that this residue, although not required for activity,
has an important role in cation interactions or selectivity (Vilsen,
1995; Feng and Lingrel, 1995).
To further examine the role of the
glutamate in Na,K-ATPase function, site-directed mutagenesis was used
to replace Glu (the equivalent residue in the rat
Na,K-ATPase) with an alanine, aspartate, or lysine residue, and the
mutant Na,K-ATPase polypeptides were expressed in Spodoptera
frugiperda (Sf9) cells using the baculovirus expression system.
All mutant sodium pumps are catalytically active, but display altered
affinities for Na
, K
, and ATP. In
addition, the Glu
Ala mutant displays an increased
affinity for Cs
, a congener of K
in
the reaction cycle. In the absence of potassium ions, the Glu
Ala mutant mediates a Na
-dependent ATPase
activity and a cellular Na
influx suggesting that
Na
is acting at the extracellular binding sites as a
surrogate for K
. An identical
Na
-dependent ATPase activity was recently reported for
the Glu
Ala mutant of the
subunit in
transfected COS cells (Vilsen, 1995), but not in HeLa cells (Feng and
Lingrel, 1995). The cause of this difference is unclear. In addition,
trypsin digestion of the Glu
Ala mutant
demonstrates that Na
alone is sufficient to stabilize
the E
(K) conformational state of the enzyme. All
together, these results suggest that in the Glu
Ala mutant, the Na
is acting at extracellular binding
sites as a replacement for K
in the catalytic cycle.
Moreover, these results suggest that Glu
is an important
coordinate of cation selectivity and activation, and may represent a
link between the extramembraneous ATP binding domain and the cation
transport regions of the Na,K-ATPase.
Na,K-ATPase activity was assayed
through determination of the initial rate of release of P
from [
-
P]ATP as
described previously (Beaugé and Campos, 1983).
Samples of 50-100 µg of total protein were assayed in a final
volume of 0.25 ml. Following a 30-min incubation period at 37 °C,
the reaction was terminated by the addition of 5.5% trichloroacetic
acid (final) and incubating on ice for 5 min. Released
P
-P
was then converted to
phosphomolybdate and extracted with isobutyl alcohol. Finally, 0.15 ml
of the organic phase were taken and radioactivity measured by liquid
scintillation counting. The ATP hydrolyzed never exceeded 15% of the
total ATP present in the sample and hydrolysis was linear over the
incubation time. Na,K-ATPase activity was determined as the difference
in ATP hydrolysis in the absence and presence of 1 mM ouabain.
For maximal Na,K-ATPase activity, the medium contained 120 mM NaCl, 30 mM KCl, 3 mM MgCl
, 3 mM ATP, 0.2 mM EGTA, 2.5 mM sodium azide, 30 mM Tris-HCl, pH 7.4, ± 1 mM ouabain. For the cations,
incubation media were the same as above except that for Na
dependence, Na
concentration was varied from 2.5
to 122.5 mM. For K
and Cs
stimulations, the respective ion concentrations were varied from
0 to 30 mM. In medium depleted of K
, flame
photometry demonstrated that the concentration of free K
was approximately 30 µM. Choline chloride was added
so that the final concentration of Na
or K
plus choline totaled 150 mM. The ATP dependence was
determined under saturating concentrations of all cations (120 mM Na
, 30 mM K
, and 3
mM Mg
). To determine the effect of different
ouabain concentrations on Na,K-ATPase activity, samples were
preincubated with the indicated concentrations of ouabain for 30 min at
37 °C in the reaction medium; the reaction was started by the
addition of ATP.
For Na transport assays, infected Sf9
cells were harvested at 48 or 72 h post-infection. Cells were
centrifuged (2,000
g) and resuspended at a density of
1
10
cells/ml in preincubation medium (150 mM NaCl, 100 µM bumetanide, 25 mM HEPES, and
2.5 mM MgCl
, pH 7.4). After a 30-min incubation on
ice, cells were centrifuged (2,000
g) and resuspended
at a density of 2
10
cells/ml in flux medium (50
mM NaCl, 75 mM choline chloride, 25 mM HEPES/Tris, 100 µM bumetanide, pH 7.4), in the
presence or absence of 1 mM ouabain. Flame photometry
demonstrated that the concentration of free K
in the
flux medium was approximately 30 µM. After an additional
5-min incubation at 37 °C, the reaction was started by adding an
equal volume of flux medium containing
Na (1 µCi/ml)
in the presence or absence of 1 mM ouabain. At prescribed
intervals, 150-µl aliquots (1
10
cells) were
removed and layered on 0.5 ml of silicone oil (Versilube F-50; Harwich
Chemical Corp.) in a 1.5-ml microcentrifuge tube and centrifuged for 30
s. Cell separation occurred in <5 s. The aqueous and oil phases were
removed by aspiration and discarded. The cell pellets were excised and
the radioactivity determined by liquid scintillation. The rates of
ouabain-sensitive sodium-uptake shown were determined at the 2-min time
point.
Figure 1:
Expression of rat Na,K-ATPase mutant
polypeptides in infected Sf9 cells. A, immunoblot analysis of
Sf9 proteins. Recombinant baculoviruses directing the coexpression of
the mutant (Glu
Asp, Glu
Lys, or Glu
Ala) and native
1
subunits were used to infect Sf9 insect cells. After 72 h Sf9 proteins
(20 µg) were separated by SDS-PAGE (7.5% gel) and transferred to
nitrocellulose. The
and
polypeptides were detected with a
polyclonal anti-
antiserum (poly(
A)) that immunoreacts
with both the rat
1 and
1 subunits. B, association
of mutant
and native
subunits coexpressed in Sf9 cells.
Proteins from mutant
infected Sf9 cells, and combined
proteins from cells individually expressing
1 and
1 (
1
+
1) were immunoprecipitated with a monoclonal antibody
(C464) against the
-subunit (IP AB). The precipitated
proteins were separated by SDS-PAGE, transferred to nitrocellulose, and
immunoblotted with the anti-
1 antiserum (IB AB). The
native
1 subunit from kidney membranes (15 µg) is shown as a
standard.
To determine if the
individual polypeptides can assemble with the
1 subunit,
infected Sf9 cells were solubilized in 1% CHAPS and immunoprecipitated
with an
-specific monoclonal antibody. If the mutant
1 and
1 subunits are in a stable detergent-resistant association, then
the
1 subunit should coimmunoprecipitate with the mutant
1
polypeptide. Using an anti-
antiserum, the
subunit can be
identified in the immunoprecipitate. As shown in Fig. 1B, the
mutants and the
1 polypeptide
can form detergent-stable complexes when coexpressed in the cells. In
contrast, when cells singly infected with either an
1 or
1
baculovirus are combined prior to solubilization, the
1
polypeptide is not identified in the immunoprecipitate (Fig. 1B, lane 2). Thus, the site-directed
mutants are expressed at high levels in the Sf9 cells and are
structurally competent as judged by their ability to stably assemble
with the
1 subunit in cells coexpressing both the
and
polypeptides.
To characterize the cation requirements of the
mutant enzymes, ATPase assays were performed in medium free of both
Na and K
, in K
-free
medium with Na
, or in medium containing both
Na
and K
. Interestingly, as shown in Table 1, the Glu
Ala mutant displays a
significant ouabain-sensitive Na
-ATPase activity in
the absence of potassium ions. This Na
-ATPase activity
represents approximately 38% of the maximal activity and is not present
at a sodium concentration of 1.5 mM. No ouabain-sensitive
ATPase activity was observed with K
alone (data not
shown).
Figure 2:
Na uptake in
(Glu
Ala)
1 infected Sf9 cells.
Ouabain-sensitive sodium uptake was measured in Sf9 cells infected for
48 or 72 h, using
Na as tracer. The transport assay was
started with the addition of the tracer and stopped at the times
indicated by centrifuging the cells through a layer of silicone oil.
Bumetanide (100 µM) was present to inhibit the
bumetanide-sensitive sodium uptake. The inset shows rates of
ouabain-sensitive sodium uptake at the 2-min point in Sf9 cells
infected with the
(Glu
Ala)
1 baculovirus
as compared to the wild-type
baculovirus. Each value is the
mean with error bars representing the standard error of
triplicate determinations.
Figure 3:
Proteolysis of the Na,K-ATPase. Membrane
proteins (200 µg) from 1
1 or
1(Glu
Ala)
1 infected cells were digested with trypsin in a
NaCl or salt-free medium, resolved on SDS-PAGE, and transferred to
nitrocellulose. The immunoblot was probed with an anti-
antiserum
(anti-NASE) that recognizes a sequence (
KNPNASEPKHLL
) common to both the 77-
(Na
) and 58-kDa (K
) tryptic
fragments. The position of the 77-kDa tryptic fragment and the 58-kDa
tryptic fragment are shown by arrows. In the controls (lanes 1 and 3), the tryptic cleavage pattern
produced in medium free of Na
and K
is shown.
Figure 4:
K, Na
,
and ATP activation of the
1(Glu
Asp)
1
mutant enzyme. A, K
activation of Glu
Asp mutant. Na,K-ATPase activity of Sf9 membranes
expressing the mutant
(Glu
Asp)
1
(
,
, and
) or wild-type
1
1 (
,
, and &cjs2105;), was determined in a reaction medium
containing: 120 mM NaCl, 3 mM ATP, 3 mM MgCl
, 0.2 mM EGTA, 30 mM Tris-HCl,
pH 7.4, and KCl as indicated, in the absence or presence of 1 mM ouabain. B, Na
activation of Glu
Asp mutant enzyme. Ouabain-sensitive ATPase activity
assays were performed as described in A but with a reaction
medium containing 30 mM KCl and varying concentrations of
Na
from 2.5 to 122.5 mM. In the Na
and K
activation experiments, ionic strength was
kept constant with choline chloride. C, ATP stimulation of the
Glu
Asp mutant enzyme. Ouabain-sensitive ATPase
activity assays were performed in the reaction medium described in A but containing constant Na
and K
(30 mM KCl, 120 mM NaCl) and the indicated ATP
concentrations. All data are expressed as percent of the maximal
Na,K-ATPase activity obtained. Each value is the mean and error
bars represent the standard errors of the mean of at least three
experiments performed in triplicate on samples obtained from different
infections.
Figure 5:
K, Na
,
and ATP activation of the
1(Glu
Lys)
1
mutant enzyme. A, K
activation of Glu
Lys mutant. Na,K-ATPase activity of Sf9 membranes
expressing the mutant
(Glu
Lys)
1
(
,
, and
) or wild-type
1
1 (
,
, and &cjs2105;), was determined as described in the legend to Fig. 4. B, Na
activation of
Glu
Lys mutant enzyme. C, ATP stimulation
of the Glu
Lys mutant enzyme. All data are
expressed as percent of the maximal Na,K-ATPase activity obtained. Each
value is the mean and error bars represent the standard errors
of the mean of at least three experiments performed in triplicate on
samples obtained from different infections.
Figure 6:
K, Na
,
and ATP activation of the
1(Glu
Ala)
1
mutant enzyme. A, K
activation of Glu
Ala mutant enzyme. Na,K-ATPase activity of Sf9 membranes
expressing the mutant
(Glu
Ala)
1
(
,
, and
) or wild-type
1
1 (
,
, and &cjs2105;), was determined as described in the legend to Fig. 4. B, Na
activation of
Glu
Ala mutant enzyme. C, ATP stimulation
of the Glu
Ala mutant enzyme. All data are
expressed as percent of the maximal Na,K-ATPase activity obtained. Each
value is the mean and error bars represent the standard errors
of the mean of at least three experiments performed in triplicate on
samples obtained from different infections.
To determine the requirement of the
Glu
Asp, Glu
Lys, and
Glu
Ala mutants for sodium, Na,K-ATPase activity
was measured at varying concentrations of Na
(2.5-122.5 mM) with K
fixed at 30
mM. In all cases the mutant Na,K-pumps display a shift in the
apparent affinity for Na
relative to the wild-type
enzyme (Panel B, Fig. 4, Fig. 5, and Fig. 6). As listed in Table 2, the conservative
substitution of glutamate with an aspartate residue resulted in
approximately a 1.5-fold reduction in Na
affinity
(compare the K
value of 23.2 mM with
16.4 mM for the wild-type Na,K-ATPase). Whereas, replacement
with an alanine resulted in a lower apparent affinity for
Na
, corresponding to a 2-fold reduction relative to
the
1
1 enzyme (K
value of 33.5 mM for the Glu
Ala mutant). Surprisingly, in
contrast to the Glu
Ala and Glu
Asp mutants, the replacement of Glu
with the
positively charged lysine residue demonstrates a modest, 1.5-fold
increase in the K
value for Na
activation (K
value of 9.5 mM).
As the lysine substitution exhibits opposing effects on cation affinity
and an undiminished level of Na,K-ATPase activity (see Table 1),
a direct role of Glu
in charge neutralization and cation
binding is unlikely.
To characterize the kinetics at the low
affinity ATP site, dose-response curves of Na,K-ATPase activity at
millimolar concentrations of ATP were performed. The ATP dependence of
the baculovirus-induced mutants is presented in Panel C of Fig. 4, Fig. 5, and Fig. 6and the K
values are described in Table 2. As shown,
all three mutant pumps display a 3-9-fold increase in apparent
affinity for ATP over the wild-type enzyme (K
values of 0.11, 0.11, and 0.05 for the Glu
Asp, Glu
Ala, and Glu
Lys
mutants, respectively, compared to 0.46 for the wild-type enzyme).
Figure 7:
Cs activation of
1(Glu
Ala)
1 and native
1
1
enzymes. Na
,Cs
-ATPase activity of Sf9
membranes expressing the mutant
(Glu
Ala)
1 (
) or wild-type
1
1 (
) was determined in
a reaction medium containing: 120 mM NaCl, 3 mM ATP,
3 mM MgCl
, 0.2 mM EGTA, 30 mM Tris-HCl, pH 7.4, and CsCl as indicated, in the absence or
presence of 1 mM ouabain. Apparent cesium affinity was
calculated as a percent of the maximal Na,Cs-ATPase activity obtained
after subtraction of the Na-ATPase activity. Each value is the mean and error bars represent the standard errors of the mean of at
least three experiments performed in triplicate on samples obtained
from different infections. Total ionic strength was kept constant with
choline chloride.
Previous chemical modification studies using DEAC have
implicated a glutamate residue of the Na,K-ATPase subunit in
cation binding (Argüello and Kaplan, 1994). In the
present study, this glutamate residue, which in the rat enzyme
corresponds to Glu
, was replaced with an aspartate,
alanine, or lysine residue and the Na,K-ATPase mutants were coexpressed
with the
1 subunit in Sf9 cells and characterized. All mutants are
able to support ouabain-sensitive ATPase activity at levels comparable
to that of the wild-type
1
1 enzyme. In addition, studies
using transfected mammalian cells demonstrate that the Glu
Ala and Glu
Gln mutants have maximal
turnover numbers similar to the wild-type enzyme (Vilsen, 1995; Feng
and Lingrel, 1995). Analysis of the kinetic properties of the mutant
Na,K-ATPases, however, revealed altered dependences for
Na
, K
, and ATP as compared to the
wild-type Na,K-ATPase. In the case of K
, all mutants
display a decrease in the apparent affinity for the cation, while for
Na
, the affinity is reduced in the Glu
Ala and Glu
Asp substitutions and,
unexpectedly, is increased slightly in the Glu
Lys
mutant. A similar decrease in the relative Na
affinity
was observed for the
(Glu
Ala)
1 enzyme
in transfected COS and HeLa cells (Vilsen, 1995; Feng and Lingrel,
1995). All three mutants exhibit a significantly higher affinity for
ATP compared to the wild-type enzyme, while sensitivity to the
cardiotonic inhibitor, ouabain, remains relatively unaltered.
The
observation that replacement of intramembrane Glu with
the positively charged lysine residue does not inactivate Na,K-ATPase
function nor exhibit a greater effect on cation affinities, argues
against a primary role for Glu
as a ligating residue
within the cation binding domain. Rather, it seems likely that the
glutamate residue participates indirectly in transport function by
contributing to the overall structural integrity of the cation binding
domain by possibly allowing the proper positioning of the ligating
residues within the binding pocket. Hence, amino acid substitutions at
position 781 that modify cation dependence are predicted to alter the
proper active site architecture required for high-affinity binding.
Furthermore, the observation that mutations of Glu
affect
both Na
and K
affinities is
consistent with the notion that the residues involved in Na
binding and transport are also involved in binding and transport
of K
. This is also supported by the finding that
K
, and to a lesser extent Na
, can
protect against modification by DEAC (Argüello and
Kaplan, 1991). In addition, the ability of all three mutants to stably
associate with the
subunit to form active enzymes, along with the
unmodified ouabain sensitivities, demonstrates that mutation of
Glu
does not introduce gross conformational changes in
the enzyme. Rather, the effect of the Glu
mutations
appears to be localized to the cation binding domain and, secondarily,
to the catalytic site.
Site-specific mutagenesis was recently used
by Vilsen(1995) to replace Glu of the rat Na,K-ATPase
subunit with an alanine residue. When expressed in COS cells, the
Glu
Ala mutant was shown to exhibit a significant
K
-independent Na
-ATPase activity,
although the exact mechanism underlying the Na
-ATPase
was not determined. A similar mutation in the sheep
1 subunit,
however, did not exhibit a detectable K
-independent
Na
-ATPase activity (Feng and Lingrel, 1995). In the
present study, using the rat
1 subunit, we report a significant
ouabain-sensitive Na
-ATPase activity in Sf9 cells
expressing the
(Glu
Ala)
1 isozyme and
provide evidence for a Na
/Na
exchange
mechanism, with Na
acting as a surrogate for
K
, to account for this aberrant activity. This
conclusion is based on the finding that (i) Na
in the
absence of K
stimulates the ATPase activity of the
Na,K-ATPase mutant, (ii) in flux assays, the
(Glu
Ala)
1 mutant can mediate the inward transport of
extracellular Na
, and (iii) trypsin digestion of the
(Glu
Ala)
1 mutant in Na
medium produces a peptide fragment (M
= 58,000) normally associated with the E
(K) conformation of the enzyme. These results
preclude both the ADP-sensitive form of
Na
/Na
exchange, which is not
accompanied by appreciable hydrolysis of ATP, and uncoupled
Na
efflux in which only intracellular sodium is bound
and transported (reviewed in Glynn, 1985).
The elevated
Na influx associated with the Glu
Ala substitution can be readily explained by a decreased capacity of
the enzyme to discriminate between Na
and K
at the extracellular loading sites. As a result, extracellular
Na
substitutes for K
, promoting its
uptake and enzyme turnover. A rearrangement of the ligating residues
within the cation binding pocket that alter the constants for
K
and Na
binding could also account
for the decreased capacity of the Glu
Ala mutant
enzyme to discriminate among alkali metal ions at the outer surface.
Consistent with a reduction in cation selectivity is the finding that
the affinity for Cs
, a weak congener of K
in enzyme activation, is increased in the Glu
Ala mutant as compared to the wild-type enzyme. This
increase in Cs
affinity gives the mutant enzyme a
nearly identical affinity for Cs
and
K
. Interestingly, this effect appears to be limited to
the extracellular loading sites, as K
alone cannot
substitute for Na
in mutant enzyme activation. This
indicates a greater cation selective capacity at the cytoplasmic
surface, and is consistent with the inability of cytoplasmic
K
to substitute for Na
in the
reaction cycle of the wild-type enzyme (Dunham and Senyk, 1977).
During the reaction cycle, binding information is transmitted
between the ATP domain in the major cytoplasmic loop and the cation
sites. Communication between the two domains is critical for
coordination of cation transport. For example, both ATP and P have been shown to stimulate deocclusion of K
,
while binding of Na
at the cytoplasmic activation site
is required for the phosphorylation by ATP bound at the catalytic
center. On the basis of the potential role of intramembrane Glu
in cation binding and its proximity to the cytoplasmic ATP
binding domain, it has been suggested that this residue may reside
within a segment that actively links the two ligand binding domains
(Argüello and Kaplan, 1994; Lutsenko et
al., 1995). Consistent with this theory, we show that replacement
of intramembrane Glu
by an aspartate, lysine, or alanine
residue results in an increase in the ATP affinity. Interestingly,
substitution of Glu
in the forth transmembrane segment
has also been shown to modify the affinities for Na
,
K
, and ATP (Vilsen, 1993). By analogy, this may also
indicate a role for the fourth transmembrane segment in the
transmission of information between the cation and ATP binding domains.
Thus, ligand-induced conformational changes in one domain appear to be
transmitted to the corresponding domain through the adjacent
transmembrane segment. It should be noted that the ATP affinities
reported here are for the low-affinity ATP site, as accurate
calculation of the ATP dependence at the high-affinity site is not
possible in our enzyme preparations.
Interestingly, our observations
using infected Sf9 insect cells are similar to studies using
site-directed mutagenesis and expression in mammalian cells (Vilsen,
1995; Feng and Lingrel, 1995). Substitutions of this glutamate in all
three studies demonstrate a modified affinity for Na and an unaltered sensitivity to ouabain. Moreover, with the
exception of the Glu
Gln mutant expressed in HeLa
cells (Feng and Lingrel, 1995), all glutamate mutants display a
decreased apparent affinity for K
. However, there are
significant differences between the studies. For example, (i) while
Glu
mutants expressed in COS or Sf9 insect cells exhibit
an increase in the apparent affinity for ATP at the low affinity site,
no change in ATP activation is observed for the equivalent mutants
expressed in HeLa cells, and (ii) the Glu
Ala mutant when
expressed in HeLa cells does not exhibit a K
independent, Na
-ATPase activity (Feng and
Lingrel, 1995). It is possible that the modified sheep
1 subunit
has different properties than the rodent subunit. This possibility will
require further investigation. Finally, an advantage of the baculovirus
expression system is that the mutant Na,K-ATPases do not have to confer
ouabain resistance to be analyzed. Thus, although the Glu
Asp substitution did not alter ouabain sensitivity in HeLa
cells (Feng and Lingrel, 1995), it is catalytically competent.
In
conclusion, Glu of the Na,K-ATPase
subunit is a
critical coordinate of cation selectivity and activation, however, this
residue does not appear to have a direct role in the binding and
occlusion of cations. Similar conclusions have been obtained studying
mutations in Glu
using transfected mammalian cells
(Vilsen, 1995; Feng and Lingrel, 1995). Additionally, the effect of
Glu
substitutions on the apparent ATP affinity suggests a
role for the fifth membrane segment in the transmission of binding
information between the ATP binding domain and the cation transport
region of the
subunit.