(Received for publication, September 19, 1996, and in revised form, December 3, 1996)
From the Department of Medicine, McGill University,
Montreal, Canada and the
Department of Pharmacology and Cell
Biophysics, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267
During kinetic studies of mutant rat
Na,K-ATPases, we identified a spontaneous mutation in the first
cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop)
which results in a functional enzyme with distinct Na,K-ATPase
kinetics. The mutant cDNA contained a single G950 to A
substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and 1 cDNAs were transfected into
HeLa cells and their kinetic behavior was compared. Transport studies
carried out under physiological conditions with intact cells indicate
that the E233K mutant and
1 have similar apparent affinities for
cytoplasmic Na+ and extracellular K+. In
contrast, distinct kinetic properties are observed when ATPase activity
is assayed under conditions (low ATP concentration) in which the
K+ deocclusion pathway of the reaction is rate-limiting. At
1 µM ATP K+ inhibits Na+-ATPase
of
1, but activates Na+-ATPase of E233K. This
distinctive behavior of E233K is due to its faster rate of formation of
dephosphoenzyme (E1) from
K+-occluded enzyme (E2(K)), as well
as 6-fold higher affinity for ATP at the low affinity ATP binding site.
A lower ratio of Vmax to maximal level of
phosphoenzyme indicates that E233K has a lower catalytic turnover than
1. These distinct kinetics of E233K suggest a shift in its
E1/E2 conformational
equilibrium toward E1. Furthermore, the
importance of the H2-H3 loop in coupling conformational changes to ATP
hydrolysis is underscored by a marked (2 orders of magnitude) reduction
in vanadate sensitivity effected by this Glu233
Lys
mutation.
The Na,K-ATPase is an integral membrane protein complex that
catalyzes the exchange of three cytoplasmic sodium ions for two extracellular potassium ions coupled to the hydrolysis of one molecule
of ATP. It is a heterodimeric protein comprising a catalytic subunit and a smaller heavily glycosylated
subunit (for reviews, see Refs. 1 and 2). Although an additional protein,
, has been found
associated with the
and
subunits (3), its function is unknown.
The Na,K-ATPase is a member of the family of P-type ATPases, which are
characteristically phosphorylated by ATP, and the phosphoenzyme
intermediate undergoes rapid turnover during the reaction cycle. The
phospho- as well as dephosphoenzymes exist in at least two distinct
conformational states.
During transport both sodium and potassium ions are occluded within the Na,K-ATPase (for review, see Ref. 4). The nature of the cation occlusion site(s) and the structures involved in gating of these sites to both the cytoplasmic and extracellular milieu are unknown. Approaches using chemical labeling (5, 6) and site-directed mutagenesis (7-12) have identified a number of functionally important carboxyl-containing amino acid residues in transmembrane regions H4, H5, H6, H8, and H9. Point mutations of putative cation binding amino acid residues that resulted in the expression of functional enzymes were characterized by an altered affinity for ATP, K+, and/or Na+.
The prediction that the highly charged amino terminus had a role as a
cation gate (13) now seems unlikely. Instead, interaction of this
region with other region(s) of the protein affects the K+ deocclusion pathway of the reaction, probably via
alteration in E2/E1
conformational equilibrium. Our recent study (14) demonstrated that the
highly charged sequence comprising residues 24-32 of the amino
terminus of the
subunit is important in modulating the
K+ deocclusion pathway of the reaction cycle. Furthermore,
this modulation is not due to the 24-32 nanopeptide per se,
but rather to its interaction(s) with other isoform-specific
region(s).
This paper describes the characterization of a spontaneous,
posttransfection mutation in the H2-H3 cytoplasmic loop of the 1
isoform of the Na,K-ATPase. It differs from the wild type enzyme by
having a lysine substituted for glutamic acid in position 233, and is
designated E233K. The mutant enzyme is functional and its apparent
affinities for sodium and potassium are unaltered. However, it exhibits
a decrease in catalytic turnover, an increase in apparent affinity for
ATP, and an increase in the rate of conversion of E2(K) to E1.
Two sets of synthetic oligonucleotide primers were prepared
for polymerase chain reaction amplification of the 5 and 3
halves of
a putative spontaneous mutant in the NH2-terminal chimeric mutant of rat
1 cDNA (
1(1-14
2) cDNA) that had been
incorporated into the HeLa genomic DNA in a pRc/CMV (Invitrogen)
construct. The 5
primer set included a 32-mer complementary to the
sequence of the sense strand of the T7 promoter of pRc/CMV and a 33-mer complementary to the antisense sequence between bases 1828-1860 of the
rat
1 cDNA. The 3
primer set included a 28-mer complementary to
the sense strand of rat
1 between bases 1684 and 1711 and a 32-mer
complementary to an antisense sequence in the SP6 promoter of pRc/CMV
at the 3
end of the rat cDNA. The 5
and 3
halves of the
1(1-14
2) cDNA were amplified from 1 µg of HeLa genomic DNA
utilizing these primer sets (10 pmol of each primer) and the TaqPlus polymerase reaction kit (Stratagene). Aliquots of
the amplified DNA, with or without digestion with HindIII
and BamHI, were analyzed on 0.8% agarose gels and compared
to products obtained by amplification of 1 ng of purified pRc/CMV-rat
1 plasmid DNA. An aliquot of each reaction mixture was then utilized
as template for additional amplification with the same primer sets.
Amplified DNA from the 5
and 3
halves of rat
1(1-14
2) was
digested with HindIII and BamHI, gel-purified,
and ligated into M13mp18/19 that had been digested with
HindIII and BamHI. Competent DH5
F
Escherichia coli were transformed with each ligation mixture
and plated, and the resultant plaques were picked and amplified in
liquid cultures. Aliquots of heat-treated supernatants of the M13
cultures were adjusted to 25 mM EDTA, 5% glycerol, and
0.4% SDS and electrophoresed on 0.7% agarose gels to identify M13
clones containing the
1 DNA inserts. Aliquots of the heat-treated
supernatants of 37
5
-containing clones and of 28
3
-containing
clones were pooled and used to infect DH5
F
for the preparation of
single-stranded M13mp18-5
and M13mp19-3
DNA. The 5
and 3
halves of the rat
1(1-14
2) DNA from the pooled clones were
completely sequenced using the Sanger dideoxy method (15), Sequenase
version 2.0 (Amersham), and synthetic oligonucleotides as primers.
After detection of a single base substitution at base 950 in the pooled
M13mp18-5 DNA, individual clones of M13mp18-5
were sequenced
to identify a clone that contained the G950
A
nucleotide substitution and no other base changes between SalI(875) and BamHI(1780). Double-stranded DNA
was then prepared from the identified M13mp18-5
clone, and a
SalI(875)-BamHI(1870) fragment containing the
G950
A substitution was isolated and ligated into both
the rat
1 wild type and the rat
1(1-14
2) cDNAs in a
modified pIBI30 shuttle vector, in place of the wild type
SalI-BamHI fragments. The presence of the mutant
sequences and the termini of the exchanged cassettes of these shuttle
vector constructs were verified by DNA sequencing. The full-length
1
cDNAs were then isolated by HindIII digestion, gel-purified, and ligated into the HindIII site of pRc/CMV,
and their orientations determined by restriction analyses. Twenty µg
of QIAGEN-purified expression plasmid DNA was used to transfect HeLa
cells with CaPO4, as described by Chen and Okayama (16). HeLa cells expressing the mutant rat
1 proteins were selected by
their ability to grow in medium containing 1 µM ouabain,
and clones were isolated and amplified as described previously
(17).
Membranes were isolated, and assays of Na,K-ATPase, Na-ATPase, and ouabain-sensitive K+(Rb+) influx were carried out as described previously (18). Assays of K+-occluded enzyme and the formation of E1 from K+-occluded enzyme are also described elsewhere (14).
During the course of studies with a rat 1/
2
NH2-terminal chimeric mutant cDNAs transfected into
HeLa cells, we observed that the Na,K-ATPase of one clone had
functional properties distinct from rat
1. The observation that the
enzymic properties of several other clones selected from the same
chimeric cDNA (rat
1(1-14
2)) transfection were
indistinguishable from those of
1 suggested that a spontaneous,
posttranfection mutation had occurred within the coding region of the
rat
1 cDNA. As described under "Experimental Procedures,"
the alteration was identified as the substitution of lysine for
glutamate at position 233 in the cytoplasmic region between
transmembrane helices H2 and H3. The functional difference between this
mutant (E233K) and the rat
1 enzyme was apparent when pump-mediated
ATP hydrolysis was measured at micromolar ATP, under which condition
ouabain-sensitive Na+-dependent ATPase activity
of the
1 isoform is inhibited by K+ (19). Accordingly,
although K+ stimulates the dephosphorylation step of the
reaction (E2P + K
E2(K) + Pi) and becomes occluded
within the pump protein, its deocclusion is extremely slow. Stimulation
of deocclusion is effected by ATP binding with low affinity
(E2(K) + ATP
ATP·E1 + K+; Ref. 19). As a consequence, K+ is an
inhibitor of the overall reaction at low ATP, and an activator at high
ATP concentration. In fact, as shown previously (14, 18, 20) and
described below, at micromolar ATP the response of Na-ATPase to
K+ is a sensitive means of characterizing isoform- or
mutant-specific differences in the K+ deocclusion pathway
of the reaction.
As shown in Fig.
1, the Na-ATPase activity of membranes isolated from rat
1-transfected cells is inhibited by the addition of 0.1-5
mM KCl, whereas the activity of E233K is markedly
stimulated. At 1 mM KCl, the Na-ATPase activities of the
mutant and
1 enzymes are 200% and 50%, respectively, of their
control activities measured in the absence of KCl.
The change in K+ sensitivity at low ATP concentration caused by the mutation is suggestive of a change in rate of a reaction step following dephosphorylation of the K+-sensitive form of phosphoenzyme commonly referred to as E2P in the Albers-Post model. According to a branched pathway of K+ deocclusion (Scheme I), it is assumed that ATP can bind with either (i) low affinity to the K+-occluded form of the enzyme, E2(K), at a step preceding the release of potassium and the formation of ATP·E1 (pathway a), or (ii) with high affinity at a step following the slow release of potassium from E2(K) (pathway b). Accordingly, potassium inhibition is dependent upon the apparent affinity of the enzyme for ATP at its low affinity site and/or the rate of release of K+ from E2(K). Analysis of kinetic parameters of pathways a and b is described below.
[View Larger Version of this Image (9K GIF file)]Scheme I.
Kinetic Analysis of the Reaction Modeled According to ATP Binding
to Low and High Affinity Sites
The kinetic parameters
KATP, the apparent affinity for ATP, and
Vmax for pathways a and b
were obtained from measurements of Na,K-ATPase activity
versus ATP concentration varied from 0.1 to 500 µM. Reciprocal plots of the data are shown in Fig.
2, and the kinetic parameters are given in Table
I. In the case of the
1 enzyme, the data points can
be fitted to a two-component system, each linear in the ranges of 1-8
µM and 25-500 µM ATP. The constants for
the apparent affinities for ATP at low and high affinity sites,
designated K
L and K
H,
respectively, are 331 ± 44 µM and 5.44 ± 1.9 µM for
1; Vmax of the high
affinity component, VH, is 0.684 ± 0.20 µmol/(mg × h) and represents 4.2% of the activity of the
Vmax of the low affinity component,
VL (16.2 ± 3.0 µmol/(mg × h)).
These values for
1 are similar to those reported previously (14). In
contrast, the reciprocal plot of the E233K mutant is a straight line
within the entire range of 1-500 µM, resulting in an
apparent affinity of 56.3 ± 14 µM and a
Vmax (V
L) of 9.90 ± 1.1 µmol/(mg × h). Although these values are taken from
representative experiments and there is some variability in
Vmax values, it was observed that the activity of membranes prepared from E233K-transfected cells is generally lower
than that of the rat
1-transfected cells.
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An additional component of activity was observed in the range of
0.1-0.5 µM ATP (data not shown), which represented
1-2% of VL. The constant for its apparent
affinity for ATP, K
VH, was similar for both
enzymes (0.19 ± 0.09 µM for
1 and 0.21 ± 0.01 µM for E233K). As suggested previously (14), this
very high affinity component may represent an alternate minor pathway
of ATP hydrolysis, involving K+ release directly from
E2(K), i.e. E2(K)
E2 + K+ followed by the conversion
of E2 to E1. The nature
of this minor component was not investigated further.
The marked difference in
effect of K+ on Na-ATPase of E233K and 1 as depicted in
Fig. 1 can be attributed to the higher affinity of E233K for ATP (lower
K
L) and/or higher rate of K+
deocclusion from E2(K) via pathway b. Since the
low specific activity of the Na,K-ATPase expressed in HeLa cell
membranes prevents the direct measurement of
E2(K) and, therefore, the K+
deocclusion pathway of the reaction, an indirect method was used as
described recently (14). In this assay, formation of the K+-occluded enzyme is reflected by the decrease in
phosphoenzyme (E32P) formed by phosphorylation
with [
-32P]ATP following equilibration of the enzyme
at room temperature (i) without and (ii) with K+. The
reduction in E32P resulting from preincubation
with K+ (
E32P) is a measure of
the amount of E2(K).
As shown in Fig. 3, and in agreement with our previous
studies (14), the maximum formation of E2(K) with the 1
isoform is achieved after preincubation of the enzyme with 1 mM KCl, whereas with the
1E233K mutant maximum
E2(K) requires at least 4 mM KCl. Using a simple model,
Bmax[S]/(K0.5 + [S]),
to describe the binding as in earlier studies of K+
occlusion in the kidney enzyme (21), the values of
K0.5 obtained are 1.0 ± 0.5 and 0.12 ± 0.03 mM for the E233K and
1 enzymes, respectively
(mean ± S.D. of three experiments). Maximum levels of
E2(K), expressed as a percentage of
phosphoenzyme formed without K+ preincubation (enzyme
preincubated without KCl minus the KCl base line) were 93% ± 10% and
97% ± 5% for E233K and
1, respectively.
The shift in the equilibrium E1 + K+
E2(K) toward E1 can be accounted
for by either a slower rate of occlusion or a faster rate of
deocclusion. Although the rate of formation of
E1 from E2(K) is not a
direct measure of K+ release from
E2(K), it is characterized by single exponential kinetics (14) and, as a first approximation, is an estimate of the rate
of deocclusion through pathway b.
In the representative experiments shown in Fig. 4, the
rate of E1 formation from
E2(K) was determined as follows: 1 and E233K enzymes were first equilibrated with 8 mM KCl to form
E2(K).
E32P, the
difference ((E32P formed following preincubation
in the absence of K+) minus (E32P
formed following preincubation with 8 mM K+))
was taken to represent 100% K+-occluded enzyme (Fig. 3).
Deocclusion was measured by following the rate of increase in
E32P at 10 °C, a temperature at which
deocclusion is sufficiently slow to permit manual assays. We showed
previously that (i) maximal E32P formed from
E1 was similar at 10 °C and 0 °C, and
remained constant over the period used to follow deocclusion at
10 °C, and that
E32P remained constant for
up to 30 s at 0 °C. Therefore, the time course of increase in
E32P at 10 °C reflects the rate of the
rate-limiting step in the sequence E2(K)
E1K
E1 at that
temperature.
As can be seen in Fig. 4, the rate of formation of
E1P from E2(K) is
significantly faster for the E233K mutant compared to the 1 enzyme;
rate constants are (s
1) 0.09 ± 0.005 and 0.02 ± 0.004 for E233K and
1, respectively. In other control experiments
carried out with
1 and E233K, deocclusion was allowed to proceed for
10 s at 10 °C in ATP-free Na+ medium, after which
the E1 formed was measured by rapid dilution and
conversion to E1P at 0 °C (5-s
phosphorylation at 0 °C). The rate constants estimated from the 10-s
decrease in E2(K) were similar to those obtained
with 1 µM [
-32P]ATP present during
deocclusion. This result indicates that the presence of 1 µM ATP during deocclusion did not significantly affect
the rate of deocclusion under these conditions.
Since
oligomycin stabilizes Na+ occlusion in
E1 (22, 23) and traps the enzyme in the
E1P state, the extent to which EP increases in the presence of oligomycin is a measure of the
steady-state distribution of the dephospho- and phosphoenzyme during
steady-state catalysis. As shown in Table II (see
legend), phosphoenzyme measured at 0 °C in the presence of
oligomycin is increased 2.4-fold in the 1 enzyme and only 1.4-fold
in E233K. Estimates of turnover, calculated as the ratio of
Vmax to EP measured in the presence of oligomycin and Na+, indicate that the E233K mutation
results in a 40% reduction in turnover at 37 °C.
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As described by Forbush and Klodos (24),
the pH-dependence of Na,K-ATPase is limited partly by the rate of
K+ deocclusion at acidic pH, by the rate of the
E1P E2P transition at
neutral pH, and by phosphorylation at pH above pH 8.0. The pH
dependence profiles of the E233K mutant and
1 enzymes shown in Fig.
5 indicate that as pH is lowered from the optimum at
pH
8.0 (room temperature) to pH 6.2, the decrease in activity
was less for E233K (20% decrease) than for
1 (50% decrease). In
contrast, the activity profile on the alkaline side of the optimum was
similar for both enzyme forms.
Apparent Cation Affinities
To determine if the substitution
of glutamate 233 with a lysine residue alters the apparent affinity of
the mutant enzyme for cations, we compared the transport behavior and
Na,K-ATPase kinetics of HeLa cells transfected with the E233K and 1
enzymes as described previously (18). Briefly, Rb86 influx
sensitive to high (10 mM) ouabain was measured in medium containing either various concentrations of sodium in the presence of
monensin to control and maintain intracellular sodium concentration and
saturating extracellular KCl concentration, or various concentrations of extracellular KCl, in the presence of monensin and 10 mM
NaCl as described previously (25). We found that the apparent
affinities for cytoplasmic Na+ (Nacyt) of the
E233K mutant and
1 enzymes are indistinguishable. In five separate
experiments with the data fitted to a three-site cooperative model, the
apparent affinities for Nacyt (K0.5)
are (mM) 20.9 ± 2.0 for E233K and 20.1 ± 2.7 for
1. Since intracellular Na+ could not be decreased
below
15 mM with monensin present (25), we also compared
the apparent affinities for Na+ in assays of Na,K-ATPase at
1 mM ATP and constant (10 mM) K+.
No difference in apparent affinities for Na+ could be
detected. K0.5 values (mM) were
2.49 ± 0.041 for E233K and 2.75 ± 0.16 for
1 (average of
three experiments).
As shown earlier by Eisner and Richards (26) in studies of Na,K-ATPase
of red cell ghosts, an increase in Kext results in an
increase in KATP, and increasing the
concentration of ATP leads to an increase in
K
Kext. Although there is no difference in
apparent affinities for either Kext or Nacyt
between
1 and E233K pumps under the conditions of the flux assays
carried out under physiological conditions of ATP concentration
(K0.5 values (mM) were 0.38 ± 0.04 for E233K and 0.37 ± 0.02 for
1 (average of five
experiments), it is predicted that as the concentration of ATP is
decreased below saturation, the apparent affinity for K+
for both enzymes would increase, and that the increase would be less
for E233K than for
1. This prediction held true as shown by the
analysis of K+ activation of Na,K-ATPase carried out at 1.0 and 0.1 mM ATP. At 1 mM ATP, with the data
fitted to a two-site cooperative model, K0.5
values (mM) for K+ are 1.11 ± 0.034 and
1.76 ± 0.12 for
1 and E233K, respectively; at 0.1 mM ATP, the K0.5 values
(mM) are 0.410 ± 0.026 and 1.31 ± 0.074 for
1 and E233K, respectively.
Vanadate interacts with the
phosphorylation site in the catalytic H4-H5 domain and inhibits P-type
ATPase. Studies with the yeast H+-ATPase have provided
evidence that structural alterations in the H2-H3 cytoplasmic loop
result in changes in vanadate sensitivity (27, 28). To assess
interaction of Glu233 with the catalytic phosphorylation
site and/or the role of the region of this mutation on conformational
coupling in the Na,K-ATPase, the effect of vanadate was tested. The
results shown in Fig. 6 indicate that the
Glu233 Lys mutation causes a dramatic decrease in
sensitivity to vanadate, with Ki values for
1 and
E233K being 8 and 2000 µM, respectively.
Although the insensitivity to vanadate conferred by the
Glu233 Lys mutation is consistent with the conclusion
that this change reflects an alteration in the steady-state
E1/E2 distribution as
suggested for the yeast H+-ATPase (27, 28), other
interpretations are plausible. The effect of this mutation is
reminiscent of the decrease in vanadate sensitivity observed in the
absence compared to presence of K+ (29), which raises the
possibility of a direct alteration in enzyme-K+
interaction. Another possibility is that binding of phosphate is
altered. The former is less likely according to the argument that
changes in K
K effected by mutations in
cytoplasmic domains are probably secondary to changes in
K
ATP as discussed below.
The experiments described in this report provide evidence for a
functional role of the H2-H3 cytoplasmic loop in conformation coupling
in the Na,K-ATPase. The changes effected by mutation of glutamate 233 to lysine are consistent with the conclusion that this substitution
alters the equilibrium between the major conformational states of the
dephospho- and phospho- forms during steady-state catalysis. An
increase in the steady-state distribution of E1
and E2 in favor of E1
effected by the Glu233 Lys mutation is apparent as (i)
an
4-fold increase in the rate constant for
E1 formation from E2(K),
accounting for the higher Km for K+
occlusion of E233K compared to
1, (ii) a 6-fold increase in the
apparent affinity for ATP at the step E2(K) + ATP
ATP·E1 + K+ when the
overall Na,K-ATPase reaction is studied at 37 °C, and (iii) a lesser
decrease in activity as pH is lowered to pH 6.8 under which condition
deocclusion of K+ from E2(K) becomes
the main rate-limiting reaction in the wild type enzyme (24). The
results are also consistent with a shift in the
E1P
E2P equilibrium
toward E1P caused by the Glu233
Lys mutation. The evidence is two-fold: the smaller increase in
E32P effected by oligomycin and lower turnover
of E233K compared to
1, at least at physiological pH under which
condition the E1
E2P
transition is partly rate-limiting (24).
Glu233 is in a region previously identified as having a
role in the E1P E2P
conformational transition (for review, see Ref. 30). When Na,K-ATPase
in the E1 conformation is exposed to cleavage at
either Leu266 or Arg262 in the H2-H3
cytoplasmic loop, as described by Jorgensen and co-workers (reviewed in
Ref. 31), the E1P
E2P
conformational transition is blocked. In the sarcoplasmic reticulum
Ca-ATPase, tryptic cleavage at Arg198 as well as
site-specific mutation of residues in the predicted
-strand domain
of the H2-H3 loop also blocked the E1P
E2P conformational transition (reviewed in Ref.
32).
As pointed out by Green and Stokes (33), the H2-H3 loop is well
conserved among P-type ATPases. In their model of P-type ion pumps,
based largely on studies of the sarcoplasmic reticulum Ca-ATPase (34),
their suggestion that the anti-parallel -strand is positioned close
to the phosphorylation site is supported by vanadate protection of
Ca-ATPase from proteolytic degradation (35). In the yeast
H+-ATPase, Perlin and co-workers (27, 28) have shown that
perturbations of residues in this region not only alters the
distribution of conformational intermediates during steady-state
catalysis, but also decreases the sensitivity of this P-type pump to
vanadate inhibition, consistent also with the conclusion that this
region interacts with the catalytic phosphorylation domain.
In studies of Na,K-ATPase, involvement of the H2-H3 loop in structural
rearrangements associated with ligand binding and phosphorylation has
been apparent in distinctive conformational changes revealed by
proteolytic cleavage patterns (36). The importance of these interactions in conformation coupling is emphasized by the remarkable vanadate insensitivity caused by the Glu233 Lys
mutation. This vanadate insensitivity suggests interaction of
Glu233 located in the putative
-strand region of the
H2-H3 loop with the phosphate binding domain within the catalytic H4-H5
loop. It is also possible that the vanadate insensitivity of E233K is the consequence of a decrease in steady-state level of
E2 required for Pi (vanadate)
binding (cf. the vanadate-insensitive mutants of PMA1;
Ref. 28).
When deocclusion of K+ from the K+-occluded
state, E2(K), is analyzed as a branched pathway
reaction (Scheme I; cf. Refs. 14 and 37), values of
Na,K-ATPase activity as a function of varying ATP concentration can be
fitted to a biphasic reciprocal plot in the case of the 1 enzyme.
The ratio of Vmax values for the high affinity
to low affinity components,
VH/VL, is 0.043 for
1.
In contrast, there is little, if any, evidence of a high affinity component (VH) (pathway b) with the
E233K enzyme. This is not unexpected, since, according to the simple
Michaelis-Menten relationship shown in Equation 1, activity attributed
to pathway a, compared to activity attributed to pathway
b, is much greater in the E233K enzyme than it is in the
1 enzyme when ATP is reduced to micromolar concentrations.
![]() |
(Eq. 1) |
It is of interest to compare this mutation with those in other
cytoplasmic regions of the 1 subunit. In the H4-H5 loop, mutation of
aspartate at the phosphorylation site of the Torpedo
californica, pig, or sheep kidney enzymes results in complete loss
of Na,K-ATPase activity (38-40), and the latter two groups reported an
increase in ATP affinity. Mutation of Lys507 of the
T. californica enzyme at the putative ATP binding site decreases both activity and ouabain binding capacity (38), and substitution of Cys513 in the rat
1 enzyme decreases ATP
affinity (41). In contrast to these mutations, a deletion mutant in
which residues 1-32 have been removed from the cytoplasmic amino
terminus of
1 results in a fully active enzyme with kinetic changes
that are similar to those observed with the E233K mutation (14). This
mutation results in a 2.5-fold increase in affinity for ATP at its low affinity binding site and an increase in rate through pathway b (14). With this mutant (
1M32), as with E233K, the rate
of conversion of E2(K) to
E1 and the apparent affinity for K+
occlusion were increased, and turnover was substantially reduced. Similarly, a NH2-terminal truncated mutant of the
Bufo marinus enzyme was recently shown to have a reduced
E1 to E2 conformational change (42). As shown elsewhere, the
2 isoform behaves similarly to
1M32 and may be regarded as a conformational variant of
1.
These cytoplasmic mutants contrast with those in which substitutions in
transmembrane helices also result in active, functionally altered
enzymes, most of which are characterized by changes in affinities for
Na+ and K+. Of these, one mutation localized to
H5 (Glu779 Gln; Ref. 11) alters (decreases) only
Na+ affinity. An Asn326
Leu mutation in H4
decreased the apparent affinity for Na+ but slightly
increased it for K+ (8). Another group of transmembrane
substitutions resulted in decreases in affinities for both
Na+ and K+. The substituted residues are, in
H4: Glu329
Gln (43) and Glu327
Leu (9);
in H5: Glu781
Ala (7, 12), with quantitatively smaller
changes noted in Glu781
Asp (12); and in H6:
Thr809
Ala (7). In contrast to these mutations,
substitution of Ser775 in H5 with either alanine or
cysteine decreases apparent K+ affinity dramatically,
31-fold in the case of Ser775
Ala and 13-fold in the
case of Ser775
Cys, with no evidence of a change in
Na+ affinity (10).
Substitutions in transmembrane helices effecting an increase in
KK are also associated with an increase in
apparent affinity for ATP. In the analysis of Eisner and Richards (26)
mentioned earlier, pump-mediated K+ influx was empirically
described by a relationship which showed that decreasing
Kext concentration (presumably equivalent to increasing K
Kext), increases the apparent affinity for
ATP. Similarly, increasing ATP concentration (presumably equivalent to
decreasing K
ATP), decreases the apparent
affinity for external K+ (and also increases
Vmax). Considering, for example, the following sequence of reactions that constitute the low affinity pathway of
K+ transport, it is likely that the various transmembrane
residues which coordinate K+ ions probably do so with
distinct affinities and/or selectivities.
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Reaction 1 |
The relationships between cation and ATP affinities of the cytoplasmic
mutations are clearly different from those of mutations in
transmembrane helices. Although the replacement of glutamate with
lysine in the present study and, to a lesser extent, the deletion of
the amino terminus described earlier (1M32) also increased the
apparent affinity for ATP, changes in K
K were
not observed as long as the ATP concentration was saturating. This behavior is consistent with the interpretation that the primary functional alteration effected by mutating Glu233 to lysine
is an increase in the rate of E1 formation from
E2(K). We suggest that this substitution in the
cytoplasmic H2-H3 loop alters its interaction(s) with other regions of
the
subunit resulting in a change in the conformational equilibria,
such that the apparent affinity for ATP is increased. Changes in
apparent affinity for K+ that were observed at suboptimal
ATP concentration are probably secondary to changes in
K
ATP, as well as to steps involved in the
conversion of conformational forms.
We thank Dr. J. B Lingrel, University of
Cincinnati College of Medicine, for the 1-transfected HeLa cells,
and Dr. B. Forbush III, for communicating unpublished results. We
gratefully acknowledge the technical assistance of Rosemarie Scanzano
and Apolonia Wilczynski.