From the Department of Biochemistry, McGill
University, Montreal, Quebec H3G 1A4, Canada and
§ Department of Pharmacology and Cell Biophysics, University
of Cincinnati College of Medicine, Cincinnati, Ohio 45267
Received for publication, November 14, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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We showed earlier that the kinetic behavior of
the The Na,K-ATPase or sodium pump is an integral membrane protein
complex found in the plasma membrane of virtually all animal cells. It
catalyzes the exchange of three intracellular Na+ ions for
two extracellular K+ ions using the energy of hydrolysis of
one molecule of ATP. Consequently, the sodium pump plays an essential
role in the maintenance of the electrochemical alkali cation gradients,
providing the driving force for the transport of various nutrients into
the cell. The Na,K-ATPase is a member of the family of P-type ATPases,
which during the course of their catalytic cycle undergo
phosphorylation and dephosphorylation of a conserved aspartate residue
located in the large catalytic loop between transmembrane segments 4 and 5 of the catalytic The One region of marked primary sequence diversity among the otherwise
homologous The experiments described in the present study were designed to extend
the chimera approach to additional domains of isoform diversity to
pinpoint regions that confer the Mutagenesis, Transfection, and Cell Culture--
All of the
mutant and chimeric
The
Chimeras
The N-terminal chimera,
The chimera in which the isoform divergent portion within the M4-M5
loop,
HeLa cells were transfected with the pRcCMV- Membrane Preparation--
NaI-treated microsomal membranes were
prepared from the mutant cells as described earlier (6, 9). Protein
content was determined with a detergent-modified Lowry assay (10).
Tryptic Cleavage--
Digestion was carried out at a
trypsin/enzyme ratio of 1200 units trypsin/mg of membranes
(L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin, Type II, Sigma) in a buffer comprising 100 mM
NaCl, 0.2 mM MgSO4, and 20 mM Tris
glycylglycine (pH 7.4) at a final enzyme concentration of 35 µg/ml.
Digestion was terminated by adding 10-fold excess (units) soybean
trypsin inhibitor (Type I-S, Sigma) in a buffer containing 50 mM choline chloride, 0.2 mM MgSO4,
and 20 mM Tris glycylglycine (pH 7.4).
Enzyme Assays--
Na,K-ATPase activity was measured as the
release of 32Pi from [ Rate of E1P Cytoplasmic Interactions of
Experiments carried out in the present study to address the question of
whether cytoplasmic interactions of
Thus, at micromolar ATP concentrations sufficient to saturate only the
high affinity phosphorylation site the response of Na-ATPase to
K+ is a sensitive means to characterize mutant-specific
differences in the K+-deocclusion pathway of the reaction
cycle [E2(K+)
As shown in Table I,
To gain further insight into the effects of these mutations on
conformational equilibrium, we investigated the sensitivity of the
Na,K-ATPase activity of the mutants to inhibition by vanadate. Inorganic orthovanadate is a transition state analog of inorganic phosphate that binds to P-type ATPases in the E2
conformation during steady-state catalysis. Consequently, sensitivity
of an enzyme to inhibition by vanadate is a measure of the proportion of enzyme in the E2 state (19). As shown by the
representative experiment in Fig. 2 and
summarized in Table I, Chimeras of the
From a comparison of the primary structure of the cytoplasmic regions
of
To assess the effect of these different regions on the
E1/E2 poise of
Fig. 5 shows the determination of
K'ATP(L) using the Lineweaver-Burk
transformation of the simple Michaelis-Menten analysis of Na,K-ATPase
activity as a function of ATP concentration, with values presented in
the inset. Only the
To further examine the contribution of the various domains to the poise
of E1/E2, we used
vanadate as a conformational probe for E2 forms
as described above. In one set of experiments, chimeras
It has been observed that the Our earlier studies showed a notable similarity between the As a preliminary step toward defining the structural basis for
As already mentioned, the primary structure of the Although it is attractive to hypothesize that the
E1 shift of It is instructive to consider likely The present kinetic analysis involves evaluation of the overall
reaction cycle under different conditions of rate limitation or ligand
perturbations as well as certain partial reactions relevant to
conformation transitions. The usefulness of this approach is indicated
by the comparison of 2 isoform of the Na,K-ATPase differs from the ubiquitous
1
isoform primarily by a shift in the steady-state
E1/E2 equilibrium of
2 in favor of E1 form(s). The aim of the
present study was to identify regions of the
chain that confer the
1/
2 distinct behavior using a mutagenesis and chimera approach.
Criteria to assess shifts in conformational equilibrium included (i)
K+ sensitivity of Na-ATPase measured at micromolar ATP,
under which condition E2(K+)
E1 + K+ becomes rate-limiting, (ii)
changes in K'ATP for low affinity ATP binding,
(iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate
of the partial reaction E1P
E2P. We first confirmed that interactions
between the cytoplasmic domains of
2 that modulate conformational
shifts are fundamentally similar to those of
1, suggesting that the
predilection of
2 for E1 state(s) is due to
differences in primary structure of the two isoforms. Kinetic behavior
of the
1/
2 chimeras indicates that the difference in
E1/E2 poise of the two
isoforms cannot be accounted for by their notably distinct N termini,
but rather by the front segment extending from the cytoplasmic N
terminus to the C-terminal end of the extracellular loop between
transmembranes 3 and 4, with a lesser contribution of the
1/
2
divergent portion within the M4-M5 loop near the ATP binding domain. In
addition, we show that the E1 shift of
2
results primarily from differences in the conformational transition of
the dephosphoenzyme, (E2(K+)
E1 + K+), rather than phosphoenzyme
(E1P
E2P).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit. During the catalytic cycle both dephospho- and phosphoenzymes undergo conformational transitions commonly referred to as E1
E2 and E1P
E2P, respectively. In addition to the large
catalytic
subunit, the Na,K-ATPase comprises a smaller, highly
glycosylated
subunit that is important for the proper folding of
and its insertion into the plasma membrane. At present, four
isoforms of
and three isoforms of
have been described,
and these are distributed in a tissue- and developmentally dependent manner.
2 isoform is located primarily in skeletal muscle and in brain,
predominantly in glial cells. Our earlier studies indicated that it
differs from the ubiquitous
1 subunit primarily in the steady-state
E1/E2 equilibrium. Thus,
compared with
1, the
E1/E2 poise of
2 is
shifted toward E1. This shift is reminiscent of the changes in
1 effected by deleting 32 residues from its N terminus (mutant
1M32) which corresponds to the
E1-shifted trypsinized kidney enzyme first
described by Jorgensen (1). Except for a modest (
1.5-fold) increase
in K'Na (12),
2 resembles
1 with respect
to apparent affinity for extracellular K+ when
ouabain-sensitive K+ influx is assayed under physiological
conditions of ATP concentration. However, marked differences between
2 and
1 become apparent when, at micromolar ATP, the
E2(K+)
E1 conversion is rate-limiting. Under these
conditions,
2* 1
resembles closely the
1M32 mutant. We showed previously (2, 3) that,
compared with
1, both
2* and
1M32 have faster rates of
K+ deocclusion as seen in K+ stimulation rather
than
1-like inhibition of Na-ATPase activity at 1 µM
ATP, with a concomitantly lower K'ATP for low
affinity ATP binding and decreased (50%) catalytic turnover.
1,
2*, and
3* isoforms is the N terminus. In
previous studies we compared the kinetics of
1/
2 chimeras in
which the first 32 residues were interchanged. The results showed that
although removal of 32 residues from the N terminus of
1 yields an
enzyme with
2*-like kinetics as mentioned above, substitution of the
first 32 residues of
1 with those of
2* is without effect.
Similarly, substituting the analogous N-terminal sequence of
1 into
that of
2* does not dramatically alter the kinetics of
2. It was
therefore suggested that either removal of the N terminus, as in the
case of
1M32, or alteration of its primary or secondary structure,
as in the case of
2*, likely results in a weakening of
intramolecular interactions between the N terminus and some other
regions of the
protein (2).
1/
2 distinct behavior. As a
prelude to this analysis, we first show that cytoplasmic interactions
that underlie the E1/E2
conformational transitions seen with
1 (3, 4) are notably similar in
2*. The subsequent analysis of
1/
2 chimeras shows that the
N-terminal segment encompassing the cytoplasmic N terminus and the
first (M2-M3) cytoplasmic loop of
2*, the so-called "Actuator"
or A domain (5) and, to a lesser extent, the most divergent portion of
the nucleotide binding or N domain within the large M4-M5 loop, are
responsible for the E1 shift of
2*.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
's used in this study were derived from the
ouabain-insensitive rat
1 and
2* cDNAs previously described
by Jewell and Lingrel (6). The E231K mutation was introduced into a rat
2* cDNA that had been excised from pRcCMV with
HindIII(81, 3282) using PCR amplification with
synthetic nucleotides and the TaqPlus polymerase reaction kit
(Stratagene). The reaction mixtures were eluted from QIAquick spin
columns (Qiagen) and digested with DpnI, EcoRI,
and SalI. The
EcoRI(780)-SalI(1202)
2* fragment was gel-purified and ligated into a modified pIBI
2* shuttle vector in place of the wild type
EcoRI-SalI fragment. Clones containing the E231K
mutation (G800
A) were identified by the presence of
adenosine at nucleotide 800, and the complete sequence of the
substituted fragment was verified before excising the full-length
2E231K cDNA from the shuttle vector and ligating it into pRcCMV
(Invitrogen). Orientation of the
2E231K cDNA in pRcCMV was
determined by restriction enzyme analysis.
2M30 mutant was constructed by introducing a 30-residue deletion
into the 5'
HindIII(66)-EcoRI(780)
restriction fragment cassette of the rat
2* cDNA as described
previously (2). The mutant cassette was then ligated into the rat
2*
cDNA in place of the wild type HindIII-EcoRI
cassette. The full-length mutant cDNA was released from the shuttle
vector by digestion with HindIII(66, 3284) and
ligated into the expression plasmid pCDNA3.1 (Invitrogen), and
orientation of the cDNA was determined by restriction analysis.
2-(1-309)/
1 and
1-(1-311)/
2 were prepared
by exchanging 5' HindIII-BssHII fragments of the
two cDNAs. The HindIII site of each cDNA is in the
5'-untranslated region, and the BssHII site splits the
codons for Ala-347 in
1 and the corresponding Ala-345 in
2. It
should be noted that the two isoforms have identical sequences in the
region between residues 311 and 346 of
1. The full-length cDNAs
for the chimeric proteins were then excised from the shuttle vectors
and cloned into pRcCMV as above.
2-(1-63)/
1, denoted as
2nt/
1, was prepared using the
oligonucleotide-directed technique of Kunkel (7). Starting with the 5'
SacI (230)-SalI(875)
cassette of
1-(1-32)
2 (3), nucleotide mutations encoding the
2 amino acid sequence from residue 33 through 63 were introduced.
This mutant 5' cassette was sequenced and ligated into an
1 shuttle vector in place of the wild type cassette, and the full-length chimeric
cDNA was transferred into pRcCMV. Because residues 64-94 of rat
2 are naturally identical to residues 66-96 of
1, the entire
N-terminal cytoplasmic tail of the
2nt/
1 protein has the
2 sequence.
2-(427-562)/
1, denoted as
2L/
1, was
prepared using the Kunkel (7) technique to introduce the
2 sequence into five short segments of the H5-H6 cytoplasmic loop of rat
1.
Three of the substituted segments are encoded by nucleotides in the
SalI(875)-BamHI(1776)
cassette: I, 429-442
1 replaced by 427-440
2; II, 457-473
1
replaced by 455-471
2; III, 489-499
1 replaced by 487-496
2. The other two segments are in the
BamHI(1777)-KpnI(2290) cassette: IV, 515-530
1 replaced by 512-527
2; and V, 552-565
1 replaced by 549-562
2. After sequence verification, the
mutated cassettes were ligated into an
1 shuttle vector in place of
the wild type cassettes, and the
2L/
1 cDNA was
then transferred to pRcCMV. The mutated 5'
SacI-SalI cassette used above to prepare
2nt/
1 was also substituted into the
2L/
1 cDNA in place of the wild type cassette to
produce
2(nt+L)/
1.
and pCDNA-
constructs using either the calcium phosphate method (8) or the LipofectAMINE technique (LipofectAMINE, Invitrogen), and cells expressing the relatively ouabain-resistant rat
enzymes were selected as previously described (6, 9). HeLa cells expressing the
mutant
enzymes were amplified in Dulbecco's modified Eagle's medium plus 10% newborn calf serum, 100 units/mg penicillin G, 100 µg/ml streptomycin, and 1 µM ouabain as described
previously (2).
-32P]ATP
as previously described (11). Briefly and unless indicated otherwise,
the membranes were preincubated for 10 min at 37 °C with all
reactants added except [
-32P]ATP. The reaction was
initiated by the addition of [
-32P]ATP. Final
concentrations for Na,K-ATPase activity measurements were 100 mM NaCl, 10 mM KCl, 3 mM
MgSO4, 20 mM histidine (pH 7.4), 5 mM EGTA (pH 7.4), and 5 µM ouabain (Sigma). 5 mM ouabain was used to determine base-line hydrolysis
activity. As in earlier studies and unless indicated otherwise, assays
of Na,K-ATPase activity were carried out using 1 mM ATP to
maintain close to saturating ATP concentration and maximize sensitivity
of assays of the relatively low activity cultured cells (cf.
Refs. 3, 9, and 12). Na-ATPase activity was measured at 1 µM ATP as described previously (2), with varying amounts
of added KCl and choline chloride to maintain constant chloride (40 mM) concentration. Base-line activity was determined with
40 mM KCl replacing NaCl. For studies of vanadate
sensitivity, inorganic orthovanadate (Fisher) solutions were prepared
before the experiment and added with the [
-32P]ATP
solution to initiate the reaction. Na,K-ATPase activities obtained at
various vanadate concentrations and expressed as the percentage of that
obtained in the absence of vanadate were analyzed by fitting the data
to a one-compartment model using a nonlinear least-square analysis of a
general logistic function, as described elsewhere (13). Curve fitting
was carried out using the Kaleidagraph computer program (Synergy). Each
experiment was carried out at least three times, with one or more
chimeras analyzed concurrently with the
2* and
1 isoforms.
E1P--
After formation
of E1P in the presence of high chloride
concentration (14), the rate of E1P
E2P was determined by measuring the rate of
disappearance of total phosphoenzyme after rapid dilution of the salt
(to allow "normal" relaxation of E1P
E2P) plus the addition of KCl to catalyze rapid
hydrolysis of E2P (14, 15). Accordingly, the
enzyme was first phosphorylated in medium containing 600 mM
NaCl to stabilize E1P, 1 mM
MgCl2, 1 mM EGTA, and 20 mM Tris-HCl (pH 7.4) with 1 µM [
-32P]ATP
for 30 s at 0 °C to obtain maximal phosphoenzyme.
Dephosphorylation was then initiated by 6-fold dilution with a
"chase" medium containing final concentrations of 20 mM
KCl, 10 µM unlabeled ATP, 1 mM EGTA, and 20 mM Tris-HCl (pH 7.4), which simultaneously lowered the NaCl
concentration to 100 mM. Samples were taken for measurement of [32P]E for periods up to 30 s. Background
phosphoenzyme levels were obtained by allowing the chase to continue
for 60 s. The data were fitted to a first-order decay model using
the Kaleidagraph nonlinear fitting program (Synergy). At least two
different membrane preparations obtained from at least two different
clones were assayed. The data presented are representative of at least
three independent experiments. Each value shown is the mean ± S.D. of triplicate determinations.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2*--
Our earlier studies showed
that the steady-state
E1/E2 conformational
equilibrium of
2*, compared with that of the ubiquitous
1
isoform, is poised toward E1. In fact, the
kinetic behavior of
2* is generally similar to that of mutants of
1 in which the poise is shifted toward E1.
These mutations include a 32-residue deletion of the cytoplasmic N
terminus (mutant
1M32) and a Glu-233
Lys replacement (mutant
1E233K) in the first cytoplasmic loop. Furthermore, the combination
of these two mutations of the so-called Actuator domain (mutant
1M32E233K) in
1 results in a remarkably synergistic shift in
poise toward E1 state(s). The findings were interpreted to indicate that interactions between these regions of the
Actuator domain (5) and the catalytic loop are critical for
conformational coupling of the Na,K-ATPase (4).
2* are analogous to those of
1 are summarized in Fig. 1 and Table
I.
Expression of
2M30 and
2E231K in
HeLa cells yielded functional enzymes capable of supporting cell growth
in 1 µM ouabain. Cells transfected with the double mutant
2M30E231K, analogous to
1M32E233K (4), failed to grow even in
elevated K+ (cf. Ref. 16). Therefore, a
comparable "mutation" was prepared by enzymatically cleaving the N
terminus of
2E231K in the E1(Na+)
conformation with trypsin (cf. Ref. 1). The single mutants and the cleaved
2E231K (
2E231K-Tryp) were then assessed for shifts in the E1/E2
equilibrium using the following criteria: (i) the effect of
K+ on Na-ATPase activity as a measure of
E2(K+)
E1
(see Ref. 2), (ii) K'ATP for low affinity
binding to E2(K+),
K'ATP(L), and (iii) sensitivity of Na,K-ATPase
activity to inhibition by inorganic orthovanadate.
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Fig. 1.
Effect of K+ on Na-ATPase
activity of 1 and
2*
cytoplasmic mutants. ATP hydrolysis was assayed in the presence of
1 µM ATP, 20 mM NaCl, and various
concentrations of KCl as described under "Experimental Procedures."
Data are presented as percent of Na-ATPase activity measured in the
absence of added KCl and in the presence of 1 mM KCl.
Results shown are the average of at least three separate
experiments ± S.D.
Kinetic analysis of cytoplasmic mutants of 1 and
2*
E1 + K+] that becomes rate-limiting
under these conditions (2, 17). As shown previously and summarized in
Table I (upper panel), a low concentration of K+ (1 mM KCl) inhibits the Na-ATPase activity of
1 but
stimulates that of
1M32 and
1E233K with a further and notably
synergistic stimulation of the double mutant
1M32E233K. The pattern
for
2* cytoplasmic mutants is remarkably similar (see Fig. 1 and the lower panel of Table I). With respect to the
2 mutants,
K+ stimulates the Na-ATPase activity of
2M30 and
2E231K, ~300 and 900%, respectively, and that of
2E231K-Tryp,
more than 2000%. In fact, the apparent stimulation may be an
underestimation of the true stimulation since trypsinolysis of
2E231K undoubtedly results in a heterogeneous pool of enzyme species
that includes untrypsinized enzyme having inherently lower sensitivity
to K+ activation. It should be mentioned that
trypsinization of
2 in the presence of Na+ increased
K+ activation such that
2-Tryp resembled
2M30 (data
not shown; cf. tryptic cleavage of
1 described in Ref.
18), which is not surprising since the region encompassing the tryptic
cleavage site (T2; see Ref. 1) is conserved between
1 and
2.
These results suggest that
2M30,
2E231K, and
2E231K-Tryp all shift the
E1/E2 equilibrium of
2* progressively further toward E1.
2M30,
2E231K, and
2E231K-Tryp also
exhibit increased affinities for low affinity ATP binding relative to
2*, with a synergistic effect of the N-terminal deletion and the
E231K mutation in the
-strand region of the M2-M3 loop. It should be
noted that, as with the K+ stimulation of Na-ATPase
activity shown above, the K'ATP(L) of
2E231K-Tryp is an overestimation of the actual value of the double mutant due to the presence of untrypsinized enzyme.
2M30 and
2E231K are both less
sensitive to vanadate inhibition than
2*, suggesting that these
mutations further shift the
E1/E2 equilibrium of
2* toward E1. We were unable to determine
accurately the IC50 for vanadate inhibition of
2E231K-Tryp because the residual Na,K-ATPase activity was
too low relative to the background (
15 nmol/mg·min).
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Fig. 2.
Vanadate sensitivity of Na,K-ATPase activity
of 2* cytoplasmic mutants. ATP hydrolysis
at varying vanadate concentrations was determined with 100 mM NaCl, 10 mM KCl, and 1 mM ATP as
described under "Experimental Procedures." Data are presented as
percent Na,K-ATPase (control) measured in the absence of vanadate.
Results shown are from a representative experiment. Each value is the
mean ± S.D. of triplicate determinations. Symbols are:
,
1;
,
2M30;
,
2E231K. 100% maximal activities are, 176.0 ± 3.3, 162.7 ± 5.1, and 127.5 ± 8.3 for
1,
2M30, and
2E231K, respectively.
1 and
2* Isoforms--
The above analysis of
cytoplasmic mutations of the
2* isoform of the Na,K-ATPase support
the notion that interactions between the cytoplasmic domains of
2*
that modulate conformational shifts are fundamentally similar to those
of
1. The analysis also suggests that it is the
2-specific
regions of the A domain and/or the catalytic domain with which A
interacts that underlie the predilection of this isoform for the
E1 state(s). Accordingly, an
1/
2 chimera approach was used to address this issue.
1 and
2* (Fig. 3B),
it is important to note that the
strand region in the M2-M3 loop of
1 encompassing Glu-233 is highly homologous to that of
2*. In
contrast, the two isoforms differ significantly in the primary sequence
of their N termini and in the sequence encompassing the ATP binding
site in the M4-M5 cytoplasmic loop. Therefore, chimeras were
constructed in which the entire N terminus (residues 1-65) and the
divergent portion of the nucleotide binding (N) domain in the large
M4-M5 loop (residues 429-565) of
1 were substituted with the
analogous residues of
2*. It should be noted that our original
intent was to investigate 5 individual divergent regions contained
within residues 429-565 (see "Experimental Procedures," cassettes
I-V) on the E1/E2
equilibrium of
1. Because the individual domains showed no effect on
the K+ inhibition of Na-ATPase of
1 relevant to the
K+ deocclusion
pathway,2 we constructed an
1/
2 chimera where all 5 cassettes of
2* were substituted into
1. Two additional chimeras were constructed. In these chimeras, the
first 311 residues of
1, which encompass the entire A domain, are
interchanged between
1 and
2* because this region comprises the
portion of the cytoplasmic domain that undergoes large rotational
displacements during
E1/E2 transitions (5,
20). A schematic representation and the designation of the chimeras are
illustrated in Fig. 3A.
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Fig. 3.
Chimeras of 1
and
2. A, schematic
representation of
1/
2 chimeras.
1/
2 chimeras were
constructed as described under "Experimental Procedures" whereby
the N terminus (
2nt/
1, residues 1-65) and a portion
of the catalytic M4/M5 loop of
1 (
2L/
1, residues
429-565) were substituted with the analogous residues of
2* either
individually or together (
2(nt+L)/
1). Two additional
chimeras were constructed in which the region encompassing the entire
Actuator domain (residues 1-311 in
1 and the analogous region of
2*) was interchanged (
1-(1-311)/
2 and
2-(1-309)/
1).
B, sequence alignment of
1 and
2. The sequences for
the regions described in A of the rat
1 and
2*
were aligned using ClustalW, namely (i) the N-terminal segment and (ii)
the L region of the N domain. Transmembrane segments are
shaded.
1, we
first investigated the effect of K+ on Na-ATPase activity
measured at micromolar ATP for each of the chimeras as described above.
As shown in Fig. 4, the Na-ATPase activities of wild type
1 and chimeras
2nt/
1,3
2L/
1, and
2(nt+L)/
1 are all
inhibited by K+. In contrast, wild type
2* and
2-(1-309)/
1 are stimulated by K+ at least up to 1 mM, consistent with a faster
E2(K+)
E1
transition and, hence, with a preponderance of the
E1 conformation. It is noteworthy that although
the
2nt/
1 construct encompasses the most divergent
portion of the primary sequence, it is only upon replacement of the
homologous N-terminal segment (residues 1-311) of
1 with that of
2*, chimera
2-(1-309)/
1, that the shift toward
E1 is observed.
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Fig. 4.
Effect of K+ on Na-ATPase
activity of 1,
2*,
and chimeras. Assays were carried out as in Fig. 1. Data are
presented as percent of Na-ATPase activity (control) measured in the
absence of added KCl. Results shown are from a representative
experiment. Each value is the mean ± S.D. of triplicate
determinations. Symbols are:
,
1;
,
2*;
,
1-(1-311)/
2; ×,
2-(1-309)/
1;
,
2L/
1;
,
2nt/
1;
,
2(nt+L)/
1.
2-(1-309)/
1 chimera, like wild
type
2*, showed an increased affinity for ATP. Thus, the
K+ stimulation of
2-(1-309)/
1 described in Fig. 4
correlates with its increased apparent affinity for ATP, consistent
with a shift in the E1/E2
poise in favor of E1. Chimeras
2L/
1 and
2(nt+L)/
1 show no
difference in K'ATP(L) relative to
1.
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Fig. 5.
Lineweaver-Burk plot of Na,K-ATPase activity
as a function of ATP concentration. ATP hydrolysis was assayed in
the presence of 100 mM NaCl, 10 mM KCl, 3 mM MgSO4, and varying ATP concentrations as
described under "Experimental Procedures" and normalized to 100%
VMAX. Each value is the mean ± S.D. of
triplicate determinations. Results are taken from a representative
experiment, with K'ATP values show in the inset.
Symbols are as in Fig. 4.
1-(1-311)/
2 and
2-(1-309)/
1 were analyzed concurrently
with
1 and
2* (Fig. 6A),
and in another, the
2L/
1 chimera was compared with
1 and
2* (Fig. 6B). As shown previously (21), the
Na,K-ATPase activity of
2* is ~25-fold less sensitive to vanadate
than
1 (Fig. 6A). Interestingly, the
1-(1-311)/
2
enzyme is 3.7-fold less sensitive to vanadate than
1 and resembles
1 with respect to K+ inhibition of Na-ATPase and
K'ATP(L), whereas
2-(1-309)/
1 is 8.5-fold
less vanadate-sensitive than
1 and resembles
2* with respect to
K+ activation of Na-ATPase and
K'ATP(L). A modest shift in conformational poise
is also indicated by the behavior of the
2L/
1
chimera. This replacement of the divergent region of the N domain of
1 by that of
2 reduces the vanadate sensitivity of
1 by a
factor of 2.3. In other experiments (not shown) a similar
2-fold
shift was effected by including the loop insertion of
2* with the
2* N terminus, i.e. a 2-fold higher IC50 for
vanadate seen for
2nt/
1 compared with
1, which was
further doubled, resulting in a 4-fold higher IC50 compared
with
1 for
2(nt+L)/
1. Taken together these
findings imply a major contribution of the N-terminal segment encompassing residues 1-309 and a smaller contribution of the L region
of
2* in effecting shifts away from E2.
View larger version (12K):
[in a new window]
Fig. 6.
Vanadate sensitivity of Na,K-ATPase activity
of 1,
2*, and
chimeras. Assays were carried out as in Fig. 2. One series of
experiments was carried out with chimeras
1-(1-311)/
2 and
2-(1-309)/
1 assayed concurrently with
1 and
2*, and a
representative experiment is shown in panel A. In a second
series, chimera
2L/
1 was assayed concurrently with
1 and
2*, and a representative experiment is shown in Panel
B. Symbols are as in Fig. 4.
2* isoform and the 32-residue deletion
mutant of
1,
1M32, are both enzymes with their
E1/E2 equilibrium shifted
toward E1 forms. Both enzymes show similar K+ sensitivities of Na-ATPase activity at low ATP,
K'ATP(L) values, catalytic turnovers, and
K+ deocclusion rates. One major difference between the two
enzymes is their sensitivity to inhibition by vanadate. Thus,
2* has a 25-fold lower IC50 than
1M32. Because vanadate
sensitivity reflects the steady-state levels of
E2, we investigated differences not only in the
E2(K+)
E1
transition rate but also the E1P
E2P transition rate. As shown previously (22)
and represented in Fig. 7,
1M32 has a
5-fold slower conversion of E1P to
E2P than
1, consistent with a preference for
the E1P form. This does not hold true for
2*; its E1P
E2P
transition rate is only slightly slower than that of
1, providing an
explanation for its higher sensitivity to vanadate compared with
1M32. The implication of vanadate sensitivity as a measure of the
E1/E2 poise during
steady-state catalysis is discussed further below.
View larger version (15K):
[in a new window]
Fig. 7.
Rate of formation of E2P from
E1P. The rate of E1P E2P was measured indirectly at 0 °C as
described under "Experimental Procedures." The results are taken
from a representative experiment with mean ± S.D. of triplicate
determinations. Symbols are
,
1;
,
2*;
,
2-(1-309)/
1; ×,
1M32.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2*
isoform of the Na,K-ATPase and the
"E1-shifted" mutants of
1, particularly
the deletion mutant
1M32. Nevertheless, the behavior of
1/
2
chimeras in which the first 32 residues of the N termini were
interchanged showed that the kinetic difference between
1 and
2*
could not be explained by their distinct N-terminal 1-32 residue
per se. More likely, those findings indicate that the
difference is due to the interaction of the N terminus with another,
isoform-distinct region(s) of the enzyme.
1/
2 differences, we have used a mutagenesis approach to obtain evidence for cytoplasmic interactions between the N terminus, the M2-M3
loop, and large M4-M5 loop of
2*, as noted previously for
1 (3,
4). As shown in Table I (lower panel), a kinetic analysis of
cytoplasmic mutants of
2*, namely
2M30 and
2E231K, show that
both mutations effect shifts in the
E1/E2 poise of
2* analogous to those seen for
1, although even further toward
E1. HeLa cells transfected with the double
mutant,
2M30E231K, analogous to
1M32E233K, failed to grow. Noting
that the catalytic turnover of
1M32E233K is
500 min
1
(4), it is likely that the catalytic turnover of
2M30E231K is much
too low to support HeLa cell growth in 1 µM ouabain.
Consequently, the N terminus of
2E231K was enzymatically cleaved by
trypsinolysis of membranes isolated from the
2E231K-transfected cells as originally described by
Jorgensen (1). Like
1M32E233K,
2E231K-Tryp showed a strong,
synergistic effect on E1
E2, shifting it even further in favor of
E1 forms. Therefore, we conclude that the
interaction of the cytoplasmic domains of
2* that modulate
conformational shifts are fundamentally similar to those of
1.
-strand region in
the M2-M3 loop encompassing Glu-231 of
2* is highly homologous to
that of
1 containing Glu-233. In contrast, the primary structures of
the two isoforms differ significantly in their N termini (domain nt,
56% identity)4 and a region
within the N domain of the M4-M5 loop and referred to here as the L
region (61% identity). It is noteworthy that for the remainder, the
sequence identity is very high, namely 86% between the end of the
nucleotide and the beginning of the L region (residues 429-565; see
Fig. 3B) and 92% from the C-terminal end of the L region to
the C terminus of the protein. Therefore,
1/
2 chimeras were
constructed in which domains of divergent primary sequence were
interchanged. Thus, the N terminus (nucleotide residues 1-65) and L
region (residues 429-565) of
1 were substituted with the analogous
regions of
2*. This was done either individually (
2nt/
1 and
2L/
1, respectively) or
in combination (
2(nt+L)/
1) (see Fig. 3A
for a schematic representation). In addition to the above, chimeras
encompassing the entire Actuator domain, i.e.
1-(1-311)/
2 and
2-(1-309)/
1), were analyzed. As shown in
recent structural studies, this domain undergoes large rotational
motions (estimated at 110° in the sarcoplasmic reticulum Ca-ATPase)
in the course of the conformational transitions (20). The present results show that although a switch of the entire N terminus is without
effect, inclusion of the entire isoform-divergent N-terminal segment up
to residue 309 of
2* is capable of conferring
2*-like kinetics to
1. This is apparent from the K+ activation of Na-ATPase
activity at low ATP when E2(K+)
E1 is rate-limiting as well as a decrease in
K'ATP(L). However, this N-terminal segment of
2* only partially decreases the vanadate sensitivity of
1
(
8.5-fold compared with 20-25-fold for
2* relative to
1). In
addition, however, the L region of
2* confers a 2.3-fold decrease in
vanadate sensitivity to
1, such that synergistic effects of the two
domains, residues 1-309 in the N-terminal segment and residues
427-562 in the N domain, can account for the
2* versus
1 differences.
2-(1-309)/
1 is due to the A
domain, one cannot exclude the possible contribution of amino acid
replacements in other regions, namely M1, M2, and M3 and the
extracellular M1-M2 and M3-M4 loops. Of these regions, it is noteworthy
that Coppi et al. (31) showed that an
2-(1-129)/
1
chimera is not E1-shifted because, like
1,
its Na-ATPase activity is inhibited by K+ at low ATP
concentration (31). This result provides a basis for eliminating M1 and
the M1-M2 loop as candidates for effecting the
E1 shift. For the rest, there are four
replacements, namely Ser-132
A in M2, His-288
Gln in M3,
and Glu-309
Gly and Thr-311
Ser in the M3-M4 loop. Experiments
are currently under way to determine whether and/or which ones of these
replacements are important or whether it is the A domain per
se that confers the
2-like E1 shift seen
with the
2-(1-309)/
1 mutant.
1/
2 differences in the
interactions of the A domain with the N and P domains in the E1 and E2 conformations
as well as interactions within regions of the A domain. With the
Na,K-ATPase, metal-catalyzed oxidative cleavage studies of domain
interactions reveal that in the E1 conformation,
the N domain docks onto the phosphorylation (P) domain, and A moves
apart; in E2, A docks onto P, and N is displaced (32), consistent with putative domain interactions deduced from crystal
structures of sarco(endo)plasmic reticulum calcium ATPase in
E1 and E2 states (5, 20,
33). Concerted effects of
1 versus
2 distinct residues
within the N-terminal segment encompassing domain A and the L region
within domain N can be explained by the recent model for regulation of
the aforementioned domain interactions regulated by the N terminus
(22). Thus, assuming that the secondary structure of the
2* N
terminus, like that of
1, has the propensity to form three short
helices (H1, H2, and H3), intramolecular interactions between helices 1 and 2 of the N terminus allow the M2-M3 loop in A and the N domain to
come together in the E2 conformation. In the
E1 conformation, H2 interacts with the M2-M3
loop to keep it apart from N. Thus, isoform diversity in the N terminus
impacts intramolecular interactions of H2 with M2-M3 within the A
domain. The minor E1 shift effected by the swap
of the isoform divergent portion of the N domain (L swap) probably
indicates interaction between N and P in the E2P
conformation mediated by the M2-M3 loop. Accordingly, the following are
likely interactions deduced from the proximity (
3Å) of backbone
carbonyl groups of sarco(endo)plasmic reticulum calcium ATPase. (i) The
N terminus may interact with the region near Asp-203 of the
sarco(endo)plasmic reticulum calcium ATPase M2-M3 loop (Glu-233 of
1) in the E1 (5) but not in E2P (vanadate-trapped intermediate) (33) or
E2 (thapsigargin-bound intermediate) (20)
conformations. (ii) The region near Asp-203 of the sarco(endo)plasmic
reticulum calcium ATPase M2-M3 loop does not interact with either the N
or P domains in E1 but could interact with both
N and P in E2P and with only N in
E2; there are no nearby sites of interaction
between the N terminus and either the N or P domains, consistent with
an intermediary role of the M2-M3 loop of the A domain in
E1
E2 conformational
changes (22).
2* and
1M32 as summarized in Table
II. Compared with
1,
1M32 but not
2*, exhibits a substantial slowing of the E1P
E2P conversion, providing an explanation for its much larger decrease in sensitivity to vanadate compared with
2*, i.e. IC50 values of
1M32 and
2 are
decreased ~500- and 20-fold, respectively, compared with
1.
Accordingly, the E1 shift in
E1/E2 poise of
2* is
due to a shift in dephosphoenzyme [E2(K+)
E1] but not phosphoenzyme
[E1P
E2P]. This
result indicates the "nonequivalence" of the two conformational
transitions and also highlights a limitation of the sole use of
vanadate as a probe of conformation since the steady-state proportion
of enzyme in the E2 state reflects the rates of
transition between both dephospho- and phosphoenzyme states.
Criteria for assessing E1/E2 poise of 1,
1M32,
and
2*
This report deals only with the structure/function analysis of
isoform-specific kinetic behavior. Isoform-specific interactions of the
pump with other proteins such as cytosolic second messengers may be
important for adapting sodium pump function to cell-specific requirements as suggested by Blanco et al. (23). Thus,
targets for protein kinase C phosphorylation present in the N terminus of 1 are absent in
2. The significance of this difference is underscored by the observation that in transfected opossum kidney cells
(24) protein kinase C
-mediated phosphorylation of
1 at these
residues promotes its translocation to the plasma membrane. Similarly,
in
1 but not
2, the presence of Tyr-5 within a consensus sequence
for phosphorylation by tyrosine kinases of the Src family has been
implicated in the insulin stimulation of pump activity in the proximal
convoluted tubules of the rat kidney (25). Furthermore, although
insulin acts via tyrosine kinases in rat proximal convoluted tubules to
stimulate pump activity by increasing the apparent Na+
affinity (26) in skeletal muscle, it promotes translocation of
2 to
the plasma membrane (27) via the action of protein kinase C (28). There
is also direct evidence for a role of the isoform-specific primary
sequence within the M4-M5 loop domain, in regulation of the pump by
second messengers. In agreement with our findings, Pierre et
al. (29) failed to detect an effect of the introduction of an
2* distinct region of the large catalytic loop (residues 489-499,
contained within the L region) on the E1/E2 conformational
equilibrium of
1. However, a role for this domain in the
isoform-specific response of transfected opossum kidney cells to
protein kinase C activation was observed in the differential response
of the isoforms to hormones via the action of second messengers. Taken
together, the foregoing studies suggest that the primary sequence
diversity may in part be relevant to isoform-specific pump regulation,
separate from a role in isoform-distinct pump kinetics.
In conclusion, we have shown that the E1 shift
in the conformational equilibrium of 2 can be largely accounted for
by the N-terminal third of the
subunit that comprises mainly the A domain, with a small contribution of the isoform-specific sequence of
the N domain within the M4-M5 loop. In addition, despite the kinetic
similarities of the
2 isoform with cytoplasmic mutants of
1, such
as
1M32, the E1 shift of
2 results
primarily from differences in the dephosphoenzyme conformational
transition, i.e. E2(K+)
E1, with its E1P
E2P transition rate similar to that of
1.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. E. Jewell and J. B. Lingrel for the rat 2* transfected cells.
![]() |
FOOTNOTES |
---|
* This work was supported by Canadian Institutes of Health Research Grant MT-3876, an operating grant from the Quebec Heart and Stroke Foundation (to R. B.), National Institutes of Health Grant HL 49204 (to L. K.), and a predoctoral fellowship from the Heart and Stroke Foundation of Canada (to L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Montreal General Hospital Research Institute, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada. Tel.: 514-934-1934 (ext. 44501); Fax: 514-934-8332; E-mail: rhoda.blostein@mcgill.ca.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M211636200
1
2* denotes the rat
2 enzyme rendered
relatively ouabain-resistant to permit its distinction from the
endogenous, ouabain-sensitive HeLa enzyme (6). The asterisk is not
shown for the
2 mutants.
2 L. Segall, L. K. Lane, and R. Blostein, unpublished information.
3
Chimera abbreviations: 2nt/
1,
2-(1-63)/
1;
2L/
1,
2-(427-562)/
1;
2(nt+L)/
1,
2-(1-63, 427-562)/
1.
4 As determined by BLAST, NCBI.
![]() |
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