(Received for publication, August 17, 1995; and in revised form, January 18, 1996)
From the
The experiments described in this report reconcile some of the
apparent differences in isoform-specific kinetics of the Na,K-ATPase
reported in earlier studies. Thus, tissue-specific differences in
Na and K
activation kinetics of
Na,K-ATPase activity of the same species (rat) were observed when the
same isoform was assayed in different tissues or cells. In the case of
1,
1-transfected HeLa cell, rat kidney, and axolemma
membranes were compared. For
3, the ouabain-insensitive
3*-transfected HeLa cell (cf. Jewell, E. A., and Lingrel,
J. B.(1991) J. Biol. Chem. 266, 16925-16930), pineal
gland, and axolemma (mainly
3) membranes were compared. The order
of apparent affinities for Na
of
1 pumps was
axolemma
rat
1-transfected HeLa > kidney, and for
K
, kidney
1-transfected HeLa > axolemma.
For
3, the order of apparent affinities for Na
was pineal gland
axolemma >
3*-transfected HeLa, and
for K
,
3*-transfected HeLa > axolemma
pineal gland. In addition, the differences in apparent affinities for
Na
of either kidney
1 or HeLa
3* as compared
to the same isoform in other tissues were even greater when the
K
concentration was increased. A kinetic analysis of
the apparent affinities for Na
as a function of
K
concentration indicates that isoform-specific as
well as tissue-specific differences are related to the apparent
affinities for both Na
and K
, the
latter acting as a competitive inhibitor at cytoplasmic Na
activation sites. Although the nature of the tissue-specific
modulation of K
/Na
antagonism remains
unknown, an analysis of the nature of the
isoform associated with
1 or
3 using isoform-specific immunoprecipitation indicates
that the presence of distinct
subunits does not account for
differences of
1 of kidney, axolemma, and HeLa, and of
3 of
axolemma and HeLa; in both instances
1 is the predominant
isoform present or associated with either
1 or
3. However, a
kinetic difference in K
/Na
antagonism
due to distinct
s may apply to
3 of axolemma (
3
1)
and pineal gland (
3
2).
The sodium potassium adenosine triphosphatase (Na,K-ATPase) or
sodium pump is responsible for maintaining the electrochemical gradient
of Na and K
across the plasma
membrane of animal cells. It normally couples the hydrolysis of one
molecule of ATP to the transport of three Na
ions out
and two K
ions into the cell (for reviews, see (1, 2, 3, 4) ). This cation pump is
a heterodimer comprised of a catalytic
subunit (
105 kDa) and
a highly glycosylated
subunit (45-55 kDa), and may (5, 6) or may not (7, 8) form larger
oligomers. The
subunit contains the binding sites for
Na
, K
, ATP, and the highly specific
cardiac glycoside inhibitors such as ouabain, as well as the site of
phosphorylation(1) . The function of the
subunit is not
completely understood; it appears to be essential for the normal
delivery and correct insertion of
into the plasma membrane (9) and to have some influence on the catalytic activity of
(10, 11, 12, 13, 14) .
A third peptide subunit known as the
subunit (6.5 kDa) appears to
exist in association with
and
, at least in certain tissues,
although its role is yet to be determined(15) .
In mammals,
three isoforms of the subunit (
1,
2, and
3) and
two of the
subunit (
1 and
2) are known to
exist(4) . Isoforms of the
subunit are expressed in a
tissue-specific manner:
1 is present ubiquitously;
2 is
detected mainly in skeletal muscle, heart, and certain neuronal cells
(neurons and astrocytes); and
3 is mainly in
neurons(4, 16) .
Earlier studies of cation
activation of the Na,K-ATPase by Sweadner (17) using rat
kidney and axolemma and later studies by Shyjan et al.(18) using kidney, brain, and pineal gland indicated a
higher affinity for Na in preparations now known to be
predominantly
3. Thus, the order of apparent affinities for
Na
in the former study was axolemma (predominantly
3) > kidney (
1 only) and in the latter, pineal gland
(predominantly
3)
brain (a mixture of
1,
2, and
3) > kidney. In contrast, Jewell and Lingrel(19) ,
using membranes isolated from HeLa cells transfected with the
individual
isoforms, reported that the order of apparent
affinities for Na
is
1
2* >
3*,
and for K
,
3* >
2*
1, where
2* and
3* denote ouabain-resistant mutants of
2 and
3, respectively. Moreover, studies of pump-mediated K
(Rb
) influx into these individual
isoform-transfected cells confirmed the general conclusions drawn from
the aforementioned work, except that considerably larger kinetic
differences among the isoforms were observed(20) .
Interestingly, in experiments carried out with kidney and axolemma
microsomal membranes delivered by membrane fusion into red cells, the
order of apparent affinities for cytoplasmic Na
and
K
resembled those of
1- and
3*-transfected
HeLa cells, respectively(20) .
The aim of the experiments
described in this study was to reconcile the discordant results
obtained in the foregoing studies, as well as numerous earlier reports,
regarding the order of apparent affinities for Na and
K
of different tissues and/or isoforms (for review,
see (4) ). In particular, the question of isoform-specific versus tissue-specific properties of the rat Na,K-ATPase has
been addressed by studying the same isoform, either
1 or
3,
in the membranes of various cells. Thus, the properties of the
1
isoform were examined in kidney, axolemma and rat
1-transfected
HeLa cells, and those of the
3 isoform, in axolemma, pineal gland,
and
3*-transfected HeLa cells. The results provide evidence for
isoform-independent, tissue-specific modulation of the kinetic behavior
of the Na,K-ATPase, the most striking being the differences in the
effects of intracellular K
as a competitive inhibitor
of Na
at cytoplasmic Na
activation
sites.
Figure 1:
Activation by Na or
K
of rat
1 Na,K-ATPase from kidney, axolemma, and
transfected HeLa cells. Membranes were prepared and assayed as
described under ``Experimental Procedures.''
Ouabain-sensitive activities are the difference between hydrolysis
measured in the presence of 10 µM and 5 mM ouabain, and are the means ± S.D. (triplicate
determinations) expressed as percentages of V
.
The curves were fitted to . Representative experiments are
shown and values of K
and n for
replicate experiments are shown in Table 1. A,
activation by Na
at 10 mM KCl (V
values are 3.72 ± 0.07, 0.260 ±
0.004, and 0.107 ± 0.003 µmol/(mg
min) for kidney,
axolemma, and HeLa cells, respectively); B, activation by
K
at 100 mM NaCl (V
values are 4.32 ± 0.14, 0.330 ± 0.030, and 0.108
± 0.003 µmol/(mg
min) for kidney, axolemma, and HeLa
cells, respectively).
, kidney;
, axolemma;
,
transfected HeLa cells. In the insets the data were fitted to .
Figure 2:
Activation by Na and
K
of rat
3 Na,K-ATPase from pineal glands,
axolemma, and transfected HeLa cells. Assays were carried out and
analyzed as described in Fig. 1, except that ouabain-sensitive
activities attributed mainly to
3 are the differences between
hydrolysis measured in the absence and presence of 5 mM ouabain (pineal gland) or the absence and presence of 10
µM ouabain (axolemma) or in the presence of 10 µM and 5 mM ouabain (
3*-transfected HeLa cells). A, activation by Na
at 10 mM KCl (V
values are 0.353 ± 0.011, 3.34
± 0.07, and 0.078 ± 0.002 µmol/(mg
min) for
pineal gland, axolemma, and HeLa cells, respectively); B,
activation by K
at 100 mM NaCl (V
values are 0.465 ± 0.008, 5.19
± 0.07, and 0.075 ± 0.003 µmol/(mg
min) for
pineal gland, axolemma, and HeLa cells, respectively).
, pineal
gland;
, axolemma;
, transfected HeLa cells. In the insets the data were fitted to .
where K is the apparent affinity for
Na
, and [Na] is the Na
concentration or (ii) the cooperative, n-site model
described by the following form of the Hill equation (cf. (32) ):
where ``cat'' represents the cation (K or Na
). was fitted to the
experimental points and the values of V
, K, and n obtained. K
representing K
or
K
was obtained from through
the relationship K
= (K)
.
The results of
kinetic experiments carried out with 1-containing membranes
isolated from rat kidney, axolemma, and rat
1-transfected HeLa
cells are shown in Fig. 1, and the results for membranes rich in
3, in Fig. 2. The data are expressed as percentages of V
and the curves are best fits to .
The insets in Fig. 1A and Fig. 2A represent the same data fitted to .
As shown in Fig. 1A, the apparent Na affinity of
1 from kidney, with the K
concentration held
constant at 10 mM, appears somewhat lower than that of
1
from either axolemma or HeLa; K
values
were 6.6 ± 0.6, 4.7 ± 0.9, and 5.0 ± 0.3 mM for the three tissues, respectively. At a higher K
concentration (20 mM; cf. (18) ), the
difference in the K
value for
1 of
kidney became greater as indicated below (see Fig. 4). In the
case of K
activation (Fig. 1B), the
order of apparent affinities (assayed at 100 mM Na
) are as follows: axolemma < kidney
HeLa, with K
values of 2.4 ± 0.5,
0.9 ± 0.1, and 1.1 ± 0.3 mM, respectively. For
the
3 isoform of the enzyme, Fig. 2A shows that
the apparent affinity for Na
of
3* from HeLa
cells is markedly lower than that of
3 from either pineal gland or
axolemma; K
values were 11.1 ±
1.5, 4.9 ± 0.8, and 5.7 ± 0.9 mM, respectively.
The K
-activation profiles of
3 pumps (Fig. 2B) show that the apparent affinity for
K
of HeLa
3* pumps is higher than that of other
tissues (K
values were 0.7 ± 0.1,
1.6 ± 0.1, and 1.4 ± 0.4 mM, respectively). The
kinetic constants and Hill coefficients (n) are shown in Table 1.
Figure 4:
Dependance of K ` on
K
concentration for rat pumps from kidney, axolemma,
pineal gland, and transfected HeLa cells. Assays were carried out as
described in Fig. 1, but at varying concentrations of KCl (5,
10, 20, 35, and 50 mM). K
were first
determined by fitting the data obtained for each
Na
-activation curve to and were then
plotted as a function of KCl concentration. Each point represents an
average ± S.D. of at least three separate experiments, and the
values of K
and K
obtained
are shown in Table 2. A,
1 pumps:
, kidney;
, axolemma;
, transfected HeLa cells. B,
3
pumps:
, pineal gland;
, axolemma;
, transfected HeLa
cells.
In other experiments (not shown), the possibility
that the results from axolemma membranes were flawed by an incomplete
distinction of 1 from
3 was tested. Thus, since axolemma
1 activity is measured as the difference in ATPase activity in the
presence of low and high ouabain concentrations, and
3, as the
difference in activity in the absence and presence of low ouabain, a
spuriously lower-than-true apparent affinity of axolemma
1 and
higher-than-true apparent affinity of
3 for K
may
have resulted from the well documented K
-mediated
decrease in ouabain binding(34) . In other words, incomplete
inhibition of
3 at high K
concentrations would
effect an apparent affinity decrease in the former and affinity
increase in the latter curves relating activity to K
concentration. In order to rule out this possibility, even though
this K
/ouabain antagonism is minimal in rat brain
preparations(34) , an experiment was carried out in which the
K
concentration was varied in the presence of
different concentrations of ouabain (5, 10, and 20 µM). It
was found that K
for the
1 enzyme
remained constant at all three ouabain concentrations. Moreover, it
should be noted that the enzyme was preincubated with ouabain in the
absence of K
(see ``Experimental
Procedures''), and that the enzyme activity measured thereafter
remained constant as a function of time.
Figure 3:
Potassium inhibition of Na,K-ATPase of
kidney, axolemma, pineal gland, and transfected HeLa cells at low
Na concentration. Membranes were prepared and ATPase
activity assays performed as described in Fig. 1and Fig. 2, but in the presence of 5 mM NaCl. The
representative experiments show the mean ± S.D. of triplicate
determinations expressed as percentages of the activity measured at 2
mM KCl. A,
1 pumps:
, kidney;
,
axolemma;
, transfected HeLa cells. B,
3 pumps:
, pineal gland;
, axolemma;
, transfected HeLa
cells.
It has been observed that the
antagonistic effect of vanadate, a potent inhibitor of the sodium pump,
is facilitated by the presence of K ions(35, 36) . To ensure that the
K
-mediated inhibition observed in this study is not
the result of vanadate present in the kidney preparation, two control
experiments were performed: (i) in one, assays were carried out in the
presence of 2.5 mM norepinephrine, which reverses the effect
of vanadate(36) , and (ii) in the other, the assay time was
reduced 10-fold and the amount of kidney microsome sample increased
10-fold; if vanadate were present in the microsome suspension, such an
increase in endogenous vanadate concentration should result in greater
inhibition of activity. Neither of these conditions altered the
K
-inhibition profiles, which argues against an
apparent K
inhibition secondary to the presence of
vanadate in the kidney preparation.
Based on the Albers-Post model
of the Na,K-ATPase reaction mechanism and, more specifically, on the
model which assumes random binding of Na and
K
to (the same) three equivalent sites on the
cytoplasmic side of the enzyme, Garay and Garrahan(31) , in
their studies on Na
efflux in red cells, and
Sachs(37) , in studies of ouabain-sensitive ATPase activity in
broken red cell ghosts, showed that activity adhered closely to the
following relationship:
where [Na] and [K]
are the cytoplasmic concentrations of Na
and
K
, respectively. In accordance with this model, the
plot of K
, the apparent affinity for sodium,
as a function of K
concentration yields the linear
relationship K
= K
(1 +
[K]
/K
) (Equation
4; see (37) ). From this plot, K
, the
apparent affinity for Na
when the K
concentration is zero, as well as K
, the
apparent affinity for K
at the cytoplasmic
Na
binding site, are readily obtained (cf. (37) ). In order to apply this analysis to our system, a series
of experiments were carried out in which K
was
determined at various K
concentrations for each of the
tissues studied in Fig. 1and Fig. 2. It should be noted
that the plots of K
shown in Fig. 4, A and B, are best fits to the model described by (see insets of Fig. 1A and
2A), which adheres to the noncooperative assumptions of Garay
and Garrahan, while the data of Fig. 1and Fig. 2were
best fits to . The values of K
and K
, representing the cytoplasmic binding constants
for Na
and K
, respectively, and
calculated from the Fig. 4plots, are shown in Table 2.
In comparing these values, it is evident that the main difference
between the 1 pumps of kidney and those of HeLa and axolemma is
the higher apparent affinity for K
as a competitive
inhibitor of Na
. In the case of the
3 isoforms,
however, the lower apparent affinity for Na
characteristic of transfected HeLa cells (Fig. 2A) is a function of both a lower affinity for
Na
as an activator as well as a higher affinity for
K
as a competitive inhibitor, as compared to either
the pineal gland or axolemma
3 enzymes. Thus, the ratio K
/K
reflects the ability of
K
to compete with Na
for the
cytosolic cation binding site and the larger the ratio, the more
susceptible the enzyme is to competitive inhibition by
K
. A large K
/K
ratio explains the greater K
inhibition, at low
Na
concentration, observed in the case of kidney pumps
and HeLa
3* pumps, as depicted in Fig. 3, A and B. As well, since K
/Na
antagonism should decrease as the Na
concentration is increased, the curves of enzyme activity versus Na
concentration shift to the right,
resulting in the lower apparent affinities for Na
, as
noted in Fig. 1A and Fig. 2A.
Figure 5:
Coimmunoprecipitation of the subunit
with
1 and
3 from rat axolemma. Membranes were prepared,
solubilized in 1% Triton X-100 and immunoprecipitated with either an
1- or
3-specific monoclonal antibody as described under
``Experimental Procedures.'' Following SDS-polyacrylamide gel
electrophoresis, the proteins were analyzed by Western blotting using
polyclonal antisera specific for: A,
1; B,
3; C,
1; and D,
2. Lanes are: 1, axolemma; 2, kidney; 3, Immunoprecipitate
from axolemma using
1-specific monoclonal antibody 6H; 4,
Immunoprecipitate from axolemma using
3-specific monoclonal
antibody M7-PB-E9; and 5, control: immunoprecipitation
performed in the absence of the primary antibody. Molecular masses are
given in kilodaltons.
When a mouse monoclonal antibody specific for
1 was used to immunoprecipitate the enzyme of Triton
X-100-solubilized axolemma membranes, the only subunit isoforms
detected on Western blots using rabbit polyclonal antisera specific for
1,
3,
1, and
2 as primary antibodies, were
1
and
1 (Fig. 5, A-D, lanes 3).
Further, when a mouse monoclonal antibody specific for
3 was used,
3 and
1 were detected along with a barely visible band
corresponding to
2 (Fig. 5, A-D, lanes
4). Control experiments carried out omitting the precipitating
antibody showed minimal amounts of nonspecific binding of the axolemma
Na,K-ATPase subunits to the protein G-linked agarose beads (Fig. 5, A-D, lanes 5). The fact that
the appearance of two bands, one of slightly higher mobility than
1 and
3 (Fig. 5, A and B, lane
3), the other at
50 kDa (Fig. 5C, lanes
3-5), reflect nonspecific reactions was evidenced in the
following controls (not shown). (i) The first band was present even
when the primary detecting antibody was omitted, thus indicating that
it is the result of nonspecific binding of the secondary blotting
antibody to the primary immunoprecipitating antibodies. (ii) The second
nonspecific band appeared even when the primary antibody (mouse
anti-
1 or -
3) was omitted (Fig. 5C) or when
the procedure was carried out in the absence of solubilized axolemma
(not shown), indicating that it probably represents a nonspecific
reaction involving the primary blotting antibody to
1 and goat
anti-mouse antibodies.
The estimate of 1:
1 stoichiometry in
axolemma was based on a comparison of the densities of the
and
bands of axolemma immunoprecipitated with anti-
1 monoclonal
antibody with those of unprecipitated kidney microsomes, following
exposures to anti-
1 and anti-
1 antisera as shown in Fig. 5, A and C (lanes 2 and 3). The ratio of
1 to
1 in precipitated axolemma was
found to be 1.08 ± 0.17 (S.E. for five independent experiments).
Because there is no tissue in which 3 and
1 have been
shown to be expressed in a 1:1 ratio, the question of possible
3-
1 versus
3-
2 associations was evaluated
as follows. The
3:
1 ratio as detected in
anti-
3-immunoprecipitated axolemma samples was compared to that
found in unprecipitated axolemma membranes, after correcting the ratio
for the proportion of
1 presumed to associate with
1. The
ratio in the immunoprecipitate of axolemma sample was found to be
reasonably close to the ``corrected'' ratio observed in the
unprecipitated membranes. Thus, if the corrected
3:
1 ratio of
unprecipitated axolemma is normalized at 1.00, the ratio of the
precipitate is 1.08 ± 0.09 (S.E. for four independent
experiments).
It should also be mentioned that in other experiments
(not shown) aimed to determine whether the detergent Triton X-100
interfered with subunit interactions, immunoprecipitations were also
carried out with a 4-fold lower concentration of Triton X-100 (0.25%)
and with 1% CHAPS. Under both conditions, the :
ratios
obtained were not significantly different from those observed with 1%
Triton X-100 (data not shown).
In this study we show that the divergent results regarding
the relative affinities of the Na,K-ATPase of the different rat
isoforms as reported in different laboratories are not simply accounted
for by differences in the experimental conditions used. Thus, we have
reproduced the relative cation affinities for 1 versus
3 as reported by Jewell and Lingrel (19) and Munzer et al.(20) on the one hand, and those of Sweadner (17) and Shyjan et al.(18) , on the other. To
gain insight into the basis for this dichotomy, we have assessed the
apparent cation affinities of pumps of the same catalytic isoform,
either
1 or
3, but from different tissues and, therefore,
membrane environments. Marked differences in the apparent affinities
for both Na
and K
were observed in
pumps of the same
isoforms isolated from different cellular
sources. These data and previous work in lamb (22) and dog (41) tissues are consistent with the conclusion that factors
other than the type of catalytic isoform influence interactions of the
pump with Na
and K
.
The most
obvious tissue-specific protein component which interacts with the pump
is the subunit. In fact, effects of different
subunits on
both K
(10, 11, 12) and
Na
(13, 14) affinities have been
described. Accordingly, one question is whether the kidney
enzyme's lower apparent affinity for Na
and
higher apparent affinity for K
as compared to other
1 pumps are the result of interactions of
1 with different
subunits. To address this question, particularly in axolemma, in
which both
1 and
2 have been identified, the nature of the
subunit which coimmunoprecipitates with the distinct
subunits was assessed. The results of these experiments indicate that
1 associates with
1 in axolemma and that the stoichiometry of
the association is close to 1.0. The determination of
/
1
stoichiometries imply that little, if any,
2 associates with
either
1 or
3. Whether
2 associates preferentially with
2 in axolemma remains to be determined. Although HeLa cells
contain human
1, while kidney cells contain rat
1, these
subunits are 95% identical(39) . A difference in both type and
amount of glycosylation has been observed between kidney and brain
1 (42) and is presumably the basis for the differences in
mobilities in immunoblots of kidney and axolemma as shown in Fig. 5C. The possibility remains that these differences
are at least partly responsible for the distinct kinetics, even though
there is evidence that the oligosaccharides are not essential for
primary function (reviewed in (43) ).
It is unlikely that
the presence of distinct s account for differences of
1 of
kidney, axolemma, and HeLa, and of
3 of axolemma and HeLa; in both
instances,
1 is the predominant
isoform present or
associated with
1 or
3. However, tissue-specific differences
in cation affinities, despite similar
pairing, does not
imply that the
subunit has no effect on function. In fact, a
kinetic difference due to the distinct
subunits is observed in
the case of
3. Thus, as shown in Table 2, the ratio K
/K
is 1.7-fold lower in
pineal gland compared to axolemma, reflecting the 1.9-fold difference
in K
. That this difference is a result of
2
association with
3 in the pineal gland and of
1 with
3
in axolemma is supported by a recent report showing a 1.6-fold higher
apparent affinity for Na
of
3
2 compared to
3
1 in Sf-9 cells transfected with these isoform pairs (14) . In that study, the Na
activation
kinetics from which the kinetic constants were obtained were carried
out in the presence of 30 mM K
so that the
difference in apparent affinity for Na
may also
reflect a difference in K
. Other kinetic
differences were not detected. Taken together, these results are
consistent with a role for the distinct
s in modulating
K
interactions at cytoplasmic Na
sites.
An important observation regarding the
coimmunoprecipitation studies presented here is the isoform specificity
of the reactions as evidenced in the coimmunoprecipitation of
with
, but not of
1 with
3. This lack of
coimmunoprecipitation between the different
isoforms is in
contradiction with reports that pumps coimmunoprecipitate as
heterodimers in rat brain and in bacculovirus infected Sf-9
cells(5) . Although it is possible that the detergent (1%
CHAPS) used in that study (5) did not fully solubilize the
membranes, or that the Triton X-100 used in this study disrupted
-
interactions, these are unlikely explanations, since we
confirmed our results using 1% CHAPS.
It can be argued that certain
methodological procedures may be responsible for some of the
differences observed for the 3 enzyme of axolemma as compared to
that of other tissues. As described above, the activity ascribed to
3 in axolemma is that which is sensitive to 10 µM ouabain. Unfortunately, the ouabain-affinities of
2 and
3, both present in axolemma, are quite similar(4) , and it
was technically difficult to distinguish the two on that basis.
However, the amount of
2 in axolemma is relatively low
(
25%)(20) , so that its effect on the kinetic behavior
cannot account for the magnitude of the differences in the observed
kinetic constants as discussed below. As well, the similar fold
difference in apparent affinity for external K
ascribed to
1 versus
3 in two separate systems
(transfected HeLa cells compared to axolemma- and kidney-fused red
blood cells; see (20) ) argues against a substantial
contribution of
2 to the behavior of the ouabain-sensitive pumps
of axolemma. For the same reasons, it is unlikely that the mutation of
3 to render it ouabain-resistant in HeLa cells alters its
behavior.
There has been some evidence that detergents, such as SDS
used here to increase the permeability of membrane vesicles to
substrates, can have an effect on cation activation kinetics of the
Na,K-pump(44) . Although such an effect was not observed in the
case of the Na-activation profile of axolemma enzyme,
or in the case of the Na
and K
activation profiles of transfected HeLa cells (data not shown),
it is entirely possible that other pumps might react differently to SDS
treatment. However, the SDS concentration and the SDS:protein
concentration ratio were identical in all of our experiments.
Therefore, if SDS affects the different enzymes to varying extents, it
should be the result of differential interactions with the surrounding
environment, which would be consistent with the notion that the
catalytic behavior of the pump does not depend solely on the isoform of
the
subunit.
The most intriguing results of this study concern
tissue-distinct K/Na
antagonism.
Differences in K
, the apparent affinity for
K
at (cytoplasmic) Na
activation
sites, underlie tissue-specific differences in the sodium-activation
profiles noted in both the present and earlier
studies(17, 18, 19, 20) . There is
evidence also of differences in apparent Na
affinity,
independent of K
concentration, between pumps of the
same catalytic isoform, although this difference was slight in the case
of the kidney enzyme compared to other
1 pumps (see Table 2). In general, the ability of K
to act as
a competitive inhibitor of Na
binding is reflected in
the ratio of K
to K
, the
cytosolic binding constants for Na
and
K
, respectively. It is apparent from Table 2that, of the membrane systems examined,
1 in the
kidney and
3 transfected into the HeLa cell have the largest K
/K
ratios when compared to
other pumps with the same
isoforms. It is these two enzyme
preparations which exhibit the greatest sensitivity to inhibition by
K
as depicted in Fig. 3. Specifically, it seems
that in the case of kidney
1, this inhibition is due to its
relatively lower K
compared to the other
1
pumps, whereas with HeLa
3 pumps, it is due mainly to a higher K
(Table 2). Whether these characteristics
are intrinsic to the enzyme, for example due to tissue-specific co- or
postranslational modification(s), or rather, the result of modulation
of the enzyme by another associated protein remains unresolved.
The
kinetic analyses of the sigmoid activation kinetics described in this
and previous studies (17, 18, 19, 20) are based on
conventional cooperative or noncooperative models. Using the
noncooperative model, the apparent affinities of 1 and
3 for
intracellular Na
and K
of the same
tissue (HeLa) derived in the present study can account for the low
affinities of
3 compared to
1 observed in studies of Munzer et al.(20) using intact cells, with the following
provisos. Those authors pointed out that their data points for
Na
activation of
3 pumps could be obtained only
in the region of the curve well below saturation due to the technical
difficulty of raising intracellular Na
above
45
mM. This precluded a reliable estimate of the kinetic
constants for
3 when using the noncooperative model. However, the
data fitted well to a cooperative model (Equation 2 in (20) )
giving K
values for intracellular
Na
of 17.6 mM for
1 and and 63.5 for
3. In the present study, the K
and K
values for rat
1 and
3 in HeLa
membranes (Table 2) were used to obtain the observed apparent
affinity, K
, at 135 mM intracellular
K
, a concentration approximating that of the intact
cells used by Munzer et al.(20) . Using these values
of K
(7.1 mM for
1 and 30.3
mM for
3) we derived curves of pump activation as a
function of varying intracellular Na
using the
cooperative model (Fig. 6). The curves (solid lines)
and the values of K
thus obtained (19.2
mM for
1 and 75.2 mM for
3) are similar to
those derived from the data of previous studies (20) with
intact cells (Fig. 6, dashed lines). Therefore, these
results indicate that the physiologically significant extremely low
apparent affinity of
3 reflects its much greater sensitivity to
inhibition by intracellular K
.
Figure 6:
Comparison of
Na-activation curves derived from kinetic constants in Table 2and those obtained in transport studies with intact
cells. Using Equation 4, K
and K
values from Table 2were used to obtain
K
for
1 and
3 at 135 mM K
(cf. (20) ). The
K
values thus obtained (7.1 mM for
1 and 30.3 mM for
3) were used to derive curves (solid lines) of percent of V
as a
function of Na
concentration using the cooperative
model () with n = 3.0 as in Munzer et
al.(20) . Data points for
1 (
) and
3
(
) were taken from Fig. 6of Munzer et al.(20) and are also expressed as percent of V
. The dashed curves were derived from
the K
values for intracellular Na
shown in Table 2of Munzer et
al.(20) .
Modulation of pump
behavior by the membrane environment is most likely the explanation for
the discrepancies among previous reports concerned with
isoform-specific behavior. A slightly higher Na affinity in axolemma compared to kidney was reported first by
Sweadner (17) , and later by others including Shyjan et al.(18) who compared brain and kidney. This observation was
replicated in the present study of kidney and axolemma Na,K-ATPase,
with values very close to those reported by Sweadner, i.e.
K
values of 0.72 mM and 1.02 mM,
respectively (Table 2) corresponding to K
values of 4.0 mM and 4.3 mM for axolemma and
kidney, respectively (not shown).
Tissue-specific as well as
isoform-specific behavior is evident not only in apparent affinities
for cytoplasmic Na and K
but also for
K
at extracellular sites (Table 1). Thus, it was
only in the same (red cell or HeLa cell) environment that a higher
affinity for extracellular K
of
3 or axolemma
compared to kidney (
1) was observed(19, 20) . It
may be relevant that exogenous kidney pumps fused into red cells and
endogenous red cell pumps behave identically with respect to the
apparent affinity for extracellular K
, K
(45) . (The greater difference
between
1 and
3 observed in studies with intact cells may
reflect the limitation of kinetic studies with unsided preparations).
The foregoing considerations argue in favor of the conclusion that
the primary structure of the isoform is not the sole determinant
of the magnitude of cation affinity/selectivity. Our observations are
consistent with the existence of some pump modulator, for example one
which interacts and effects a greater sensitivity of
1 to
K
inhibition in the kidney compared, for example, to
1 of axolemma; in the microsome-fused red cell system, association
of the
subunit with the putative regulator may be interrupted
following its association with other components of the new (red cell)
environment. This kind of regulation is reminiscent of the effects of
an intrinsic red cell membrane (blood group) antigen, Lp, found in
genetically low potassium sheep red blood cells. This protein or
glycoprotein interacts with the pump and effects
K
-inhibition (for review, see (46) ).
Interestingly, when kidney pumps are delivered from microsomes into low
potassium sheep red cells, the Lp antigen effects susceptibility to
K
inhibition(47) , which supports the idea
that fusion into red blood cells confers a new membrane environment for
the pump.
The study described in this report provides evidence in
support of the conclusion that factors in addition to the primary
structure of the isoforms dictate the kinetic behavior of the
Na,K-ATPase. Likely candidates include other membrane-bound components
or modulation by co- or post-translational modifications of either
subunit.