From the Department of Medicine, McGill
University, Montreal, Quebec H3G1A4, Canada, § INSERM U478,
Institut Federatif de Recherche 02, Faculte de Medecine X. Bichat, BP
416, 75870 Paris Cedex 18, France, and the ¶ Department of
Biological Chemistry, Weizmann Institute of Science,
Rehovot 761001, Israel
Received for publication, November 30, 2000, and in revised form, March 6, 2001
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ABSTRACT |
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The A small membrane protein, On SDS-polyacrylamide gel electrophoresis, the Although earlier studies showed that the The functional role of the Studies in our laboratory (8, 11) have shown that Based on the recent structural analysis of Kuster et al.
(10) showing two main Microsome Preparation--
Rat kidney outer medulla microsomes
(3-4 units/mg of protein) or partially purified Na,K-ATPase (10-15
units/mg of protein) were prepared as in Ref. 19.
Antibodies--
Two preparations of anti-
Immunocytochemical experiments were performed with affinity-purified
anti- Immunolocalization of the Cloning and Transfection of Rat Enzyme Assays and Kinetic Analysis of Data--
Membranes were
prepared from HeLa cells as in Ref. 21. Unless indicated otherwise,
Na,K-ATPase and Na-ATPase assays were carried out as described
previously (11). Na-ATPase activity refers to activity measured at low
(1 or 10 µM) ATP concentration, in the absence of
K+. For experiments with HeLa cells expressing the
ouabain-resistant rat
Data for Na+ and K+ activation of Na,K-ATPase
activity were expressed as percentages of Vmax
using the Kaleidagraph computer program (Synergy Software) with the
noninteractive model of cation binding described by Garay and Garrahan
(24),
Evaluation of K+ antagonism of Na+ activation
at cytoplasmic sites was determined by analyzing Na+
activation profiles as a function of K+ concentration. As
in our previous study (26) and based on the Albers-Post model with the
assumption that Na+ and K+ bind randomly at
three equivalent (noninteractive) cytoplasmic sites, the data were
analyzed using the relationship described by Garay and Garrahan
(24),
All experiments shown or described are representative of at
least three similar experiments, except for experiments summarized in
the inset of Fig. 1 and those shown in Fig. 6, in which
cases the means of at least three independent experiments are shown. For the representative experiments shown, each data point is the mean ± S.D. of three replicate samples.
Functional Studies
The experiments described below were carried out to extend
previous investigations of the functional role of Expression in Cultured Cells
We showed recently that expression in cultured human cells
(wild-type HeLa cells and HEK cells), of cDNA encoding the two individual Effects of Fig. 1 confirms the earlier finding
that subunit of the Na,K-ATPase is a member of
the FXYD family of type 2 transmembrane proteins that probably function
as regulators of ion transport. Rat
is present primarily in the kidney as two main splice variants,
a and
b, which differ only at their extracellular N termini
(TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu,
H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish,
S. J. D. (2000) J. Biol. Chem. 275, 18441-18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between
a and
b. At least two singular
effects are seen, irrespective of whether the variants are expressed in
HeLa or rat
1-transfected HeLa cells, i.e. (i) an
increase in apparent affinity for ATP, probably secondary to a left
shift in E1
E2 conformational equilibrium and (ii) an
increase in K+ antagonism of cytoplasmic Na+
activation. Antibodies against the C terminus common to both variants
(anti-
) abrogate the first effect but not the second. In contrast,
a and
b show differences in their
localization along the kidney tubule. Using anti-
(C-terminal) and
antibodies to the rat
subunit as well as antibodies to identify
cell types, double immunofluorescence showed
in the basolateral
membrane of several tubular segments. Highest expression is in the
medullary portion of the thick ascending limb (TAL), which contains
both
a and
b. In fact, TAL is the only
positive tubular segment in the medulla. In the cortex, most tubules
express
but at lower levels. Antibodies specific for
a and
b showed differences in their
cortical location;
a is specific for cells in the macula densa and principal cells of the cortical collecting duct but not
cortical TAL. In contrast,
b but not
a is
present in the cortical TAL only. Thus, the importance of
a and
b may be related to their partially
overlapping but distinct expression patterns and tissue-specific
functions of the pump that these serve.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, first described over 20 years ago
in purified kidney Na,K-ATPase preparations (1, 2) associates, in
approximately equimolar amounts, with the
and
subunits (3, 4).
Molecular cloning of the
subunits of rat, mouse, cow, and sheep
indicated a molecular weight of ~6500 (5). Cloning and sequencing of
the human (6) and Xenopus laevis (7)
subunits have also
been reported. Comparison of sequences shows ~75% homology among
subunits of the aforementioned different species but is much higher
(93%) for only mammalian sequences. Further structural analysis has
shown that
comprises a single transmembrane domain and has an N
terminus-out, C terminus-in topology (7, 8). In addition, two major
forms have been recently identified at the molecular level as described below.
subunit runs as a
doublet (apparent molecular masses of ~8 and ~9 kDa) (5, 8),
and a doublet is observed following expression in tissue culture cells
(8, 9) and in in vitro expression in the presence (5) but
not absence of pancreatic microsomes (5, 7). Recent mass spectrometry
of the
chains of rat kidney Na,K-ATPase showed that
a (upper band on SDS-polyacrylamide gel electrophoresis) has a mass of 7184.0 ± 1 Da (carbamidomethyl cysteine) (10), corresponding closely to that for the published sequence without the
initiator methionine (11), while
b (lower band) has a
mass of 7337.9 ± 1 Da. Tryptic peptide mapping and sequencing by
mass spectrometry reveals that the seven N-terminal residues of
a, TELSANH, are replaced by Ac-MDRWYL in
b, but otherwise the two chains are identical. These
sequences are identical to those obtained by searching the expressed
sequence tag data base (12). Expression of
a or
b cDNAs in human embryonic kidney
(HEK)1 as well as HeLa cells
was analyzed by Western blotting using antiserum raised against a
peptide representing the C-terminal 10 residues of the
subunit. The
results showed clearly that the major bands expressed correspond to
a or
b of the renal Na,K-ATPase.
Additional minor bands seen after transfection, namely
a' in HEK and
b' in HeLa cells imply that
these are cell-specific posttranslational modifications (10).
subunit is co-expressed
with
and
ATPase subunits in kidney and not at the surface of
Xenopus oocytes in the absence of
and
subunits (7), Jones et al. (13) have reported expression of
in the
absence of the sodium pump on the apical surface of mouse blastocysts. In contrast with the ubiquitous localization of
and
subunits, however, the
subunit is expressed in a limited number of organs (8). Earlier studies showed identical expression patterns of
and
in renal proximal tubules and collecting ducts as well as
co-immunoprecipitation of the
subunit with both the
and
subunits in kidney membranes (5).
subunit has only recently begun to be
investigated. Although it was previously shown that the
peptide is
not necessary for function (4, 14, 15) and
subunit mRNA could
not be detected in many tissues in both mammals (5, 6) and amphibia
(7), recent experiments have shown that
has an important functional
role in some systems. Thus, treatment of mouse blastocysts with
subunit antisense oligodeoxynucleotide reduced the amount of expressed
subunit and caused a reduction in ouabain-sensitive
86Rb+ transport as well as delayed blastocoel
formation (13). A recent report describing a mutation in
in a
family with dominant renal hypomagnesemia suggests a role of
in
magnesium reabsorption (16).
is a
tissue-specific regulator of the Na,K-ATPase and that it causes an
increase in the apparent affinity of the enzyme for ATP in a manner
that is reversible by anti-
antiserum. The specific effect of
anti-
on ATP affinity implies a specific structural interaction
whereby the
subunit counteracts short term changes in ATP
concentration in kidney cells in which ATP utilization is high. A role
of the
subunit in interactions of the Na,K-ATPase with
K+ was suggested on the basis of findings that the
subunit is a component of the protein complex found in so-called
"19-kDa membranes," the product of tryptic digestion following
occlusion of K+ or Rb+ by the enzyme to form
E2(K) (17), and partial protection by K+ ions
against tryptic digestion of the
subunit in renal microsomes (8).
In addition, experiments on cRNA-injected Xenopus oocytes have shown that the
subunit has an influence on the apparent affinity of the Na,K-ATPase for K+ in a complex
Na+- and voltage-dependent fashion
(7), although the interpretation of these results remains unclear. A
recent report has attributed modulation of Na+ and
K+ affinities to the
a subunit when
expressed in kidney cells (9). The
subunit has also been shown to
induce ouabain-independent ion currents in injected Xenopus
oocytes and 86Rb+ and
22Na+ influx in baculovirus-infected Sf-9 cells
(18).
species in rat kidney (
a and
b), it is now clear that all aforementioned functional
studies (7, 8, 9, 11, 14, 15) examined effects of only the
a variant, thus raising the critical issue of whether
the two variants have distinct roles. The study described in this paper
addresses two aspects of the role of
in regulation of Na,K-ATPase.
One set of experiments extends the functional studies and examines the question of whether, and to what extent, the two variants have distinct
effects on Na,K-ATPase kinetic behavior. The other set of experiments
concerns the localization of the two
variants along the nephron.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(C-terminal) were
raised in rabbits. One is against both bands of the pig kidney
subunit excised from a gel. The antibody was purified on an affinity
column of the peptide KHRQVNEDEL corresponding to the last 10 residues
of the
subunit; thus, it is referred to as anti-
(C-terminal). The other is
C33, described in Ref. 8. It was raised against a
synthetic peptide consisting of the last 10 residues and used for
enzyme assays without further purification. Anti-
a and
anti-
b antibodies were raised against N-terminal
peptides TELSANHC and MDRWYLC, respectively, after coupling to keyhole
limpet hemocyanin. Anti-
a was purified on an affinity
column of TELSANHC. Attempts to affinity-purify anti-
b
were unsuccessful. For affinity purification, peptides were coupled to
Poros activated affinity chromatography columns (epoxide or amino), and
the antibodies were purified on a BioCad Perfusion Chromatography
apparatus. The antibody was eluted off the column in a solution of 0.2 M Tris-HCl, pH 2, neutralized immediately with Tris base,
and diluted 1:1 with glycerol, and 0.02% sodium azide was added for preservation.
(C-terminal) diluted 1:200 and anti-
a
antibodies (1:100) and anti-
b antiserum (1:200). In
addition, the following polyclonal antibodies (as described in Ref. 20)
have been used for co-localization: (i) anti-aquaporin 2 (AQP2)
antiserum, (1:100); (ii) anti-Tamm-Horsfall protein antibody (sheep
anti-oromucoid from Biodesign International, Kennebunk, ME;
1:50); (iii) antibody against the alpha subunit of Na,K-ATPase
(1:50).
Subunit of Na,K-ATPase in the
Kidney--
Rat kidneys were frozen in liquid N2.
Immunolocalization was performed as previously described (20) by
incubating cryostat sections with the different anti-
subunit
antibodies (
-C-terminal,
a and
b) and
with a secondary antibody (goat anti-rabbit Fab fraction, Jackson;
1:200) coupled to the fluorochrome CY3 (red fluorescence). For
colocalization experiments (20), the CY3-labeled sections were
incubated with antibodies against different protein markers (see above)
and then overlaid with fluorescein isothiocyanate-labeled (green
fluorescence) goat anti-rabbit IgG Fab fraction or donkey anti-sheep
antibody. The specificity of the anti-
antibodies in
immunofluorescence studies was established in competition experiments by including their respective peptides in the treatment of the kidney
sections (data not shown).
a and
b cDNAs--
The cloning and transfection of rat
a and
b cDNAs were carried out
exactly as described for wild-type HeLa cells (10), except that a
stable cell line expressing rat
1 was used (
1-HeLa cells obtained
as a gift from E. Jewell and J. B Lingrel; see Ref. 21). After
transfection, the cells were cultured for 3 weeks in Dulbecco's
modified Eagle's medium containing 10% newborn calf serum and
selected by including 400 µg/ml hygromycin B and 1 µM
ouabain (10) in the medium. Western blot analysis was carried out as
described previously (8) except using antibodies against the C terminus
(
C33) and against the N termini of
a and
b. Quantitative PhosphorImaging was carried out using a
Storm PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
1 catalytic subunit (
1-HeLa), the cells are
grown in 1 µM ouabain and assayed in medium containing a
low (5 µM) ouabain concentration. Activities shown are
the differences in ATP hydrolysis measured in the presence of low (5 µM) and high (5 mM) ouabain concentrations.
For studies with wild-type HeLa cells, activities shown are differences
measured in the absence and presence of 5 mM ouabain. For
studies of sensitivity to vanadate, at each concentration of vanadate,
Na-ATPase assays were carried out in sets of triplicates, without and
with vanadate, with low and high ouabain added to one set of
each (cf. Ref. 22). For practical purposes, either
a- or
b-transfected
1-HeLa were
compared, on the same day, with control
1-HeLa membranes. Data for
the vanadate sensitivity of Na-ATPase activity, expressed as a
percentage of that obtained in the absence of vanadate, were analyzed
by fitting the data to a one-site model using a nonlinear least squares fit (Kaleidagraph computer program) to a general logistic function as
referred to previously (23).
where v represents the rate of the reaction,
V represents the maximal rate, K'cat
represents either K'Na or
K'K (the apparent affinity for Na+
or K+, respectively), [cat] represents the concentration
of cation (either Na+ or K+), and n
represents the number of binding sites (either three in the case of the
Na+ activation experiments or two in the case of
K+). Values of Vmax and
K'cat were obtained from this fitting procedure.
(Eq. 1)
Where [Na] and [K] represent the cytoplasmic concentrations
of Na+ and K+, respectively; v and
V have their usual meaning; KNa is
the affinity for Na+ binding at cytoplasmic activation
sites in the absence of K+; and
KK is the affinity for K+ acting as
an antagonist of Na+ binding at cytoplasmic sites. This
equation predicts a linear relationship between the apparent affinity
constant for Na+, K'Na, and
K+ concentration according to the following
relationship.
(Eq. 2)
(Eq. 3)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and to compare the effects of the two variants,
a and
b.
This was done primarily by comparing the kinetic behavior of
a- and
b-transfected cells with
mock-transfected HeLa cells and, where indicated, by analyzing the
effect of anti-
antibodies on the renal enzyme. Experiments were
initially carried out with
-transfected wild-type HeLa cells and
then repeated with
-transfected
1-HeLa to assure that effects, or
absence thereof, were not specific to the species of
, either rat or
human. Similar findings were obtained with both cell lines. Only those
from either wild-type or rat
1-transfected HeLa cells are shown.
Furthermore, we noted that selection and growth of
1
HeLa cells in
a low concentration of ouabain (cf. Ref. 21) down-regulates
endogenous human
1 such that expressed rat
1 predominates
(experiments not shown).
variants revealed additional bands (see Fig. 5 of Ref.
10). That these additional bands, termed
a' and
b', are due to posttranslational modifications was
evidenced by the cell-specific manner of their appearance following
transfection into cultured cells. Thus,
a' appears
primarily in HEK cells, and
b' appears primarily in HeLa
cells. In the present study, identical results were obtained for
a and
b expressed in rat
1-transfected
HeLa cells (not shown). As in the case of wild-type HeLa, a conspicuous
b' is visible in
b-transfected
1-HeLa,
and only one band is visible in
a-transfected
1-HeLa
cells. Densitometry of the
and
subunits of membranes from
clones of
a-HeLa,
a-
1-HeLa, and
b-HeLa and
b-
1-HeLa, in which
expression is relatively high and used for functional analysis, showed
that the
/
ratios are ~50% that of rat kidney (density of both
bands).
on Apparent Affinity for ATP
a decreases K'ATP for ATP
(low affinity binding) and shows further that a similar (~2-fold)
decrease in K'ATP (low affinity binding) is
effected by
b. Furthermore, this effect appears
to be independent of the presence (HEK cells; Ref. 11) or absence (HeLa
cells; this study) of
a'. It was also noted earlier that
this effect of
is abrogated by anti-
antibody as evidenced in
the increase in K'ATP following preincubation of
either
a-transfected HEK cells or the rat kidney enzyme
with the antiserum
C33 raised against the last 10 residues of the C
terminus. Similar effects of
a and
b were
observed in other experiments (not shown) in which the cDNAs of
these variants were transfected into
1-HeLa cells. Although anti-
(C-terminal) decreases the Vmax of kidney enzyme
(8), variation in specific activity among different membrane
preparations from the HeLa cells (see the legend to Fig. 1) precluded
the determination of
effects on Vmax.
View larger version (21K):
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Fig. 1.
Effects of
a and
b on ATP affinity. Membranes
isolated from
a- and
b-transfected as
well as control mock-transfected HeLa cells were assayed for
Na,K-ATPase activity at varying ATP concentrations (50, 100, 200, 300, 500, and 1000 µM) in the presence of 100 mM
NaCl, 10 mM KCl, and 40 mM choline chloride as
described under "Experimental Procedures." Base-line values (5 mM ouabain added) were subtracted. Values ± S.D.
shown are the differences in activities obtained after subtraction of
the base-line values. Data were analyzed by the Michaelis-Menten
formulation using the linear Lineweaver-Burk transformation.
Inset, K'ATP values ± S.D shown are the averages of three or four separate experiments,
each of which was carried out with membranes of either
a- or
b-transfected HeLa and control HeLa
membranes. K'ATP values for both
a and
b compared with control are
considered statistically significant (p < 0.01).
Average Vmax values ± S.D. (nmol/mg/min)
for control and
a- and
b-transfected
cells were 254.5 ± 12.7, 168.5 ± 13.8, and 225.1 ± 10.1, respectively.
Effect of on the Steady-state Conformational Equilibrium:
Studies with Vanadate
The question of whether the effect of on
K'ATP is primarily on interaction of the enzyme
with ATP and/or secondary to an alteration in the conformational
equilibrium was tested by using inorganic orthovanadate as a probe of
the E2 conformation. Accordingly, vanadate
sensitivity of ATPase activity was tested under conditions in which its
effect should not be secondary to an alteration in Kext interaction with the enzyme. This was done
by measuring the steady-state hydrolysis of the Na-ATPase activity with
ATP added at low concentration (1 µM) to assess turnover
of the enzyme in the absence of K+ as in Ref. 22 with
modifications described in the legend to Fig. 1. In one series of
experiments, we tested the effect of anti-
(C-terminal) on the renal
enzyme. As shown in Fig. 2A, the sensitivity to vanadate is increased; the I50 is
decreased ~2-fold (see legend to Fig. 2). As predicted, the opposite
effect was observed with the two
variants. In the typical
experiment shown for
a in Fig. 2B, it is
evident that, compared with control (mock-transfected)
1-HeLa cells
(see "Experimental Procedures"), the sensitivity to vanadate is
decreased. The I50 for vanadate was increased ~2-fold.
Similar results were obtained for
b-transfected
1-HeLa cells (not shown; p also <0.01). These results
are consistent with the conclusion that both
a and
b shift the E1
E2 equilibrium in favor of
E1 form(s) as suggested in earlier studies (8, 11, 25). Those experiments showed that (i) inhibition of the kidney
enzyme by anti-
is greatest at acidic pH (pH 6.2) and least at
alkaline pH (pH 8.9), under which conditions the
E2 (K)
E1
transition is either the major rate-limiting or a non-rate-limiting step of the reaction, respectively.
|
Effects on Cation Interactions
K+ Interactions Relevant to the E2P
E2 (K)
E1 Pathway--
Treatment of
the rat kidney enzyme with anti-
decreases the
K'K for K+ activation ~25%, at
least at suboptimal ATP concentration (25). Those results are
consistent with the conclusion that effects on
K'K are secondary to effects of
on the
E1/E2 conformational equilibrium. However, similar experiments aimed to show the presumably opposite effect (increase in K'K) of
a and
b on
1-transfected HeLa cells
were equivocal due to the large variances in small increases (
25%)
in K'K values due, presumably, to much lower specific activity and higher nonspecific ATP hydrolysis of HeLa membranes. In other experiments (not shown) in which the
ouabain-sensitive influx of
86Rb+(K+) was assayed as in Ref.
23, a significant difference in the apparent affinity for extracellular
K+ between either
a- or
b-transfected and mock-transfected
1-HeLa cells could
not be detected (experiments not shown). Nonetheless, Fig.
3 shows clearly that when the ATP
concentration was reduced to 10 µM so that the
K+ deocclusion reaction becomes strongly rate-limiting,
both
a and
b increased the extent of
K+ activation of Na-ATPase of HeLa cells. Similar results
were obtained with
1-HeLa cells (experiments not shown). This result
is consistent with the conclusion that both variants increase the rate
of the rate-limiting E2(K)
E1 reaction, an effect abrogated by
anti-
.
|
K+ Antagonism of Na+ Activation--
One
of the notable differences between 1
1 pumps of kidney compared
with many other tissues is the notably lower apparent affinity for
Na+ (increased K'Na) in kidney,
which is readily seen at high K+ concentrations (
20
mM; Ref. 26). In that study, we showed that this increase
in K'Na is accounted for by the higher apparent affinity
for K+ (KK) as an antagonist at
cytoplasmic Na+ activation sites rather than a difference
in Na+ affinity, per se. At face value, the
notion that this effect is due to interaction with
seems unlikely,
since this K+/Na+ antagonism is even more
dramatic in heart tissue (26) that is devoid of
(8).
Nevertheless, to determine whether the
subunit is relevant to this
phenomenon in the kidney, the following experiments were carried out:
(i) an analysis of the sensitivity of the enzyme to inhibition by
K+ at low (5 mM) Na+ concentration
(Fig. 4) and (ii) an extensive
kinetic analysis to determine
K'Na as a function of K+
concentration. The results for
transfected
1-HeLa cells are shown in Figs. 5 and
6 and Table
I. Similar results were obtained with
transfected wild-type HeLa cells (not shown). Fig. 4 shows that,
compared with the control, cells transfected with either
a or
b are more inhibited by
K+ at low Na+ concentration compared with
mock-transfected control cells. Since stabilization of
E1 by the
subunit, described above, should itself have the effect of lowering the apparent affinity for
K+ ions as antagonists of Na+ ions at
cytoplasmic sites, the result in Fig. 4 shows that there must be an
additional effect of
on the cation sites.
|
|
|
|
Fig. 5 is a kinetic analysis of the effect of varying Na+
as a function of K+. The results shown are representative
comparisons of a- and
b-transfected and
control mock-transfected
1-HeLa activity profiles determined at 20 and 100 mM K+. The data were analyzed by
fitting the Na+ activation curves to a noncooperative model
of Na+ activation (cf. Ref. 24). As indicated in
the legend to Fig. 5, both
a and
b
decrease the apparent Na+ affinity, and the decrease is
greater at the higher K+ concentration. Fig. 6 shows the
results of a large series of similar experiments carried out for both
variants at varying K+ concentration. The plots show the
relationship between K'Na and K+
concentration for control (mock-transfected) and
a- and
b-transfected
1-HeLa cells. As described under
"Experimental Procedures," the data were analyzed according to
Equation 3, which predicts a linear relationship between
K'Na and cytoplasmic [K+] whereby
KNa and KK are the
affinity constants for Na+ (extrapolated to
[K+] = 0) and for K+ at cytoplasmic site(s),
respectively. At each K+ concentration, the data point
shown is the mean of at least three separate determinations of
K'Na. The linearity of the plots indicates that
this relationship provides a valid basis for using our data to estimate
KNa and
KNa/KK. As shown by the
data summarized in Table I, both
a and
b
have similar effects. Both increase
KNa/KK with no
significant effect on KNa, consistent with the
conclusion that the effect of both variants is mainly due to a decrease
in KK, i.e. an increase in the
affinity for K+ acting as an antagonist of Na+
binding at cytoplasmic sites. It is noteworthy that (i) the kinetic constants for control
1-HeLa taken from Fig. 6 are virtually identical to those reported earlier (26), indicating the high reproducibility of the assays, (ii) the same effects of the two variants were observed in a similar series of experiments carried out
with wild-type HeLa cells (experiments not shown), and (iii) the
magnitude of the change in
KNa/KK (~50% increase)
effected by
is notably similar to the higher (50%)
KNa/KK of kidney compared with
1-HeLa cells reported earlier (26).
As summarized in Table II, antisera
raised against the cytoplasmic C- and extracellular N-terminal regions
of failed to abrogate the effect of
on
K+/Na+ antagonism. Thus, treatment of the rat
kidney enzyme with antiserum
C33 raised against the C terminus
failed to change the magnitude of K+ inhibition noted at
low Na+ concentration (75% inhibition in both the presence
and absence of 100 mM K+); the same holds true
of anti-
a raised against the N terminus of
a (see below).
|
Are Interactions Involving the N Terminus Relevant to the Effects
of ?
This issue was addressed using anti-a antiserum
raised against the extracellular N terminus. As summarized in Table II,
anti-
a failed to affect the activity at either limiting
or nonlimiting concentrations of either ATP, Na+, or
K+ or affect the concentration dependence of
inhibition by ouabain (experiments not shown).
Immunolocalization
The intrarenal pattern of expression of the subunit of
Na,K-ATPase was examined by immunofluorescence in the renal cortex and
outer and inner medulla (Fig. 7). The
anti-
(C-terminal) antibody alone (CY3, red fluorescence), decorates
several tubules (Fig. 7, A-C). All regions show staining,
but it is particularly strong in outer medulla. Double
immunofluorescence experiments were performed using antibodies against
the
subunit of Na,K-ATPase (Fig. 7, D-F), against AQP2
(apical membrane of principal cells of the collecting duct) in
G-I and against Tamm-Horsfall protein (apical membrane of
the thick ascending limb of Henle's loop) in J-L, all
evidenced by their fluorescein isothiocyanate-green fluorescence.
Colocalization with the
subunit appears in yellow. The
subunit of Na,K-ATPase (Fig. 7, D-F) is apparent in all tubular segments, as expected: proximal tubules, loop of Henle, and
distal nephron. In outer and inner medulla, there is extensive overlap
of
and
subunits (E and F), but in the
latter some nephron segments express
but no
subnit
(F). In cortex, some cells (Fig. 7D) clearly show
colocalization (arrow). Since they lie in close vicinity to
the glomerulus, they may be macula densa cells; this is documented
further in Fig. 8. Colocalization of AQP2
and the
subunit (Fig. 7, G-I) was apparent in
the cortex only, indicating that the collecting duct, in its cortical
portion only, expresses the
subunit. Tamm-Horsfall protein (Fig. 7, J-L) colocalizes with the
subunit in medullary TAL
(mTAL) (K, in yellow), with respective apical and
basolateral expression. Thus, these experiments show that the
subunit of Na,K-ATPase has predominant expression, at the protein
level, in the mTAL, with few positive cortical TAL (cTAL), and faint
labeling of proximal tubules and cortical collecting ducts. Its
expression is more restricted than that of the
subunit of the
sodium pump, indicating that both subunits are not systematically
coexpressed.
|
|
The recent identification of a splice variant of the subunit of
Na,K-ATPase leads to the notion that the two forms,
a
and
b, may have different cellular expression and
function. In order to establish whether
a and
b have distinct localizations, immunofluorescence experiments were performed using antibodies specific for each
variant, namely anti-TELSANH, and anti-MDRWYL representing the two
extracellular N-terminal sequences that differ in
a and
b, respectively. Fig. 8 documents the expression of
a and
b in the cortex (A-H)
and medulla (I-P). These antibodies were used either alone
(A, E, I, and M, in
red) or together with antibodies (in green)
raised against either the
subunit, anti-AQP2, or anti-Tamm-Horsfall
protein, as indicated in the legend to Fig. 8. In the cortex,
a is expressed in most cortical tubules (proximal tubules, cortical collecting ducts) together with the
subunit of
the pump (except in cTAL, appearing in green), while
colocalization with
b is detectable only in few tubules
(i.e. cTAL) in yellow. In particular, the
a form is clearly present together with the
subunit
in the macula densa (MD) cells (Fig. 8B, arrow)
(i.e. at the very end of the cTAL (labeled by an
asterisk)); both subunits colocalize in the MD basolateral
membrane lining the glomerulus. In contrast, the
b form
was never found in MD cells. In the cortical collecting duct (labeled
cd), as identified by its apical expression of AQP2 in
principal cells (in green in Fig. 8C), the
a variant is in the opposite membrane (i.e.
in the basolateral membrane of principal cells), while these cells have
only background fluorescence for
b (Fig. 8G);
proximal tubules (labeled p) express both
forms at low
levels. Of particular interest is the observation that the cortical
portion of the thick ascending limb of the loop of Henle (cTAL)
expresses only
b (Fig. 8H), not
a (Fig. 8D), as shown by selective
colocalization of
b with Tamm-Horsfall protein.
Panels I-P illustrate the immunofluorescence for
similar immunocytochemistry carried out with sections at the junction of the outer medulla and inner medulla. Antibodies specific for
a and
b show that each has a restricted
pattern of expression. This expression is superimposed on that of the
subunit of the Na,K-ATPase in some (mTAL), but not all tubules
(Fig. 8, J and N), since medullary collecting
tubules (mcd) are positive only for the
subunit, while
others (mTAL; labeled by an asterisk) express
together
with either
a (Fig. 8J) or
b
(Fig. 8N) although with lower intensity. The absence of
a and
b in the medullary and papillary
collecting duct is shown by the distinct localization of AQP2 and
either
a or
b (Fig. 8, K and
O). All along the medullary thick ascending loops
(identified with anti-Tamm-Horsfall protein antibody) there is evidence
of both
a (Fig. 8L) and
b
(Fig. 8P).
It is relevant that other
studies2 showed that (i)
anti- immunoprecipitated both
a and
b
from C12E8-solublized membranes and (ii) either
anti-
a or anti-
(C-terminal) immunoprecipitated the
subunit.
However, anti-
a did not immunoprecipitate
b. This result suggests that
/
subunits are
present either as the complex
/
/
a or
/
/
b, but not
/
/
a/
b, but does not, of course,
exclude the possibility of
/
subunits without associated
subunits.
On the whole, these results indicate that the subunit of
Na,K-ATPase has a restricted pattern of expression along the nephron, as compared with the
subunit, with prevalent expression in the mTAL, and, at lower levels, in the proximal tubule, and the
collecting duct in its cortical part only. The
a form is
in the basolateral membrane of MD cells and of cortical collecting duct
principal cells as well as in the medullary portion of the thick
ascending limb of Henle's loop. The
b form is
selectively expressed all along the thick ascending limb of
Henle's loop (Table III).
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DISCUSSION |
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---|
Effects of a and
b on Catalytic
Functions--
In earlier studies we showed that anti-
antiserum
raised against the C terminus of
inhibits Na,K-ATPase of the renal
enzyme but not of tissues that do not express
(8). Further analysis showed that anti-
decreases the apparent affinity for ATP probably by stabilizing the E2 form(s) of the enzyme.
Thus, the pH dependence of the anti-
-mediated inhibition of activity
is consistent with a role of anti-
in shifting the equilibrium of
the K+ deocclusion reaction (E2(K)
E1) toward E2(K). It
was hypothesized that anti-
mediates its effects by disrupting
interactions between the Na,K-ATPase complex and the
subunit, such
that the role of the
subunit is to shift the equilibrium toward
E1. By transfecting one of the two
variants
(
a) into HEK cells, it was then shown that this is
indeed the case, at least for
a (11). The studies described in this paper show that the same holds true for the
b variant. Thus, as we show here, both
variants decrease K'ATP to an equal extent, and
furthermore this effect is abrogated by anti-
(C-terminal) for both
variants (summarized in Table II). The present observation that
anti-
treatment of the renal enzyme increases sensitivity to
vanadate and, conversely, that
transfection decreases sensitivity
to vanadate, strongly support the conclusion that the change in
K'ATP reflects primarily an effect on the
E1/E2 conformational equilibrium. The result in Fig. 3 is consistent with the
assumption that
a and
b stabilize
E1 by accelerating the rate of the
conformational transition E2(K)
E1.
The other new finding concerning the function of is that it has not
one but at least two distinct effects on the catalytic function of the
Na,K-ATPase. In addition to an increase in apparent ATP affinity,
induced an increase in K+/Na+ antagonism, which
results in a reduction in the effective affinity of Na+
ions for activating Na,K-ATPase. As mentioned above, the mechanism cannot be explained on the basis of stabilization of
E1 and thus implies an additional effect of
on intrinsic binding of K+ ions at cytoplasmic sites
(perhaps on one of the two K+ sites). This effect of
on
K+/Na+ antagonism is seen equally with both
variants, and, interestingly, this function of
is not altered by
antibodies raised against either the C terminus or N terminus
(summarized in Table II). Overall, it is evident from this study that
both
variants alter the kinetics similarly, with no evidence of a
significant difference between the two on catalytic function. It may
also be noted that the functional effects do not depend on
tissue-specific posttranslational modifications of the
subunit,
although such modifications can be observed in HeLa cells
(
b') or HEK cells (
a'). Expression of
a in NRK-52E kidney cells has been reported to modulate
(decrease) Na+ and K+ affinities (9). Although
the effect on K'Na appears similar to that
described here for
a, there are puzzling experimental differences. First, the level of
expression was much lower in their
experiments (15-20%) than in the present ones carried out with the
HeLa transfectants (
50%) despite the similar functional effect.
Second, the increase in K'Na was only
observed in
a-transfected NRK-52E cells expressing a
doublet, and not in clones expressing a single species the identity of
which, in light of later studies (10), is not clear. These issues are
difficult to reconcile with the present work with
a-transfected HeLa cells that express only a single
a species. As far as K'K is
concerned, we could not detect a significant difference either with
membrane fragments (ATPase assays at high ATP concentrations) or in
86Rb+(K+) influx studies in
ATP-replete (millimolar ATP) cells, despite the higher level of
expression in the HeLa cells. The reason for these discrepancies
remains enigmatic.
The recognition that the subunit induces at least two functional
effects, only one of which is abrogated by anti-
(C-terminal), suggests that there must be more than one region of interaction between
the
and
subunits on which the functional sites reside. Presumably, the effect on the
E1/E2 equilibrium and
apparent ATP affinity involves the C-terminal sequence KHRQVNEDEL, and
the K+/Na+ antagonism is mediated by other
sequences in the molecule. Our observations that antibodies raised
against the N-terminal sequence of
a, TELSANH, do not
affect any kinetic function (summarized in Table II) imply that this
sequence is not responsible for functional interactions. (Antibodies
raised against the N terminus of
b are reactive only
with SDS-denatured enzyme, which precludes meaningful interpretation of
its failure to alter the effects of
b on
1
1 pumps.) Nevertheless, the distinctness of the two effects is
underscored by the observation (26) that polyethylene glycol-mediated
fusion of kidney pumps into cells devoid of pumps (dog erythrocytes) abrogates the kidney-specific increase in
K+/Na+ antagonism but not anti-
-mediated
inhibition of overall activity. Definition of the regions of
interaction of
and
subunits is a question to be addressed in
the future.
Physiological Role--
The physiological significance of the subunit could be that it provides a self-regulatory mechanism for
maintaining the steady-state activity of the pump in the kidney. This
notion is underscored by its abundance in mTAL (see below), suggesting
that its functional effects are tailored to meet the requirement of Na+ and K+ homeostasis in the prevailing
environmental conditions and in particular by the observations of the
dual effects on the kinetic properties, the one on K'ATP
and the other on KK, the affinity of the pump
for K+ acting as an antagonist of cytoplasmic
Na+. The effect of
on K'ATP was
discussed recently in terms of its importance in maintaining pump
activity under putative anoxic parts of the medulla, i.e. to
increase ATP utilization and maintain optimally high intracellular
K+ and low Na+ under energy-compromised
conditions as discussed previously (11, 25, 27). Such a regulator of
K'ATP should alter the pump's affinity for the
nucleotide only moderately, for an excessive increase would affect even
greater decreases in ATP concentration, thus leading to compromised
cell viability. mTAL is characterized by a rapid transcellular
Na+ flux, and the cellular Na+ concentration
should reflect the balance of rates of passive Na+ entry
and active Na+ efflux. The ability of
to increase
K+/Na+ antagonism at the cytoplasmic surface as
shown in this study may provide a means of acute regulation of the
steady-state Na+ concentration. A lowered effective
cytoplasmic Na+ affinity for activating the pump, due to a
regulatory interaction, may be tailored to fit cells in which the
steady-state Na+ concentration is higher than in cells that
lack the regulator, but that, nevertheless, must respond to changes in
Na+ entry. Thus, the optimal affinity for cytoplasmic
Na+ ions should be one at which there is plenty of reserve
capacity for responding to changes in cell Na+ at the
prevailing set point of Na+ concentration. The recent
report of a putative dominant-negative mutation (G41R) in the
subunit of the Na,K-ATPase may be relevant to a role of
in
maintaining intracellular Mg2+ secondary to elevating
intracellular Na+. Such a relationship between
intracellular Na+ and Mg2+ was seen not only in
sublingual mucous acini (28), but also in renal tubular
cells.3
Localization of --
In this study, we have used
immunocytochemistry to identify the tubular cells that express the
subunit of the sodium pump in cortex and outer and inner medulla and to
define the cellular localization of
. Interestingly, antibodies
specific for the
a or
b forms of the
subunit of Na,K-ATPase revealed clear differences in their expression
(Table III). Both are in the mTAL (which reabsorbs sodium chloride at
high rates) and to a lesser extent in the proximal tubules. The
a variant appears specific for cells in the region of
the MD (a sensor of tubular sodium chloride) and for principal cells of
the cortical collecting duct, but not cTAL, while the
b
form is present in the cTAL and absent from MD and cortical collecting
duct. Altogether, these findings extend the immunocytochemical
observations of Arystarkova et al. (9) both spatially
(providing information about
expression in medulla and papilla, in
addition to cortex, with identification of tubular segments) and in
terms of specific expression of each
form in tubules. They also
show that both forms of the
subunit are in the basolateral
membrane, i.e. in close vicinity to the pump.
The subunits of Na,K-ATPase share homologies with other small
molecules, such as CHIF (channel-inducing factor), phospholemman, and
phospholamban, which are thought to be regulators of ion transporters or channels. Among these, CHIF also has a restricted pattern of expression, in the surface cells of the distal colon epithelium and in
the terminal portions of the nephron (20, 29). More specifically, CHIF
is found to some extent in the cortical collecting duct but essentially
in its medullary and papillary portions. As for the
subunit of
Na,K-ATPase, CHIF is located essentially at the basolateral membrane. A
specific interaction between CHIF and Na,K-ATPase in colon membranes
has been demonstrated recently (30).
Although we cannot absolutely exclude the possibility of different
functional effects of a and
b, the lack
of a notable difference between
a and
b
with respect to their effects on pump kinetics may not be surprising.
The two variants differ with respect to only the six or seven residues
at the extracellular amino terminus. Accordingly, the two variants may
influence differentially such properties as membrane targeting, pump
turnover, or basolateral signaling and affect the rate of active
Na+ and K+ transport by altering the density of
pumps in the basolateral membrane. It is also plausible that each of
these variants may influence differentially, in a cell-specific manner,
some interactions of the pump with the extracellular matrix. Of
particular relevance is the proposal that extracellular hensin
influences directly polarity of collecting duct intercalated cells
(31).
![]() |
ACKNOWLEDGEMENT |
---|
The excellent technical assistance of
Rosemarie Scanzano and Ania Wilczynska is gratefully acknowledged. We
thank Dr. J. B Lingrel for the rat 1-transfected HeLa cells.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Canadian Institutes for Health Research (to R. B.), the Kidney Foundation of Canada (to R. B.), INSERM (to N. F.) and the Weizmann Institute Renal Research Fund (to S. J. D. K.).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.
The last two authors contributed equally to this work.
** To whom correspondence should be addressed: Montreal General Hospital, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada. Tel.: 514-937-6011 (ext. 4501); Fax: 514-934-8332; E-mail: Rhoda. Blostein{at}mcgill.ca.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.M010836200
2 S. J. D. Karlish and R. Goldshleger, unpublished results.
3 G. Quamme, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HEK, human embryonic kidney; TAL, thick ascending limb of Henle's loop; cTAL and mTAL, cortical and medullary portions, respectively, of the thick ascending limb of Henle's loop; MD, macula densa; AQP2, aquaporin 2.
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