1 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel; 3 Institut National de la Santé et de la Recherche Médicale U-478, Institut Federatif de Recherche 02, Faculte de Medecine X. Bichat, 75870 Paris Cedex 18, France; and 2 Department of Medicine, McGill University, Montreal, Quebec, Canada H3G1A4
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ABSTRACT |
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Like the -subunit of Na-K-ATPase, the
corticosteroid hormone-induced factor (CHIF) is a member of the FXYD
family of one-transmembrane-segment proteins. Both CHIF and two splice
variants of
,
a and
b, are expressed
in the kidney. Immunolocalization experiments demonstrate mutually
exclusive expression of CHIF and
in different nephron segments.
Specific coimmunoprecipitation experiments demonstrate the existence in
kidney membranes of the complexes
/
/
a,
/
/
b, and
/
/CHIF and exclude mixed complexes
such as
/
/
a/
b and
/
/
/CHIF.
CHIF has been expressed in HeLa cells harboring the rat
1-subunit of Na-K-ATPase. 86Rb flux
experiments demonstrate that CHIF induces a two- to threefold increase
in apparent affinity for cytoplasmic Na
(K'Na) but does not affect affinity for
extracellular K (Rb) ions (K'K) or
Vmax. Measurements of Na-K-ATPase using isolated
membranes show similar but smaller effects of CHIF on
K'Na, whereas
K'K and
K'ATP are unaffected. The functional
effects of CHIF differ from those of
. An implication of these
findings is that other FXYD proteins could act as tissue-specific
modulators of Na-K-ATPase.
corticosteroid hormone-induced factor; sodium-potassium-adenosine 5'-triphosphatase; tissue-specific modulator
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INTRODUCTION |
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THE
-SUBUNIT OF NA-K-ATPASE is a 65-amino acid type I
membrane protein associated with the
complex and is expressed
almost exclusively in the kidney (6, 18, 25). It is
expressed as two splice variants,
a and
b, with different extracellular sequences and
localization along the nephron (15, 21, 24). Functional
interactions of the
-subunit have been detected either by
coexpressing it with
subunits in Xenopus laevis
oocytes and transfected mammalian cells or by neutralizing interactions in kidney membranes using a specific anti-
antibody (1, 6, 21, 25, 26). In oocytes, there are effects on extracellular K
affinity, which vary with voltage (6). In cultured cells,
raises the apparent affinity for ATP by shifting the E1-E2
conformational equilibrium toward E1 and reduces apparent affinity for
cytoplasmic Na ions by making cytoplasmic K a better competitor
(21, 26). The first effect but not the second is abrogated
by an anti-
antibody, implying that
and
interact at more
than one position. Overall, modulation of Na-K-ATPase activity by the
-subunit appears to be a homeostatic mechanism that maintains active
Na and K pumping rates in varying conditions of the cell (ATP, Na, K,
etc.). The
-subunit has been reported to be required for normal
blastocyst formation, but the mechanism is unknown (13). A
mutation in its transmembrane segment, which causes defective routing,
is associated with primary hypomagnesemia (17).
The -subunit shares sequence and topological homology with six other
proteins, some of which were shown to play a role in the regulation or
mediation of ion transport (24). Together, they are termed
the FXYD family, after the invariant motif FXYD located in their
extracellular domains. One member of this group is the corticosteroid
hormone-induced factor (CHIF; FXYD 4). CHIF was cloned as an
aldosterone-induced gene and is expressed only in the kidney and colon
(3, 9, 23). In addition to its regulation by
corticosteroids, CHIF mRNA and protein are induced by Na deprivation
and K loading (23, 27, 28). Suppression of CHIF by low-K
intake is independent of plasma aldosterone (28). Like
, CHIF is expressed only in the basolateral membrane of target
epithelia. However, the distributions of the two homologous proteins
are different. Distal colon and kidney collecting duct have CHIF and
lack
, whereas the thick ascending limb of Henle's loop and
proximal nephron have
but no CHIF (21, 23). It has
recently been reported that CHIF can modulate Na-K-ATPase activity when
expressed with
in X. laevis oocytes (5).
Taken together, the above data suggest that CHIF could be another
epithelia-specific auxiliary subunit of the pump with functional
effects that are different from those of
. The objective of the
present study has been to test this hypothesis and elaborate on it
using native epithelia and transfected mammalian cells. Structural and
functional interactions between a mammalian modulator and the pump
detected in the native environment or mammalian cells are likely to
have physiological significance.
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MATERIALS AND METHODS |
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Membrane preparation.
Rats were killed by cervical dislocation, and distal colons and kidneys
were excised and rinsed in ice-cold PBS. Kidneys were dissected into
cortices, inner medulla, and outer medulla, and microsomal membranes
were prepared as described before (14). Distal colon
surface cells (colonocytes) were isolated by a modification of the
procedure described elsewhere (22). In brief, colonic tubes were flushed three times with 10 ml of ice-cold PBS plus 2 mM DTT
and inverted (lumen out). The inverted colons were tied at one end,
filled with DMEM plus 10% FCS, 2 mM DTT, and 2 mM EDTA, and then tied
at the other end as well. The filled colons were suspended in 25 ml of
the above DMEM-EDTA medium and incubated at 37°C for 40 min with
shaking at 140 rpm. Colonocytes were collected from the medium by
centrifugation and stored at 70°C. HeLa cell membranes were
prepared as described before (12).
Antibodies and immunochemistry.
A polyclonal antibody to the COOH tails of rat CHIF was raised against
the synthetic peptide CKVTPLITPGSAST as detailed elsewhere (23). Antibodies to the COOH tail of and the
NH2 terminus of
a have been described before
(15). Two antibodies to the
1-subunit of
Na-K-ATPase have been used. The first (6H) is a monoclonal antibody
directed against the NH2-terminal segment of the
-subunit, kindly provided by Dr. M. J. Caplan (Yale University School of Medicine). The second antibody, raised against the
COOH-terminal sequence KETYY, was kindly provided by Dr. J. Kyte
(University of California at San Diego, La Jolla, CA). Colocalization
of CHIF and
in kidney segments was done as described previously
(21, 23).
Detergent solubilization, immunoprecipitation, and immunoblotting
of CHIF, , and
.
Colonocytes, microsomal kidney membranes, and HeLa cell membranes were
suspended in a buffer containing 25 mM imidazole and 1 mM EDTA, pH 7.5, and either 20 mM Tris · HCl, 20 mM NaCl plus 0.1 mg/ml
oligomycin (incubated for 25 min at room temperature), or 10 mM RbCl
plus 5 mM ouabain (incubated for 20 min at room temperature). Membranes
were solubilized at 0°C by adding C12E10 to a
final concentration of 1 mg/ml and a final protein concentration of
~0.5 mg/ml. The detergent-solubilized membranes were centrifuged for
30 min at 100,000 g, the supernatant was collected, and
Tris · HCl, NaCl, or RbCl was added to a final concentration of
100 mM. An ~40-µg aliquot of protein was removed (total protein
samples), and the rest was subjected to immunoprecipitation. Total
protein samples were delipidated by adding 4 vol of methanol-ether
(2:1) and incubation at
20°C overnight. The suspensions were spun
down, and the pellets were dried and dissolved in SDS-PAGE sample buffer.
Transfection and selection of HeLa cells.
The coding region of rat CHIF was subcloned into the
BamHI/BstXI site of the mammalian expression
vector pIRES hyg (Clontech). HeLa cells overexpressing the rat
1-subunit of Na-K-ATPase (HeLa-
1 cells;
kindly provided by Dr. J. B. Lingrel, University of Cincinnati College of Medicine) were transfected using lipofectamine (GIBCO BRL)
according to the manufacturer's instructions. Colonies overexpressing CHIF were selected in 400 µg/ml hygromycin B.
Membrane preparations and Na-K-ATPase assays.
Membranes were prepared from transfected rat HeLa-1
cells, and kinetic assays of Na-K-ATPase were carried out in triplicate as described previously (21).
K'ATP and Vmax
values represent the least squares fit of the data of at least three
separate paired experiments fitted to a simple Michaelis-Menten model.
K'Na and K'K, apparent affinities for Na and K,
were similarly analyzed but with the data fit to the Garay-Garrahan
noncooperative three-site (Na+) or two-site
(K+) model for Na and K activation, respectively.
86Rb flux assays.
86Rb uptake was measured as described elsewhere
(20). HeLa-1 cells stably transfected with
CHIF or empty vector were cultivated in 24-well plates. Confluent
monolayers were washed twice at 37°C with 0.5 ml/well of a solution
containing the desired amount of NaCl and KCl, a complementary amount
of choline chloride to achieve a total salt concentration of 145 mM, 5 mM glucose, and 5 mM HEPES-Tris (pH 7.4). The final medium consisted of
465 µl of the above buffer plus 0.5 mM CaCl2, 0.5 mM
MgCl2, 1 mM BaCl2, RbCl, and KCl as indicated
(see RESULTS) and 0.1 mM furosemide. To block endogenous Na-K-ATPase activity, 10 µM ouabain was added to one-half of the wells, and 5 mM ouabain was added to the other half to block the transfected Na-K-ATPase. Monensin was added to a final concentration of
10 µM, and the cells were incubated for 15 min at 37°C under 95%
O2-5% CO2. Gassing was then stopped, and
uptake was initiated by the addition of 25 µl 86RbCl (100 µCi/ml) to each well. The reaction was stopped 15 min later by
aspirating the radioactive medium and rinsing the wells four times with
0.5 ml of an ice-cold PBS buffer containing 5 mM BaCl2. The
cells were lysed with two 0.4-ml volumes of 1 M NaOH, the cell lysates
were collected, and lysate radioactivities and protein contents were
determined. Data were expressed as nanomoles Rb taken up per milligram
protein per minute, and data from three wells on the same plate were averaged.
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RESULTS |
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Figure 1 provides evidence for a
specific interaction between the -subunit of Na-K-ATPase and CHIF by
coimmunoprecipitation from colonocyte membranes. Because preliminary
experiments failed to show immunoprecipitation of CHIF by
anti-
-subunit antibodies, we conducted a series of experiments to
optimize coimmunoprecipitation of
- and
-subunits from renal
Na-K-ATPase, assuming that similar conditions would hold for CHIF and
. Coimmunoprecipitation of
- and
-subunits solubilized from
renal microsomes
with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate has been described (18), but the efficiency was low. We
have now found that the efficiency of coimmunoprecipitation depends strongly on the nature of the antibody, the type of detergent used, and
ionic conditions. Of several anti-
-subunit antibodies tested, only a
monoclonal antibody (i.e., 6H) directed against the
NH2-terminal segment of the
-subunit was effective, and
with the following detergents the order of efficiency was found to be
C12E10 > 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate > dodecyl maltoside. In unrelated work, we have found that when pig
kidney Na-K-ATPase is solubilized with either
C12E10 or dodecyl maltoside, in the presence of
Rb ions plus ouabain or Na ions plus oligomycin, the solubilized
protein retains the ability to occlude Rb or Na ions, respectively.
Also, specific Fe-catalyzed oxidative cleavage is preserved, implying
that native structure is retained in these conditions (9a). In
contrast, on Na-K-ATPase solubilization in the absence of these ligand
combinations, the ability to occlude cations is lost, implying that the
native structure is not maintained. Figure 1A shows that
when the renal enzyme was solubilized with
C12E10 in the presence of Rb ions plus ouabain or Na ions plus oligomycin, the
-subunit (i.e., both
a- and
b-subunit splice variants) was
coimmunoprecipitated quite efficiently by 6H, whereas in a medium
containing only Tris ions the efficiency was lower. Thus it appears
that a native structure is required for the most efficient
coimmunoprecipitation. In optimal conditions, the efficiency of
immunoprecipitation of
by anti-
was estimated to be
15-20%. (The poor efficiency of dodecyl maltoside in
coimmunoprecipitation experiments implies that the detergent disrupts
-
interaction despite maintenance of cation occlusion and the
Fe-cleavage pattern.) The experiment in Fig. 1B shows that
6H effectively immunoprecipitates CHIF from colon membranes solubilized
with C12E10 in media containing Rb ions plus
ouabain or Na ions plus oligomycin, whereas immunoprecipitation in the
Tris-only medium is barely detectable. In contrast, the
-subunit was
effectively precipitated in all three media. The conclusion is that the
interaction between
and CHIF is preserved in the presence of Rb
ions plus ouabain or Na ions plus oligomycin and that CHIF can be
immunoprecipitated but is largely disrupted in media lacking these
ligands. Note that the effect of pump ligands is not indicative of
conformational dependence as such but reflects the ability of Rb plus
ouabain or Na plus oligomycin to protect against denaturation and
preserve an intact protein structure. In optimal conditions, the
efficiency of immunoprecipitation of CHIF by anti-
was estimated to
be ~10%. Immunoprecipitation of CHIF by a specific anti-
antibody, with an efficiency approaching that of
and dependence on
ligand conditions, excludes the possibility that the detected
association is adventitious. On the contrary, these features provide
strong evidence for specificity of the
-CHIF interaction.
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At least three FXYD proteins can be detected in kidney cells:
a,
b, and CHIF. Thus the question arises,
whether the complexes with
- and
-subunits contain only one or
more than one member. The experiments in Figs.
2 and 3
examined this question. In Fig. 2 (top), microsomes from rat
renal medulla were dissolved in C12E10 in a
medium containing Rb ions plus ouabain, and immunoprecipitation was
carried with 6H, an anti-
a-specific antibody raised
against the sequence TELSANH at the NH2 terminus, and an
anti-
COOH-terminal antibody that recognizes both
a
and
b. The blot was then probed with the anti-
COOH-terminal antibody. The result is clear-cut. While both anti-
and anti-
COOH-terminal antibodies immunoprecipitate both
a and
b (seen as a doublet at apparent
Mr values of ~9 and 10), the
anti-
a antibody immunoprecipitates only
a
and not
b. All three antibodies immunoprecipitated the
-subunit as detected with anti-KETYY (not shown). The experiment
proves that
a and
b cannot be present
together in the same complex. In some cultured cells transfected with
a, a modified form, referred to as
a', is
detected in addition to
a (1, 15, 21).
Modified forms of
a or
b were not
detected in
-subunit extracted from rat kidney membranes, when
analyzed by mass spectrometry (15). Because only one band
of
was detected after immunoprecipitation with anti-
a, the experiment depicted in Fig. 2 supports the
conclusion that modified
a-subunits are not present in
significant amounts in rat kidney membranes (15). CHIF is
expressed primarily in the collecting duct and can be detected readily
in microsomes prepared from renal papilla (23), and
is
also detected in renal papilla microsomes. In the experiment depicted
in Fig. 2 (bottom), microsomes from rat renal papilla were
dissolved as described above and anti-CHIF or anti-
COOH-terminal
antibodies were used for immunoprecipitation. The blots were probed
with anti-CHIF or anti-
COOH-terminal antibodies or with anti-KETYY to detect the
-subunit. The answer again is clear-cut. The anti-CHIF antibody immunoprecipitates CHIF and the
- but not the
-subunit, and the anti-
antibody immunoprecipitates
and
but not CHIF. (The band running just above the
-subunit is a contaminant in the
antibodies.) The conclusion from Fig. 2 is that no mixed complexes such
as
/
/
a/
b,
/
/
a,
or
b/CHIF can be detected in renal membranes. Thus the
kidney cells contain only the complexes
/
/
a,
/
/
b, and
/
/CHIF (or possibly
/
complexes without
or CHIF).
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In Fig. 3, using immunofluorescence we examined the degree of overlap
of expression of and CHIF in different sections of rat kidney. The
striking finding is that there is absolutely no overlap of fluorescence
deriving from anti-
(green) or anti-CHIF (red), although both
proteins are found in all sections except the inner zone of the inner
medulla that stains for CHIF but not
. Thus CHIF and
are
expressed in different subpopulations of cells, consistent with their
distribution in different nephron segments (21, 23, 29)
(see also DISCUSSION). The result shows that
and CHIF
cannot participate in mixed complexes (such as
/
/
/CHIF), in
agreement with the immunoprecipitation experiments. It also eliminates
a remote possibility that we failed to detect an
/
/
/CHIF
complex by immunoprecipitation, based on the unlikely assumptions that
CHIF hinders anti-
binding and
also hinders anti-CHIF binding.
HeLa cells expressing the rat Na-K-ATPase 1-subunit were
stably transfected with CHIF, and cells were then selected by growth in
a medium containing hygromycin B. Membranes were prepared from cells
transfected with CHIF or empty vector. As seen in Fig.
4A, the latter express
but
not CHIF whereas the former express both
and CHIF. The relative
intensities of CHIF and
in HeLa cells were compared with those
found in native colonocyte membranes. The signal intensities were
proportional to the amount of applied protein up to 9 µg. From a
comparison of the intensities of CHIF and
, we estimate that the
CHIF/
ratio in HeLa cells is ~70% of that in colonocytes. This
level of expression is sufficient to enable detection of functional
effects of CHIF, as described below. Immunolabeling of the transfected
cells shows that CHIF is directed to the plasma membrane (Fig.
4B). CHIF is immunoprecipitated by the monoclonal anti-
antibody as well as by the anti-CHIF antibody (Fig. 4C).
Thus the HeLa cells expressing CHIF appeared to be a good model system
by which to study the effects of CHIF on the functioning of
Na-K-ATPase.
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As one approach to the functional characterization of the effects of
CHIF, we have compared active 86Rb uptake into the
transfected and control cells. Preliminary transport experiments showed
that in the normal growth medium containing ~5 mM K ions, a large
fraction of K (86Rb) uptake is inhibited by ouabain
(50-90%) and the ouabain-inhibited 86Rb uptake is
linear for at least 20 min. Thus the flux assay appeared to be suitable
for looking at the cytoplasmic Na and extracellular K concentration
dependencies of the active flux. The method used for altering the
cytoplasmic Na concentration, normally 10 mM, involves preincubation
of the cells with the ionophore monensin in media containing different
Na concentrations. Monensin catalyzes an exchange of Na for H, and thus
equilibration of internal and external Na ion concentration should be
associated with large fluxes of protons. Within certain limits of a
changed cytoplasmic Na concentration, the cell's own pH-regulating
systems are assumed to be able to maintain the cellular pH near normal
(see also footnote 1). Preliminary experiments showed that
for all concentrations of monensin between 2.5 and 10 µM, the
extracellular and cytoplasmic Na concentrations were equilibrated, as
judged by the increase or decrease in ouabain-inhibited
86Rb uptake when the Na concentration in the medium was
changed. Monensin at 20 µM produced some nonspecific inactivation.
Figure 5A presents a
representative experiment that examines the Na ion concentration
dependence of the active K (86Rb) uptake between 2 and 50 mM Na for control and CHIF-transfected cells. The inset
shows that ouabain produced a large inhibition of the 86Rb
uptake at all Na concentrations. The flux rate increased as Na was
raised from 2 to 50 mM, and evidently the CHIF-transfected cells were
activated at significantly lower concentrations of Na ion compared with
the control cells.1 The Na
activation curves were fitted to simple Michaelis-Menten functions,
because the flux could not be measured at sufficiently low Na
concentrations to detect the normal sigmoidal shape of such curves.
Table 1 presents the derived
Vmax and K0.5 values for
three full experiments, which show that the apparent affinity for Na
ions was increased two- to threefold in the CHIF-transfected cells
whereas Vmax was not significantly affected. The
average value of
Vmax(CHIF)/Vmax(empty
vector) for the three experiments is 1.05 ± 0.14. The observation
that Vmax was not changed excludes the
possibility that the expression of CHIF affected the cell surface
expression of the pump. The data for the three Na activation curves can
be normalized for differences in absolute values of the fluxes by
plotting the V/Vmax vs. Na
concentration. Figure 6A
presents the average value of V/Vmax
for the three experiments. The fitted values for
K0.5 for cytoplasmic Na are 6.3 ± 2.0 mM for control cells and 1.9 ± 0.4 mM for the CHIF-transfected
cells. It may be noted that the effect of CHIF measured in the
transport assay is distinctly greater than that measured in ATPase
assays with broken membranes.
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The dependence of the flux on extracellular Rb ion concentration
between 0.1 and 5 mM was measured in cells bathed in a medium containing 30 mM Na and treated with monensin. The monensin treatment raises the cytoplasmic Na to a saturating concentration (see Fig. 5).
Two full experiments showed no significant difference in the Vmax and Rb activation curves of control and
CHIF-transfected cells. The curves were fitted to the Hill equation to
derive values of the Hill coefficient, K, and
Vmax. The average value of
V/Vmax for the two full experiments
for the control and CHIF-transfected cells is plotted against the Rb
concentration in Fig. 6B. Clearly, the curves are
superimposable. Values of the fitted parameters for either control or
CHIF-transfected cells are 0.25 ± 0.05 mM (K);
1.54 ± 0.13 (Hill coefficient); and 0.41 ± 0.08 mM (the corresponding
value of K0.5 for Rb ions).
As a complementary approach to the measurements of active
86Rb uptake into the intact cells at 37°C, we have
measured the kinetics of ATP, Na, and K activation of Na-K-ATPase in
isolated membranes (21). Results from ATPase assays
summarized in Table 2 indicate that CHIF
caused a modest but significant increase in
K'Na (K'Na decreased 30%; P < 0.01) without affecting either
K'ATP or
K'K.
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DISCUSSION |
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This paper provides evidence for a specific structural and
functional interaction of CHIF with the -subunit of Na-K-ATPase in
colon and kidney membranes and in cultured HeLa cells transfected with
CHIF. The demonstration of modulation of the Na affinity of the pump by
CHIF in a mammalian cell at 37°C provides important support for the
hypothesis that this functional effect of CHIF has physiological
significance. In the kidney, CHIF and the
-subunit are expressed in
different nephron segments, and the two splice variants of
are also
expressed differentially in different nephron segments in renal cortex
(21). In kidney membranes, the coimmunoprecipitation experiments show the existence of
/
/
a/,
/
/
b, and
/
/CHIF complexes but no mixed
complexes containing
a plus
b or
plus CHIF. The segregation of CHIF and
to separate complexes is
attributable to their different cell-specific expression. On the other
hand,
a and
b are expressed in the same
cells, at least in the thick ascending limb of the loop of Henle.
Therefore, the lack of mixed complexes indicates that the 1:1:1
-to-
-to-
ratio is the true form of the molecular complex (see
also Ref. 15). In contrast to the unequivocal result in
Fig. 2 excluding the possibility of mixed complexes such as
/
/
a/
b, coimmunoprecipitation of
a with
b and
was recently reported
(2). Membranes from rat kidney medulla were dissolved with
C12E8 at room temperature in a buffer
containing 25 mM imidazole, pH 7.3, and 1 mM EDTA, before the
immunoprecipitation reaction. As emphasized above, these conditions of
detergent solubilization do not maintain the Rb occlusion or a native
protein strucure intact, as judged by the Fe-catalyzed
cleavage. Thus complexes detected in these conditions should
reflect the inactivated state. Membrane proteins denatured by excess
detergent are known to undergo irreversible aggregation, particularly
at raised temperatures. It cannot be excluded that individual soluble
complexes
/
/
a and
/
/
b
underwent aggregation and were then coimmunoprecipitated.
After transfection of CHIF into the HeLa cells expressing the rat
1-subunit to a level comparable to that in native colon membranes, the apparent affinity of cytoplasmic Na for activating the
pump-mediated active K (Rb) uptake was raised two- to threefold compared with that in control cells (K0.5
6.3 ± 2.0 mM for control cells and 1.9 ± 0.4 for the
CHIF-transfected cells). In contrast, the Vmax
values and apparent affinity for activation by extracellular Rb were
unaffected by expression of CHIF (K0.5 for Rb
ions,
0.41 ± 0.08 mM; Hill coefficient,
1.54 ± 0.13 for
both sets of cells). The raised affinity for cytoplasmic Na ions is
similar to the effect reported recently after expression of CHIF in
oocytes, although the magnitude of the effect is larger in the HeLa
cells (5). In the oocyte study, CHIF was also found to
decrease by about twofold the apparent affinity for extracellular K
ions at large negative membrane potentials (up to
150 mV) in the
presence of extracellular Na ions (90 mM) but not in the absence of
extracellular Na ions. The lack of effect of CHIF on the apparent
affinity for extracellular Rb ions in the HeLa cells is different from
the oocyte result. However, this is most likely due to different
conditions of the measurements. The membrane potential in the cultured
cells is unlikely to exceed
50 mV, a potential at which the effect on
K affinity was quite small in the oocytes (see Fig. 7B in
Ref. 16). Furthermore, the extracellular Na concentration
was only 30 mM in our study compared with 90 mM in the oocyte
experiments. In oocytes, the effect of CHIF on
K0.5 for K ions was shown to depend on
extracellular Na concentration and was interpreted as an effect of the
membrane potential on the affinity of extracellular Na ions for
competition at the K sites (5). The effect could be
expected to be reduced even more at the lower Na used in our experiments (30 mM) compared with the 90 mM used in the oocyte experiments, and, as mentioned before, it disappeared altogether in the
absence of extracellular Na ions. Overall, the effects in oocytes and
HeLa cells are not inconsistent with each other.
In assays of Na-K-ATPase activity using isolated membranes, CHIF
significantly reduced K'Na from 5.37 ± 0.67 to 3.76 ± 0.93 mM with no significant change in
K'K or
K'ATP. Thus qualitatively the effect of
CHIF in reducing K'Na with no effect on
K'K is the same in isolated membranes and
whole cells. However, the magnitude of the effect on
K'Na in isolated membranes (CHIF/control
0.69 ± 0.12) is distinctly lower than in whole cells
(CHIF/control 0.3 ± 0.12). This finding could imply that
CHIF-pump interactions are relatively weak and are partially disrupted
by the experimental manipulations involved in isolating membranes.
Other evidence that suggests that CHIF-pump interactions in isolated
HeLa cell membranes are relatively weak compared with -pump
interaction may reflect a greater sensitivity to disruption by raised
C12E10 concentrations and greater dependence on
the ionic conditions of solubilization in coimmunoprecipitation assays
(results not shown).
The CHIF-induced increase in affinity for cytoplasmic Na ions, with no change in Vmax or apparent affinity for extracellular Rb (K) ions in transport assays, and similar if smaller effects on K'Na, with no effects on K'K or K'ATP observed in ATPase assays, has an interesting mechanistic implication. In principal, a change of two- to threefold in apparent cytoplasmic Na affinity could be the result of an effect on either the intrinsic binding affinity of Na sites or the rate constants of the catalytic cycle that stabilize the E1 conformation of the protein to which the cytoplasmic Na ions bind. In the latter case, one could also expect to observe an effect on Vmax and, in particular, a reduced apparent affinity for extracellular Rb (K) ions due to a reduced steady-state level of the E2P conformation to which the Rb (K) ions bind and, conversely, an increased K'ATP. Because none of the latter three effects was observed, the simple explanation is that CHIF does not affect the E1-E2 conformational equilibrium but raises the intrinsic affinity for cytoplasmic Na sites. Of the three cytoplasmic sites for Na ions, the third site, which does not bind K ions and is selective for Na ions, could be a good candidate for such an effect.
The functional effects of CHIF, described here and elsewhere
(5), including the increased apparent cytoplasmic Na
affinity and decreased extracellular K affinity at high negative
membrane potentials in Na-containing media, are quite different from
those of the -subunit described previously (1, 19, 21,
26). The effects of
include 1) an increased ATP
affinity, due to stabilization of the E1 conformation; 2) an
increased K vs. Na antagonism at the cytoplasmic surface, leading to a
reduced apparent cytoplasmic Na affinity at the physiological cytosolic
K concentration; 3) a raised apparent affinity for
extracellular K at high negative membrane potential in a Na-containing
medium; and 4) induction of unselective cation channels. In
all cases, the kinetic effects on the pump are of moderate magnitude,
at most two- to threefold changes in apparent affinities of ligands.
The physiological significance of the kinetic effects of CHIF and
on pump kinetics must focus on the cytosolic Na concentration, which is
limiting in normal physiological conditions, or on the ATP
concentration, which may fall in anoxia. As mentioned above, whether
changes in extracellular K affinity, observed in some conditions, are
physiologically significant is unclear because the extracellular K
concentration is normally close to saturating. Similarly, the
physiological significance of
-induced channels is not clear.
Presumably, the different functional modulation of the Na-K pump by or CHIF serves different physiological needs of the cells in which they
are expressed. In the kidney, the
-subunit is expressed in the
medullary thick ascending limb of Henle's loop at a high
concentration, in the distal convoluted tubules, and at lower levels in
proximal tubules and macula densa (21). The medullary
thick ascending limb of Henle's loop segments are characterized by a
very high rate of Na pumping and transepithelial Na reabsorption, which
serve their role in generating renal salt gradients. We have argued
previously that an increased ATP affinity allows maintenance of Na-K
pump rates in response to rapid falls in ATP levels accompanying anoxic
episodes, whereas a decreased cytoplasmic Na affinity may allow the
pump to respond sensitively to increases in cytoplasmic Na at higher
set point levels of cytosolic Na (21, 26). In contrast to
, CHIF is expressed exclusively in cortical and medullary collecting
ducts and could be expected to serve the different special needs of
these segments, particularly the crucial role of the cortical
collecting duct in K homeostasis and its regulation by
mineralocorticoids. A physiological rationale for the observed
functional effects of CHIF must, of course, fit the evidence for a
physiological role of CHIF in K homeostasis, based on the raised
expression induced by high serum K and a low serum Na concentration
(28). The Na-K pump rate both responds to changes in the
cytosolic Na concentration and is responsible for setting the normal
set point cytosolic Na concentration, in conjunction with the passive
Na entry systems. A higher Na affinity of the Na-K pump could lead to a
reduced cytosolic set point Na concentration and thus maintain the
driving force for Na entry and transepithelial potential, which drives
transepithelial Na reabsorption from the luminal fluid, already
depleted of Na. A CHIF-induced increased Na affinity should adapt the
pumping rate to the low cellular Na resulting from the low luminal Na
and allow the rate, and thus net Na reabsorption, to respond
effectively to a rapid increase in cytosolic Na associated with
mineralocorticoid-induced Na permeability at the luminal surface.
It has been noted previously that the affinity of the Na-K pump
for cytosolic Na in cortical collecting ducts is higher than in other
segments of the nephron (4). This may now be attributed to
the regulatory interaction of CHIF with the pump.
An obvious implication of the findings in this paper and related
results (5) is that other FXYD proteins may serve as
regulators of the Na-K pump in different tissues in which they are
specifically expressed. The different proteins of the FXYD family may
modulate Na-K pump function so as to optimally adapt it to the
requirements and environment of the cells in question. The distinct
functional effects on pump kinetics of and CHIF fit well with the
evidence for their complementary expression pattern along the nephron, which implies distinct physiological roles. Of interest in this respect
is the recent finding that expression of the
-subunit is induced in
kidney cells adapted to grow in 600- or 900-mosmol/kgH2O solutions (8). Another example of a specific FXYD
protein-pump interaction concerns a phospholemman-like protein in the
shark rectal gland Na-K-ATPase, which modulates the enzyme activity in
a manner regulated by phosphorylation by PKC (16). We have also observed coimmunoprecipitation of phospholemman and the
-subunit from bovine cardiac sarcolemma vesicles (Füzesi M,
Garty H, and Karlish SJD, unpublished observations). Thus the
investigation of possible regulatory roles of the different FXYD
proteins is an attractive subject for future investigation. The
experimental systems described here can now be exploited for further
analysis and comparison of the structural and functional interactions
between the
-subunit and different FXYD proteins.
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ACKNOWLEDGEMENTS |
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This study was supported by research grants from the Minerva Foundation (Germany), the S. Epstein Research Fund, the Weizmann Institute Renal Research Fund (H. Garty and S. J. D. Karlish), the Canadian Institutes for Health Research, and the Kidney Foundation of Canada (R. Blostein).
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FOOTNOTES |
---|
A portion of the data has appeared before in abstract form (Biophys J 80: 501a, 2001).
Address for reprint requests and other correspondence: S. J. D. Karlish, Dept. of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel (E-mail Steven.Karlish{at}weizmann.ac.il).
1 In other experiments, the rate was measured at Na concentrations up to 75 mM and was constant between 30 and 75 mM. This finding provides indirect evidence that any cytoplasmic pH changes associated with equilibration of the Na by Na/H exchange are not great enough to affect the rate of pumping. If the increased rate of Na/H exchange required to equilibrate 75 mM compared with 30 mM Na had produced a significant increase in cellular pH, one could have expected the flux rate to fall between 30 and 75 mM Na.
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.
April 16, 2002;10.1152/ajprenal.00112.2002
Received 21 March 2002; accepted in final form 10 April 2002.
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