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The Journal of Neuroscience, March 15, 2003, 23(6):2161
Phospholemman, a Single-Span Membrane Protein, Is an Accessory
Protein of Na,K-ATPase in Cerebellum and Choroid Plexus
Marina S.
Feschenko1,
Claudia
Donnet1,
Randall K.
Wetzel1,
Natalya K.
Asinovski1,
Larry R.
Jones2, and
Kathleen J.
Sweadner1
1 Laboratory of Membrane Biology, Neuroscience Center,
Massachusetts General Hospital, Charlestown, Massachusetts 02129, and
2 Krannert Institute of Cardiology, Indiana University
School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
Phospholemman (FXYD1) is a homolog of the Na,K-ATPase  subunit
(FXYD2), a small accessory protein that modulates ATPase activity. Here
we show that phospholemman is highly expressed in selected structures
in the CNS. It is most abundant in cerebellum, where it was detected in
the molecular layer, in Purkinje neurons, and in axons traversing the
granule cell layer. Phospholemman was particularly enriched in choroid
plexus, the organ that secretes CSF in the ventricles, where it
colocalized with Na,K-ATPase in the apical membrane. It was also
enriched, with Na,K-ATPase, in certain tanycytes or ependymal cells of
the ventricle wall. Two different experimental approaches demonstrated
that phospholemman physically associated with the Na,K-ATPase in
cerebellum and choroid plexus: the proteins copurified after detergent
treatment and co-immunoprecipitated from solubilized crude membranes
using either anti-phospholemman or anti-Na,K-ATPase antibodies.
Phospholemman antibodies precipitated all three Na,K-ATPase  subunit
isoforms (1-3) from cerebellum, indicating that the interaction
is not specific to a particular isoform and consistent with the
presence of phospholemman in both neurons and glia. Antibodies against the C-terminal domain of phospholemman reduced Na,K-ATPase activity in vitro without effect on Na+
affinity. At least two other FXYD family members have been detected in
the CNS, suggesting that additional complexity of sodium pump regulation will be found.
Key words:
Na,K-ATPase; phospholemman; FXYD; cerebellum; choroid plexus; Purkinje cell; Bergmann glia
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Introduction |
Phospholemman was first discovered
as a readily phosphorylated protein in sarcolemma preparations from
cardiac and skeletal muscle (Walaas et al., 1977 ; Jones et al., 1979).
It is a 72-amino acid membrane-spanning protein with a 20-amino acid
cleavable signal sequence (Palmer et al., 1991). It is phosphorylated
by protein kinases A and C in the heart in response to  - and
-adrenergic stimulation and in response to insulin in skeletal
muscle (Presti et al., 1985a,b; Walaas et al., 1988, 1994). It is also
a target of myotonic dystrophy and NIMA (Never in mitosis, gene
A) kinases (Lu et al., 1994; Mounsey et al., 1999, 2000). This suggests
that phospholemman has a role in signal transduction to integrate
converging regulatory pathways. The link between phospholemman
phosphorylation and its physiological role is not yet understood,
however. Phospholemman induces ion and taurine permeability in oocytes
and lipid bilayers (Moorman et al., 1992; Mounsey et al., 2000), but
evidence is presented here that it is an accessory protein of
Na,K-ATPase.
Phospholemman and the subunit of the Na,K-ATPase are members of the
FXYD gene family, which has seven members in mammals (Sweadner and
Rael, 2000). Several FXYD proteins, the subunit (FXYD2), PLMS (a
dogfish shark FXYD family member), CHIF (FXYD4), and FXYD7, have
been shown to associate with Na,K-ATPase (Mahmmoud et al., 2000;
Therien and Blostein, 2000; Beguin et al., 2001, 2002), and while this
manuscript was being revised, Crambert et al. (2002) independently
showed that phospholemman interacts with Na,K-ATPase when expressed in
Xenopus oocytes and in cardiac and skeletal muscle
sarcolemma. The greatest sequence homology in the gene family is in a
stretch of 35 amino acids that includes the transmembrane span, a
portion of the intracellular domain, and a portion of the extracellular
domain with the FXYD motif (PFxYD or Pro-Phe-x-Tyr-Asp) that identifies
the family. Mutations in the PFxYD motif of and CHIF were shown to
disrupt interaction with the Na,K-ATPase (Beguin et al., 2001). When
the FXYD family transmembrane span amino acid sequences are displayed
on a helical wheel, they have invariant and nearly invariant residues
all clustered on one face, with small and bulky residues aligned
separately to form a ridge-and-groove structure (Sweadner and Rael,
2000). This is consistent with right-handed helix packing and suggests similar specificity in protein-protein interaction in the membrane, such as by interacting with the same site on Na,K-ATPase.
Phospholemman mRNA expression has been detected in the CNS (Chen et
al., 1997; Bres et al., 2000; Bogaev et al., 2001), and phospholemman
protein has been detected in cerebellar astrocytes cultured from
8-d-old rats (Moran et al., 2001). We set out to determine the
distribution of phospholemman in the CNS and to test the hypothesis
that it forms a complex with Na,K-ATPase.
Parts of this paper were published previously in abstract form
(Sweadner et al., 2001).
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Materials and Methods |
Immunofluorescence microscopy. Adult outbred rats
were used for immunofluorescence microscopy. The methods for fixation,
tissue sectioning, and double-label immunofluorescence have been
described in detail elsewhere (Wetzel and Sweadner, 2001). The antibody used to detect phospholemman was an affinity-purified polyclonal rabbit
antibody against the C terminus (now called PLM-C1; Moorman et al.,
1992). This antibody also appropriately stains the sarcolemma of
cardiac and skeletal muscle (results not shown). To detect Na,K-ATPase
isoforms, we used monoclonal antibodies specific for rat Na,K-ATPase
1 subunit (McK1; Arystarkhova and Sweadner, 1996) and for 2
(McB2; Pacholczyk and Sweadner, 1997). A monoclonal antibody specific
for 3 (XVI-F9G10; MA-319; Affinity BioReagents) was
also used to stain cerebellum in experiments not shown. Confocal microscopy was performed with a Nikon (Melville, NY) TE300
fluorescence microscope equipped with a Bio-Rad (Hercules,
CA) MRC 1024 scanning laser confocal system.
Membrane preparation and Na,K-ATPase purification. Similar
procedures for membrane preparation were used for both cerebellum and
choroid plexus. First, a crude microsome preparation was made by
differential centrifugation. Tissue (bovine in most experiments) was
homogenized in 0.315 M sucrose, 20 mM TrisCl, and 1 mM EDTA, pH 7.4 (SET), for cerebellum or (in mM): 250 sucrose, 30 imidazole, and 1 EDTA, pH 7.3, for choroid plexus.
Centrifugation was for 15 min at 5800 × g; pellets
were collected, rehomogenized, and pelleted again; and the supernatants
were combined and centrifuged for 1 hr at 140,000 × g.
The final pellets were collected and resuspended in SET. These
fractions were used for immunoprecipitation experiments.
For partial purification of the Na,K-ATPase, the SDS-extraction
procedure was used (Jørgensen, 1988). Bovine choroid plexus microsomes
were suspended at 1.4 mg/ml in buffer containing (in mM): 2 ATP, 30 histidine, and 2 EDTA, pH 7.4, and SDS was added slowly with
stirring from a stock solution to a final concentration of 0.56 mg/ml.
The mixture was allowed to stir for 30 min at room temperature and then
loaded onto 36 ml 7-30% sucrose gradients in (in mM): 20 Tris-Cl and 1 EDTA, pH 7.4. The gradients were centrifuged for 6 hr at
27,000 rpm in a Sorvall 627 swinging bucket rotor.
Fractions were collected from a puncture at the bottom of the tube,
diluted, and concentrated by centrifugation at 140,000 × g for 30 min. Canine or rat brain and choroid plexus tissue was used in some experiments with equivalent results.
Canine cardiac sarcolemma, used as a Western blot-positive control, was
isolated as previously described (Jones, 1988). Rat kidney (renal
medulla) Na,K-ATPase used as a control in activity measurements was
purified by the Jørgensen SDS-extraction procedure above.
Immunoblot. SDS-gel electrophoresis was on Tricine gels made
with 12.5% polyacrylamide (Schagger and von Jagow, 1987). To detect
Na,K-ATPase subunits in blots of bovine tissue, anti-KETYY (a gift from Dr. J. Kyte, University of California, San Diego, CA) was
used as a pan-specific probe that detects the 1-3 isoforms equally well. To detect each isoform individually, monoclonal antibody
6F was used for 1 (Developmental Studies Hybridoma
Bank); McB2 was used for 2; and XVI-F9G10 was used for 3
(described above). In Figure 5, the subunit was stained with K1, a
rabbit antiserum against rat 1 cut from a gel (Sweadner and
Gilkeson, 1985), and in Figure 6, 5, a monoclonal antibody that
reacts with all three isoforms, was also used for either
immunoprecipitation or staining of blots (Developmental Studies
Hybridoma Bank). Affinity-purified antibodies against the C
terminus of phospholemman, PLM-C1 (described above), or the
extracellular portion, monoclonal antibody PLM-B8 (Moorman et al.,
1995), were used to detect phospholemman.
Immunoprecipitation. To immunoprecipitate the Na,K-ATPase
subunit, we used either monoclonal antibody 6H (a gift from Dr. M. J. Caplan, Yale University Medical School, New Haven, CT) or monoclonal antibody 5 (described above). To immunoprecipitate phospholemman, the antibodies against either the N terminus (mouse monoclonal PLM-B8) or the C terminus (PLM-C1) were used (described above). Membranes from either choroid plexus or cerebellum (bovine) at
2 mg of protein/ml were solubilized with 6 mg/ml n-dodecyl octaethylene glycol monoether detergent
(C12E8;
Calbiochem, La Jolla, CA) for 10 min at room temperature
in a buffer containing (in mM): 140 NaCl, 25 imidazole, and 1 EDTA, pH 7.3. The extract was diluted with an equal
volume of detergent-free buffer, and insoluble material was sedimented
by centrifugation for 30 min at 20,000 × g at 4°C.
The pellet was checked for residual Na,K-ATPase, which was very low.
The supernatant was incubated with primary antibodies or control IgG
(1-2 µg/ml) overnight at 4°C with rocking. The immune complexes
were collected after 2 hr of incubation with 40 µl of secondary goat
anti-rabbit or goat anti-mouse IgG antibodies covalently bound to
agarose beads (Sigma, St. Louis, MO). Immunoprecipitates were collected by centrifugation at 9300 × g for 10 min at 4°C and washed four times with solubilization buffer
containing 0.05% C12E8.
After the final wash, the pellet was resuspended in 40 µl of 1×
electrophoresis sample buffer. Samples were incubated for 20 min at
room temperature and centrifuged at 9300 × g for 10 min. Supernatants were saved. Pellets were washed with an additional 20 µl of electrophoresis sample buffer and centrifuged again. The
supernatants were combined and heated for 10 min at 65°C to dissociate IgG before loading on the gel. The excess IgG heavy and
light chains, a trace of undissociated IgG dimer, and some other
contaminants originating in the antibody can be detected in the blots
in Figures 6A and 7.
Epitopes recognized by the PLM-C1 and PLM-B8 antibodies were mapped
using cellulose-bound PepSpots (Jerini Bio Tools) by
methods described previously (Kobayashi and Jones, 1999). The PepSpots sheet contained a series of immobilized 13 mer synthetic peptides, each
overlapping by 11 residues, that covered the primary structure of
canine phospholemman. PLM-C1, which was raised against a peptide with
the sequence GTFRSSIRRLSTRRR, required the sequence FRSSIRRL for
binding. PLM-B8, which is a monoclonal antibody raised against recombinant phospholemman expressed in Sf21 insect cells (Chen et al.,
1998), required the N-terminal sequence EHDPFTYDY for binding. Although
this epitope contains the conserved FXYD motif, the antibody is quite
specific. PLM-B8 does not recognize rat phospholemman, which differs
only by a P for H substitution in the epitope and two amino acids (HT
substituting for QS) immediately after it. We also did not see
cross-reactivity of PLM-B8 with the homologous sequence in FXYD2
(TENPFEYDY; results not shown).
Antibody-induced changes in Na,K-ATPase activity.
SDS-purified bovine choroid plexus Na,K-ATPase was used to measure the
effects of incubation with anti-phospholemman antibodies. The specific activity of the preparation (ouabain-sensitive ATPase activity) was 85 µmol of ATP
hydrolyzed · hr 1 · mg of
protein1, measured in (in
mM): 130 NaCl, 20 KCl, 1 ATP, 1 EDTA, 5 EGTA, 4 MgCl2, and 30 Tris-HCl, pH 7.4. To assess the
effect of antibodies on activity, the same medium was used but with
either 5 or 100 mM NaCl. The hydrolysis of ATP
was measured at 37°C by a colorimetric assay, and assays were
performed with and without 2 mM ouabain. The
antibodies used were PLM-C2, a newer rabbit antiserum that is
equivalent to PLM-C1 (a gift from Dr. J. Cheung, Geisinger Medical
Center; Song et al., 2002), PLM-B8 (described above), or control rabbit
or mouse IgG as appropriate (Sigma). Before use, all
antibodies were diluted with 4-5 volumes of (in
mM): 50 NaCl and 20 Tris-HCl, pH 7.5, concentrated with a centrifugal filter (Amicon, Beverly,
MA; 30 kDa cutoff), and washed three times with three volumes of the
same buffer. This procedure controlled NaCl concentration and removed
any phosphate buffer that would interfere with the assay, as well as
Tris and glycine present in the affinity-purified antibody stock.
Antibody protein was added in a 10fold excess over sample protein.
For Na+ affinity measurements with PLM-C2
antibodies or control IgG, the assay buffer contained the same basic
constituents as the normal assay medium with 20 mM
K+ and different concentrations of
Na+ from 2.2 to 80 mM. The
ionic strength was kept constant by addition of choline Cl such that
[Na+] + [K+] + [choline+] = 168 mM. ATP
hydrolysis was measured with
[-32P]ATP. Preincubation with
antibodies was for 4 hr at 4°C in (in mM): 44 NaCl and 17 Tris-HCl, pH 7.5. This relatively low NaCl buffer was used because of
the need to minimize the amount of NaCl added to the assay mixture
during Na+ affinity measurements. The
final concentration of Na+ contributed by
the addition of enzyme preincubated with antibody or control IgG was
0.23 mM.
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Results |
Figure 1 shows the aligned sequences
of human phospholemman and Na,K-ATPase subunit. For the portion
extending from the end of the signal sequence of phospholemman to the
end of the sequence, the identity is 51%, and the similarity is
64%. For mouse, the identity is 48%, and the similarity is 64%, and
for rat, the identity is 41%, and the similarity is 61% (data not shown). The circles represent residues that are highly
conserved in the entire FXYD family, and the dots represent
additional residues that are identical between phospholemman and .
This strong similarity led to the hypothesis that phospholemman
associates with Na,K-ATPase.
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Figure 1.
Alignment of phospholemman and the subunit.
The sequences of human phospholemman and a are compared to display
their similarity. One gap was allowed in phospholemman to allow
alignment of three identical amino acids in the N-terminal region. The
leader sequence of phospholemman and the alternatively spliced N
terminus of are underlined and
italicized. Following these two structures, the homology
begins. As shown previously (Sweadner and Rael, 2000), there is a set
of highly conserved amino acids that characterizes the entire FXYD
family; these are marked with circles.
Lines mark additional residues that are identical
between phospholemman and . The known phosphorylation sites in the
phospholemman C terminus are indicated; this segment has no counterpart
in .
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Phospholemman expression in brain
Figure 2 shows the distribution of
phospholemman immunoreactivity in the rat CNS. This montage of images
from a rat brain sagittal section shows abundant immunoreactivity in
the molecular layer of the cerebellum and in the choroid plexus in the
lateral, third, and fourth ventricles. In addition, bright
immunoreactivity was seen in cells of a portion of the ependymal lining
of the lateral ventricle on its rostral surface posterior to the
caudate putamen. Other areas showed much less immunoreactivity,
although a detailed examination of all brain areas has not been
performed.
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Figure 2.
Phospholemman immunoreactivity in adult rat brain.
A section of rat brain (without the olfactory bulb) stained with
antibody against the C-terminal end of phospholemman, PLM-C1, is shown.
The brightest immunoreactivity was in the cerebellar molecular layer
(ml) and in the choroid plexus
(CP), whereas the cerebellar granular layer
(gl) was almost unstained. Stained choroid
plexus in (left to right) lateral, third,
and fourth ventricles can be seen, as well as in presumptive tanycytes
of the ependyma of the lateral ventricle (asterisk).
Occasional folds in the section produced irregular brighter
lines. The image is a montage of many individual confocal
images.
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Higher-magnification images of the cerebellar cortex double-labeled for
the 1 subunit isoform of Na,K-ATPase and phospholemman are shown in
Figure 3A-C and
double-labeled for 2 and phospholemman in Figure 3D-F.
Immunoreactivity for 1, 2, and 3 (results not shown) and
phospholemman in the molecular layer was diffuse or fine-grained and
was absent from the cytoplasm of the major Purkinje cell somas and
dendrites, as expected for a membrane protein. Purkinje cell somas were
completely unstained for 1 but exhibited surface ring staining for
phospholemman. Purkinje cell somas were also usually unstained for 2
but were often in close contact with 2-stained astrocytes. The
basket cell processes under the Purkinje cells were not visible with
immunoreactivity for 1 or 2, but we confirmed that they are
phospholemman-negative in other experiments using a double label with
antibody to 3, which stains the basket cell processes well (results
not shown) (for 3 distribution, see McGrail et al., 1991; Peng et
al., 1997). In the granule cell layer, the only prominent
immunoreactivity for phospholemman was in axons passing through. The
antibodies visualized other cell types in the granular layer;
immunoreactivity for 1 highlighted the glomeruli (brightest), and
each granule cell was ring-stained. The antibody to 2 stained the
astrocytes, which send irregular processes between and around the
granule cells. Phospholemman immunoreactivity with granule cells and
glomeruli was below the level of detection, and the stain for granular
layer astrocytes was very light. With this distribution, the most
likely cells producing high levels of phospholemman in the cerebellum
were the Purkinje neurons, the Bergmann glia (which are the specialized astrocytes of the molecular layer), or both. No other cell type has
this distribution of arborization.
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Figure 3.
Phospholemman immunoreactivity in cerebellar
cortex, choroid plexus, and ventricular wall. A-C,
Cerebellar cortex double-labeled for phospholemman
(green), the 1 subunit of Na,K-ATPase
(red), and the combined images. ml,
Molecular layer; gl, granular layer. Immunoreactivity
for 1 in the glomeruli (g) in the granular
layer was particularly bright and the granule cell bodies
(gc) were also stained, as was the molecular
layer. Antibody to phospholemman stained the molecular layer and
ring-stained Purkinje cells (pc) and axons in the
granular layer. D-F, Same structures double-labeled for
the 2 isoform (red) and phospholemman
(green). Purkinje cells occasionally stained for
2, as reported previously (Peng et al., 1997); astrocytes in the
granular layer (astr) were stained prominently for 2
but very lightly for phospholemman. G-I, Section of rat
brain more lateral than that of Figure 2 double-labeled for Na,K-ATPase
1 (red) and phospholemman
(green). Both proteins were confined to the
apical surface of the polarized epithelium of the choroid plexus
(CP), which lies here in a narrow ventricular space
ventral to the chamber seen in Figure 2. On the rostral surface of the
ventricle, a cuboidal epithelium of ependymal cells, presumed tanycytes
(e), is seen that was prominently stained for both
Na,K-ATPase and phospholemman at its apical surface.
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The choroid plexus is a simple cuboidal epithelium of polarized cells,
and the Na,K-ATPase is located on the apical membrane, where its net
outward transport of Na+ drives the
secretion of CSF (Wright, 1978). Figure 3G-I shows that
phospholemman colocalized with the 1 subunit of the Na,K-ATPase in
the apical membrane. Similar colocalization of Na,K-ATPase and
phospholemman was seen in bovine choroid plexus (results not shown).
Figure 3G-I also shows strong immunoreactivity for
Na,K-ATPase and phospholemman in the apical membrane of some of the
cells lining the ventricular wall. The distribution of Na,K-ATPase to the apical membrane predicts that these cells, like choroid plexus itself, may have a secretory function. On the basis of position, morphology, and apical specializations, it seems likely that these cells are tanycytes (Bruni, 1998).
The fact that phospholemman immunoreactivity in the cerebellar
molecular layer appears to be brighter than 1 or 2
immunoreactivity is not evidence that there is an excess of
phospholemman for several reasons: only one isoform at a time was
stained; the exposure intensity for the subunits was adjusted to
avoid overexposing the structures stained in the granular layer; and
comparison of immunoreactivity of two antibodies is in any event not
quantitative. The difference in the proportion of immunoreactivity for
and phospholemman between molecular and granular layers, however, does imply that there is much less phospholemman in the granular layer
relative to subunits. Figure 4 shows
that this was reflected in the ratio of phospholemman to assessed
with immunoblots. Bovine tissue was used instead of rat here because of
the ease and yield of dissection. To detect all Na,K-ATPase isoforms equally, an antibody against a shared determinant at the C
terminus, KETYY, was used. Two different anti-phospholemman antibodies, against the N- and C-terminal ends of the protein, gave the same result. The ratio of phospholemman to was much higher in membrane fractions isolated from choroid plexus than in membrane from whole cerebellar cortex and was if anything higher than the ratio seen in the
cardiac sarcolemma fraction used as a control.
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Figure 4.
Ratio of phospholemman to Na,K-ATPase. Although
the true ratio of phospholemman to the Na,K-ATPase subunit cannot
be determined by comparing the amount of immunoreactivity with two
different antibodies, nonetheless, differences in the ratio between
samples can be assessed. Here, two identical blots were prepared with
samples of canine sarcolemma as a positive control for the antibodies
(C-SL), cerebellar membranes (Cb), and
choroid plexus membranes (CP). The blots were cut in
half. The top halves were stained for Na,K-ATPase subunit with the pan-specific KETYY antibody, and the bottom
halves were stained with phospholemman antibodies against the
C- and N termini, respectively. It can be seen that, relative to the
amount of immunoreactivity for , there was much less phospholemman
(PLM) in the cerebellum sample than the choroid
plexus sample. Molecular weight markers are indicated. We have observed
that in some gels, phospholemman runs at 15 kDa, as reported by others,
but in these (12% acrylamide Tricine gels), it runs at ~8 kDa
(compared with Bio-Rad Rainbow molecular weight markers),
which is close to its predicted size.
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Copurification of phospholemman with Na,K-ATPase
The Na,K-ATPase can be purified from highly active tissue sources
by a relatively simple technique that exploits its unusual resistance
to denaturation by the strong detergent SDS (Jørgensen, 1988;
Sweadner, 1988). In the presence of ATP or ADP to stabilize the enzyme
at the active site, SDS (at ratios of up to 0.4 mg/mg of protein)
extracts contaminating proteins and leaves the Na,K-ATPase in the lipid
bilayer. The membranes are separated from the extracted proteins and
from free SDS by sedimentation on sucrose gradients. The subunit of
the kidney copurifies with Na,K-ATPase and subunits by this
method; therefore, it was tried with phospholemman-containing membranes
from choroid plexus and brain. Figure 5
illustrates the result with bovine choroid plexus. Essentially all of
the phospholemman cosedimented with the Na,K-ATPase in the gradient, with a peak at ~18% sucrose, as shown by blots of gradient fractions stained for and phospholemman. No free phospholemman fraction was
detected. The same copurification of phospholemman with the Na,K-ATPase, without a free phospholemman fraction, was obtained with
canine choroid plexus and with microsomes from whole rat brain (data
not shown).
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Figure 5.
Copurification of phospholemman with Na,K-ATPase.
Membranes from bovine choroid plexus were extracted with SDS and
sedimented on a 7-30% sucrose gradient. The bottom 8 ml were
discarded, and 14 2 ml fractions were collected. Fraction samples were
electrophoresed on a Tricine gel, and the blot was cut in half for
staining for (top; K1 antiserum) and phospholemman
(bottom; PLM-C1). Essentially all of the phospholemman
(PLM) sedimented into the gradient with the
Na,K-ATPase. The final specific activity was 200 µmol · mg of
protein1 · hr1 in this
experiment. The control lane is a sample of canine
sarcolemma (C-SL).
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Coimmunoprecipitation of phospholemman with Na,K-ATPase
To determine whether phospholemman and Na,K-ATPase form a stable
complex, immunoprecipitation was performed with
C12E8 detergent-solubilized crude membranes obtained from either choroid plexus or cerebellum. Figure 6 shows the excellent yield of
solubilization under the conditions used and also shows controls in
which nonimmune IgG was used for a mock precipitation. This control is
important to show for immunoprecipitation, because some proteins
sediment with the adsorbent, leading to false-positive signals. Two
different Na,K-ATPase -specific antibodies were used, as well as two
different phospholemman antibodies. Although the two -specific
antibodies gave the same result, the two phospholemman antibodies
showed differences that may be informative about the structure of the Na,K-ATPase-phospholemman complex.
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Figure 6.
Coimmunoprecipitation of phospholemman with
Na,K-ATPase. Canine sarcolemma samples were positive controls for the
antibodies (C-SL). As indicated, samples were
precipitated with normal IgG as a negative control, 5
or 6H antibodies against Na,K-ATPase subunit, and
anti-phospholemman N- and C-terminal antibodies (PLM-N,
PLM-C). Immunoprecipitates and their controls were
resolved by electrophoresis. Blots were cut in half for staining for
Na,K-ATPase (top; K1, 5, or anti-KETYY in
different panels) and phospholemman
(bottom; PLM-C1). A, Membranes from
bovine choroid plexus. B, Membranes from bovine
cerebellum. From both tissue sources, anti- antibodies
coprecipitated phospholemman (PLM), but
phospholemman C-terminal antibody coprecipitated well, and
phospholemman N-terminal antibody coprecipitated it poorly.
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Figure 6A shows that both anti- antibodies were
effective at coprecipitating phospholemman from bovine choroid plexus
membranes, as was the antibody against the phospholemman C terminus
(PLM-C1, the same antibody used for immunofluorescence above). The
PLM-B8 antibody against the phospholemman N terminus, however,
precipitated phospholemman well but only weakly. A similar result
is seen in Figure 6B, where the membranes were from
bovine cerebellum instead. Despite the presence of less phospholemman,
the antibodies and the anti-C-terminal phospholemman antibody
coprecipitated and phospholemman very well, whereas the
anti-N-terminal phospholemman antibody again precipitated phospholemman
much better than . The anti-N-terminal antibody is against the
conserved FXYD domain, and mutagenesis studies with other FXYD family
members have suggested that FXYD is important for interaction with
Na,K-ATPase (Beguin et al., 2001). The N terminus of phospholemman in
sarcolemma is also very resistant to proteases, indicating its
inaccessibility (Chen et al., 1998). We propose either that the
anti-N-terminal antibody cannot bind to the extracellular portion of
phospholemman when it is tightly associated with Na,K-ATPase, or that
the antibody disrupts the phospholemman-Na,K-ATPase interaction in
detergent. In contrast, the C terminus of phospholemman is not
constrained in the same way. Because the C terminus has the
phosphorylation sites, it should be accessible to approach by other
proteins, including antibodies.
To determine which Na,K-ATPase isoforms interact with
phospholemman, immunoprecipitation from cerebellar membrane fractions was performed using the PLM-C1 antibody (Fig.
7). Each isoform was detected with a
well characterized isoform-specific monoclonal antibody. The result was
that all three Na,K-ATPase isoforms coprecipitated. The lack of in controls using normal rabbit IgG demonstrates that the precipitation
was not an artifact. The conclusion is that phospholemman interaction
with the Na,K-ATPase is not isoform-specific, and the implication is
that phospholemman is expressed in both 3-containing neurons and
2-containing glia. This conclusion differs somewhat from that of
Crambert et al. (2002), who reported that assembly with 2 was less
efficient than with 1 in oocytes. The discrepancy may be
attributable to a lack of 2 in the oocytes, the most abundant
partner of 2 in the brain.
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Figure 7.
Coimmunoprecipitation of phospholemman with
Na,K-ATPase isoforms. Control lanes include both
canine sarcolemma (C-SL) and samples of the bovine
cerebellar membrane-starting material (B-Cb).
Immunoprecipitation was performed with normal rabbit IgG as a negative
control and the antibody against the C terminus of phospholemman
(PLM-C). Three identical blots were prepared and cut in
half. The bottom halves were all stained with the
phospholemman antibody, but the top halves were stained
with isoform-specific anti-Na,K-ATPase subunit antibodies: antibody
6F for 1, McB2 for 2, and XVI-F9G10 for 3.
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Functional interaction of phospholemman with Na,K-ATPase
To test the hypothesis that phospholemman regulates the activity
or properties of the Na,K-ATPase, we incubated purified choroid plexus
Na,K-ATPase (the preparation with the highest proportion of
phospholemman) with anti-phospholemman antibodies. Similar experiments
with antibodies to the Na,K-ATPase subunit gave some of the first
evidence for its functional interaction with the pump (Therien et al.,
1997, 1999). PLM-C2 is an antibody directed against the C-terminal
portion of phospholemman similar to PLM-C1. Figure
8 shows that PLM-C2, measured at a range
of Na+ concentrations in vitro,
produced a significant decrease in activity without affecting the
apparent affinity for Na+. Table
1 summarizes additional data. PLM-C2
showed an average 20% reduction of choroid plexus Na,K-ATPase activity
measured in multiple experiments with either 5 or 100 mM NaCl, whereas it had no effect on kidney
Na,K-ATPase, which has the subunit instead of phospholemman. There
was no inhibition of the ouabain-insensitive Mg2+-ATPase of either preparation. The
PLM-B8 antibody directed to the N-terminal domain of phospholemman did
not have any effect on activity, which may be attributable to an
inability to bind its target if the N-terminal portion of phospholemman
is engaged in interaction with the Na,K-ATPase. We verified that
phospholemman was not phosphorylated in the conditions of our
experiments (data not shown).
View larger version (17K):
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Figure 8.
Effect of PLM-C2 antibody on Na,K-ATPase.
Ouabain-sensitive Na,K-ATPase activity was determined as a function of
Na+ concentration after 4 hr of preincubation at
4°C with either normal rabbit IgG (filled
circles) or PLM-C2 antibody (open circles).
Inset, The following equation was fitted to the data:
v = Vmax
[Na+]nH/(KNanH + [Na+]nH), where
Vmax is the specific activity at a
nonlimiting Na+ concentration;
KNa is the Na+
concentration giving half-maximal activity; and nH is
the Hill coefficient. Best-fitting values for these parameters are
given as mean ± SEM, average of two experiments performed in
duplicate.
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Discussion |
FXYD family members as sodium pump regulators
Phospholemman is the fifth FXYD family member to exhibit
interaction with Na,K-ATPase. There are precedents for the regulation of other transport ATPases by small membrane-spanning accessory proteins. Phospholamban, a 52-amino acid protein not homologous to FXYD
proteins, regulates the sarcoplasmic reticulum
Ca2+-ATPase (SERCA2a) of the heart. It is
phosphorylated by protein kinase A and mediates -adrenergic
stimulation of contractility by reversing the inhibitory effect it has
on the affinity of SERCA for Ca2+
(Simmerman and Jones, 1998). Its homolog sarcolipin is expressed in
skeletal muscle with a different isoform of SERCA (Odermatt et al.,
1998). Yeast have small membrane proteins that regulate the plasma
membrane ATPase (Navarre et al., 1994). Ion channels also have
accessory proteins that modulate functional properties (Suessbrich and
Busch, 1999). Thus regulation of ion transport via small membrane
proteins is becoming an important theme.
Three lines of evidence presented here support the hypothesis
that phospholemman interacts specifically with the
Na,K-ATPase: the two proteins copurify after SDS-extraction;
they coimmunoprecipitate with antibodies against either
component; and an anti-phospholemman antibody reduces Na,K-ATPase
activity. Crambert et al. (2002) observed coprecipitation of
phospholemman with Na,K-ATPase from cardiac membranes or oocytes, and
in oocytes, phospholemman decreased the apparent affinity for
Na+ (and K+).
Because we observed inhibition of activity without effect on Na+ affinity, at first glance, these
results may seem contradictory. The emerging evidence for functional
effects of FXYD proteins on Na,K-ATPase is quite complex, however.
Effects on ATP hydrolysis, Na+ affinity,
and other properties appear to be experimentally separable and
sometimes subject to modulation. The bottom line is that FXYD proteins,
now including phospholemman, can affect functional properties of the
Na,K-ATPase, fine tuning its properties.
The inhibitory effect of antibody suggests that phospholemman might be
an enzyme activator. In similar experiments on the subunit,
antibodies against the C terminus reduced activity (Therien et al.,
1997), decreased apparent affinity for ATP (Therien et al., 1999), and
increased the IC50 for vanadate inhibition (an experimental test of the distribution between two principal enzyme
conformations, E1 and E2; Pu et al., 2001). Apparent affinity for ATP
was increased in transfectants of human embryonic kidney 293 and
HeLa cells. Most recently, truncation of the C terminus was shown to
abrogate the effect of on apparent affinity for ATP (Pu et al.,
2002). Together, these and other data suggested that the anti-
antibody favored E2, antagonizing a normal tendency of to favor E1.
These apparent activating effects were not the only effects of the subunit, however. There were decreases in apparent affinity for
Na+, K+, or
both in enzyme partially purified from transfected mammalian cells
(Arystarkhova et al., 1999, 2002; Pu et al., 2001, 2002) and in oocytes
(Beguin et al., 1997, 2001). This should reduce activity in
vivo, because the effects occur in the physiological range.
Anti- antibodies, however, did not interfere with the effect on
Na+ affinity, nor did truncation of the C
or N terminus (Pu et al., 2002). For these reasons, the lack of effect
of anti-phospholemman antibody on Na+
affinity reported here does not necessarily contradict the reduction in
Na+ affinity reported in oocytes (Crambert
et al., 2002). Modulation of ion affinity may require the central
domain and not the C terminus.
Further complexities in FXYD protein effects are also known. The
expression of N-terminal splice variants, as well as
post-translational modifications, changed how the subunit altered
Na,K-ATPase affinities for Na+ and
K+ in NRK-52E cells (Arystarkhova
et al., 2002) but not in HeLa cells (Pu et al., 2001). Thus different
domains of may be responsible for different effects, and the
effects may be modified by factors specific to the cell type.
Interestingly, CHIF (FXYD4) increased the affinity of Na,K-ATPase for
Na+ (the opposite of the effect of and
phospholemman; Beguin et al., 2001; Garty et al., 2002), whereas FXYD7
decreased K+ affinity at the extracellular
site (Beguin et al., 2002). These proteins are homologous to both and phospholemman in the central domain, yet each has a subtly
different effect on Na,K-ATPase properties. There is wide sequence
variation in the extracellular and intracellular domains, providing a
further basis for different functional roles. Because of the complexity
of Na,K-ATPase kinetics, it is going to take considerable effort to
fully understand the properties of these proteins in regulating the
sodium pump.
Phospholemman also presents the special problem that it has four
potential phosphorylation sites in the cytoplasmic domain, which are
expected to be important in regulating its properties. The FXYD protein
found in shark, PLMS, reduced Na,K-ATPase activity, and its
phosphorylation increased activity (Mahmmoud et al., 2000). Preliminary
experiments on phospholemman phosphorylation in vitro produced both stimulatory and inhibitory effects, suggesting that it
matters which sites are phosphorylated (our unpublished observations).
We have independent evidence that phospholemman may play a role as an
activator of Na,K-ATPase. The specific activity of Na,K-ATPase in
cardiac membranes of a phospholemman knock-out mouse was significantly lower than in its control (L. G. Jia, C. Donnet, R. C. Bogaev, R. J. Blatt, C. E. McKinney, K. H. Day, S. S. Berr, L. R. Jones, J. R. Moorman, K. J. Sweadner, and
A. L. Tucker, unpublished results). This is consistent with
the hypothesis that the PLM-C2 antibody acts as an
interfering antibody that antagonizes a normal stimulatory effect of phospholemman.
FXYD proteins in the CNS
The association of phospholemman with Na,K-ATPase in
choroid plexus is highly suggestive of a role in the regulation of CSF secretion. Secretion and Na,K-ATPase activity are both known to be
regulated by hormones and transmitters through protein kinases (Fisone
et al., 1995, 1998; Ellis et al., 2000). Given that there is
uncertainty about the universality of the Na,K-ATPase protein kinase C
site and the accessibility of the protein kinase A site, phospholemman
could be the critical kinase target.
Phospholemman has a unique distribution in cerebellum. The
stained axons passing through the granular layer were most likely axons
of Purkinje cells, because the cell bodies were clearly labeled.
Similar immunoreactivity has been seen for the Na,K-ATPase 3 subunit
(Peng et al., 1997), which is expressed in Purkinje neurons (Watts et
al., 1991). Although climbing fibers cannot be ruled out, mossy fibers
can be ruled out, because the glomeruli, which contain their club-like
termini in the granular layer, were unstained. The possibility that the
stained axons could be granule cell axons seems unlikely. This would
require strict subcellular routing to axons and not somas or dendrites,
which were unstained in the granular layer. Furthermore, the axons
extended into the white matter (data not shown). If Purkinje neurons
were the only phospholemman-containing cells, however,
coimmunoprecipitation with 2 would not be expected, because that
isoform is expressed mostly in astrocytes and Bergmann glia (Watts et
al., 1991; Peng et al., 1997). Astrocytes in the granular layer did not
stain for phospholemman, but the diffuse immunoreactivity in the
molecular layer could be in the plasma membranes of Bergmann glia as
easily as in those of Purkinje neurons, because the two are closely
interdigitated. Immunoreactivities for Na,K-ATPase isoforms 1-3,
1, and 2 all have a similar diffuse distribution in the molecular
layer (McGrail et al., 1991; Cameron et al., 1994; Peng et al., 1997), which reflects the small size and intimate contact of neuronal and
glial processes. Phospholemman has been detected in cultures of
cerebellar astrocytes from postnatal rats (Moran et al., 2001), something we have confirmed (data not shown). They presented evidence that reduction of phospholemman expression reduced astrocyte
volume-sensitive taurine release. However, Bres et al. (2000) detected
phospholemman mRNA in neurons but not in the astrocytes of the ventral
glia limitans of the supraoptic nucleus and concluded that
phospholemman was not important for volume-sensitive taurine release
there. More investigation is needed.
We can speculate that association with FXYD family member
accessory proteins is a common theme for Na,K-ATPase modulation. If so,
proteins other than phospholemman must play this role in other parts of
the brain. mRNA for FXYD6, variously called phosphohippolin (without
evidence yet for phosphorylation), phospholemman-like protein, or
vascular endothelial cell-specific protein (GenBank accession number
AB030908), has been detected by in situ hybridization in
cells of the hippocampal pyramidal cell layer and the granular layer of
the cerebellum (Saito et al., 2001; Yamaguchi et al., 2001). From
Northern blots (Beguin et al., 2002) and the sources of expressed
sequence tags (ESTs; Sweadner and Rael, 2000), FXYD7 is expressed
almost exclusively in the CNS, and ESTs for FXYD3 (mammary tumor marker
8) and FXYD5 (related to ion channel) have also been obtained from CNS
libraries, predicting the presence of as many as five FXYD family
members. Combined with the three Na,K-ATPase isoforms and three isoforms that are expressed in different brain cell types in different
combinations of and (McGrail et al., 1991; Watts et al., 1991;
Cameron et al., 1994; Peng et al., 1997; Martin-Vasallo et al., 2000),
the potential subunit diversity of the sodium pump is considerable. In
our opinion, the physiological significance of Na,K-ATPase regulation
in the CNS is much neglected.
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FOOTNOTES |
Received Nov. 4, 2002; revised Dec. 20, 2002; accepted Dec. 20, 2002.
This work was supported by National Institutes of Health Grant
R01-NS27653 (K.J.S.) and a Claflin distinguished scholar award (M.S.F.).
Correspondence should be addressed to Kathleen J. Sweadner, 149-6118, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. E-mail: sweadner{at}helix.mgh.harvard.edu.
| |
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ABSTRACT
Introduction
