Multiplicity of expression of FXYD proteins in mammalian cells: dynamic exchange of
phospholemman and gamma subunit in response to stress.
Elena Arystarkhova 1, 3, Claudia Donnet 1, Ana Muñoz-Matta 2, Susan Specht 2, and
Kathleen J. Sweadner 1
1Laboratory of Membrane Biology, Massachusetts General Hospital/Harvard Medical
School, Boston, Massachusetts, 02114, and the 2 Department of Pharmacology and
Toxicology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico 00936.
Na,K-ATPase responds to hypertonicity via FXYD proteins
Dr. Elena Arystarkhova
Laboratory Membrane Biology,
415 Thier Research Building,
Massachusetts General Hospital,
55 Fruit St., Boston, MA 02114
Phone: (617) 726-8560
FAX: (617) 726-7526
Page 1 of 60Articles in PresS. Am J Physiol Cell Physiol (October 18, 2006). doi:10.1152/ajpcell.00328.2006
Copyright © 2006 by the American Physiological Society.
Functional properties of Na,K-ATPase can be modified by association with
FXYD proteins, expressed in a tissue-specific manner. Here we show that expression of
FXYDs in cell lines does not necessarily parallel the expression pattern of FXYDs in the
tissue(s) the cells originate from. While being expressed only in lacis cells in the
juxtaglomerular apparatus and in blood vessels in kidney, FXYD1 was abundant in renal
cell lines of proximal tubule origin (NRK-52E, LLC-PK1, and OK cells). Authenticity of
FXYD1 as a part of Na,K-ATPase in NRK-52E cells was demonstrated by co-
purification, co-immunoprecipitation and co-localization. Induction of FXYD2 by
hypertonicity (500 mOsm with NaCl for 48 h or adaptation to 700 mOsm) correlated with
down-regulation of FXYD1 at mRNA and protein levels. The response to hypertonicity
was influenced by serum factors and entailed first, dephosphorylation of FXYD1 at Ser68
(1-5 hours), and second, induction of FXYD2a and decrease in FXYD1 with longer
exposure. FXYD1 was completely replaced with FXYD2a in cells adapted to 700 mOsm
and showed a significantly decreased sodium affinity. Thus dephosphorylation of
FXYD1 followed by exchange of regulatory subunits is utilized to make a smooth
transition of properties of Na,K-ATPase. We also observed expression of mRNA for
multiple FXYDs in various cell lines. The expression was dynamic and responsive to
physiologic stimuli. Moreover, we demonstrated expression of FXYD5 protein in
HEK293 and HeLa cells. The data imply that FXYDs are obligatory rather than auxiliary
components of Na,K-ATPase, and their interchangeability underlies responses of Na,K-
ATPase to cellular stress.
Key words: Na,K-ATPase; cellular stress; regulation; FXYD1; FXYD2
Page 2 of 60
The Na,K-ATPase catalyzes active efflux of Na+ and uptake of K+ ions, thus
establishing and controlling ionic gradients across the plasma membrane in virtually
every animal cell. It is the receptor for cardiac glycosides, drugs commonly used in
treatment of heart failure. Recent findings suggest that binding of ouabain to Na,K-
ATPase, besides a direct inhibition of the pump activity, initiates a signaling pathway that
ultimately leads to changes in cell hypertrophy and proliferation.
Regulation of Na,K-ATPase occurs at several levels. It may entail regulation of
gene expression of the obligatory α and β subunits (long-term regulation, which may
serve as an adaptation response) or recruitment/internalization of the active pump units
to/from plasma membrane in response to certain physiological stimuli, which can serve
as an immediate regulatory response. On top of this, multi-layered regulation of Na,K-
ATPase includes modulation of the intrinsic properties of the enzyme via physical
association with the so-called FXYD proteins [reviewed in (29,31)]. This is a family of
small (6.5-17 kDa) single-span membrane proteins with structural homology (61). The
family includes: phospholemman (PLM) (FXYD1), the major plasma membrane
substrate for PKA and PKC phosphorylation in heart (49); the γ subunit (FXYD2) (45),
which is expressed primarily in kidney in at least two splice variants (6); Mat 8
(FXYD3), a gene up-regulated in epithelial cells by oncogenic Ras and Neu and
expressed in human breast and prostate tumors (46); CHIF (corticosteroid hormone-
induced factor) (FXYD4) (7); dysadherin, or RIC (related to ion channel) (FXYD5)(27);
FXYD6 (phosphohippolin) (61); and brain-specific FXYD7. Most of the homology is
within the transmembrane segment and the signature motif PFXYD (61).
Page 3 of 60
To date, 6 members of the family have been demonstrated to associate with the
Na,K-ATPase: FXYD1 (20,26); FXYD2 (6,51); FXYD3 (22), FXYD4 (10), FXYD5
(43), and FXYD7 (11). Remarkably, association of Na,K-ATPase with each of the FXYD
proteins resulted in a somewhat different modulation of the kinetic properties of the
Na,K-ATPase. FXYD4 (10,30) and FXYD5 (43) stimulated Na,K- ATPase activity
either by increasing the affinity for Na+ or Vmax, whereas FXYD3 (22), FXYD7 (23), and
FXYD2 exhibited an inhibitory effect by either decreasing the affinity for Na+ and/or K+,
or reducing the Vmax of the pump (2,42,67). Overexpression of FXYD1 reduced apparent
affinity for Na+ and K+ in heterologous expression systems (4,20) and decreased the Vmax
of Na-pump in adult rat myocytes (72). Phosphorylation of FXYD1, on the other hand,
seemed to release the inhibition with a subsequent activation of the pump (13,28).
Some of the FXYD family members exhibit a high degree of tissue specificity.
FXYD1 is highly expressed in heart and brain (cerebellum and choroid plexus) (20,26),
but in kidney it is limited to the juxtaglomerular apparatus and blood vessels (68).
Conversely, FXYD2 is only found in kidney in healthy animals (45). In stomach,
expression of FXYD3 is in mucous cells (22), while FXYD4 is expressed in distal colon
and collecting duct in kidney (18). Whether expression of FXYD6 and FXYD7 in CNS
overlaps is not known yet. Expression of FXYD5 in normal tissue is preferentially in
kidney, intestine, spleen and lung (43). In kidney FXYD5 is at the basolateral membrane
of connecting tubules and the intercalated cells of collecting duct, but it was also detected
on apical membranes in thin limb of Henle, suggesting a potential role in addition to
being part of the Na,K-ATPase complex.
Page 4 of 60
Tissue/cell-specific expression of the regulatory FXYD subunits of Na,K-ATPase
is not static, however, and may be changed to adapt to a given physiological or
pathological situation. For instance, significant up-regulation of FXYD1 was observed in
response to cardiac infarction (57) and nerve injury (19). Expression of FXYD1 is also
stimulated in muscle with exercise (54). Gene array studies revealed expression of
FXYD2 in hippocampus in response to chemical insult (38). Expression of FXYD3 may
be induced by ras- and neu-oncogenes (46), while expression of FXYD4 is regulated by
corticosteroid and K+-deprivation (66). FXYD2, which is normally limited to kidney, can
be also induced in cells of a variety of different origins in culture by exposure to different
kinds of cellular stress (hypertonicity, heavy metals, exogenous oxidation, heat shock)
Here we present evidence that expression of FXYDs is even more plastic in cells
in culture, and cells of different origin may express more than one FXYD at a time (at
protein and/or mRNA level). Moreover, we show that cellular stress (hypertonicity)
initially caused dephosphorylation with a subsequent down regulation of endogenously
expressed FXYD1 followed by its replacement with a newly synthesized FXYD2a within
the Na,K-ATPase complex in NRK-52E cells. As we showed previously (67), induction
of FXYD2a by 48 hour exposure to hypertonicity correlated with a significant reduction
of Na,K-ATPase activity. Here we observed about 20% reduction in ATPase activity
even after 5 hours of hypertonic challenge. Since no FXYD2a was synthesized yet at this
point (67), but dephosphorylation of FXYD1 was dramatic, we suggest that the latter
event is employed as a first line of cell defense to survive an apoptotic insult under stress-
related conditions. The data support the hypothesis that alterations of Na,K-ATPase
Page 5 of 60
properties entail exchanges of FXYD subunits, and this phenomenon represents an
important adaptive mechanism.
Some preliminary data have been presented (24).
MATERIALS and METHODS
Antibodies and Cell Lines.
Polyclonal antisera K1 or K3 (60) were used for detection of the α1 subunit of
Na,K-ATPase on blots. The VG4 monoclonal antibody (3) was employed for
immunoprecipitation of α1. The RCT-G1 polyclonal antibody raised against the C-
terminal peptide (5) was used to identify both splice variants of FXYD2. On blots, non-
phosphorylated FXYD1 (PLM) was detected with the PLM-C2 rabbit antiserum raised
against the C-terminal sequence of phospholemman (the generous gift of Dr. J. Cheung,
Milton S. Hershey Medical Center, Pennsylvania State University) (59). The form of
FXYD1 that is phosphorylated at Ser68, was detected with the CP68 antibody provided
Dr. J. R. Moorman, University of Virginia Health System)(55). For immunoprecipitation
of FXYD1, PLM-C1, an affinity-purified polyclonal rabbit antibody against the C
terminus, was employed (a kind gift of Dr. L. R. Jones, Indiana State University). The
polyclonal antibody against FXYD5 (anti-dysadherin, ab2248) was from AbCam Inc.
NRK-52E, C6, MDCK, LLC-PK1, Caco-2, OK, HEK293, HeLa, and SH-SY5Y
cell lines were purchased from ATCC and grown according to the company’s
Page 6 of 60
recommendations. mIMCD3 cells were a kind gift of Dr. J. Capasso (Colorado Health
Sciences Center). Differentiation of SH-SY5Y cells was with retinoic acid (10 µM).
Cell Culture and Hypertonicity Treatment.
The normal rat kidney epithelial cell line (NRK-52E) was grown to 85–90%
confluency in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (300
mOsm). The medium was then replaced with either control or hyperosmotic (500 mOsm
total) medium supplemented with NaCl. In the case of serum reduction, cells were
incubated overnight in a medium containing 0.5% FBS before hypertonicity treatment in
the same concentration of serum. Flasks were washed with Dulbecco’s phosphate-
buffered saline with Ca2+and Mg2+at various times and frozen at -80°C.
Adaptation of NRK-52E cells to high hypertonicity was similar to what was
described for mIMCD-3 cells (15). Briefly, hypertonicity was raised in the medium by
successive increments of 50 mOsm (with NaCl). Cells were allowed to adapt to each new
medium for 2-3 weeks before the next increase in osmolality. Passaging was performed
when cells reached confluency.
Membrane Preparations and Enzyme Purification.
Crude membrane preparations were obtained from scraped cells by
homogenization and differential centrifugation (5). Isolation of membranes from rat
kidney outer medulla was performed as described elsewhere (60). Partial purification of
Na,K-ATPase from cell membranes (1.4 mg/ml) was with SDS extraction (0.56 mg/ml)
and sedimentation on 7–30% sucrose gradients. Typical specific activity of Na,K-ATPase
was about 50–150 µmol Pi/mg of protein/h in preparations from NRK-52E cells.
Page 7 of 60
To obtain cell lysates, a buffer containing 50 mM Tris-Cl, pH 8.0, 5 mM EDTA,
1% NP-40 and protein phosphatase inhibitors cocktail I (Sigma) (1:100, v/v), was used.
Cells were scraped, triturated, and insoluble material was removed by centrifugation at
3,000 x g for 10 min (Sorvall, SS-34).
Na,K-ATPase activity was measured in SDS-purified preparations from either
control or adapted cells as a function of Na+concentration in media containing 3 mM
Tris-ATP, 4 mM MgCl2, 30 mM histidine, pH 7.4, and in the presence of 20 mM K+. All
the reactions were performed at 37 °C for 30 minutes with and without 3 mM ouabain,
and ouabain-sensitive Pi release was measured colorimetrically by using the Fiske-
Subbarow method. Data were analyzed by nonlinear regression using Sigma Plot Graph
System (Jandel Scientific). Na+-activation curves were fitted according to the Hill model
for ligand binding.
Activity in crude membranes was measured using the so-called “yellow method”,
which tolerates more protein and lipid. After quenching the ATPase reaction with acid
and molybdenum as in the Fiske-Subbarow method, the modification involves the
extraction of liberated Pi as the unreduced (yellow) phospho-molybdenum complex into
an organic phase (water-saturated n-isobutanol), and reading the absorbance at 380 nm.
Protein phosphatase inhibitor was added throughout the isolation of membranes and
Immunoprecipitation. Plasma membrane-enriched fractions from NRK-52E cells (1
mg/ml) were solubilized with 3 mg/ml n-dodecyl octaethylene glycol monoether
detergent (C12E8; Calbiochem) (6) for 10 min at room temperature in buffer A, containing
Page 8 of 60
140 mM NaCl, 25 mM imidazole, and 1 mM EDTA, pH 7.3. At the end of the
incubation, the solubilized material was diluted with 2 volumes of buffer A and
sedimented by centrifugation for 30 min at 20,000 × g at 4°C. The supernatant was
incubated with primary antibodies (PLM-C1 to immunoprecipitate phospholemman or
VG4 to immunoprecipitate the α subunit) or control IgG (rabbit or mouse, respectively,
at 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
antibodiescovalently bound to agarose beads (Sigma). Immunoprecipitateswere collected
by centrifugation at 9300 × g for 10 min at 4°C and washed four times with buffer A
containing 0.05% C12E8. After the final wash, the pellet was resuspended in 1×
electrophoresis sample buffer.
Membrane fractions were resolved either on SDS-Tricine or on 4-12% MES-SDS
gels (NuPage system, Invitrogen). Proteins were transferred to nitrocellulose, incubated
with antibodies, and detection was with chemiluminescence. For positive control samples
membrane preparations from kidney or canine cardiac sarcolemma (37) were used.
Quantification was with ImageQuant TL image analysis software (Amersham).
NRK-52E cells, either wild type or adapted to 700 mOsm, were seeded into 38-
mm2 wellsof 96-well flat-bottomed plates (5 x 103/ well) in quadruplicate and allowed to
adhere overnight. Cell proliferation over the following 2-6 days was assayed based on the
cleavage of the tetrazolium salt WST-1 (Roche Diagnostics) by mitochondrial
Page 9 of 60
dehydrogenases in viable cells. Quantification was with a microtiter plate reader at 450
Immunocytochemistry was performed as described elsewhere in more detail (6).
Briefly, cells were fixed with 2% periodate/lysine/paraformaldehyde solution for 30 min
at room temperature followed by 5 minute incubation with 1% SDS in PBS, several
washes with PBS, and blocking with 5% goat serum in PBS to prevent nonspecific
binding of secondary antibodies. McK1 antibody was used for α1 detection, whereas
PLM-C1 (described above) and CP68 antibodies were used to probe non-phosphorylated
or phosphorylated FXYD1, respectively. The secondary antibodies were either Cy3-
conjugated goat anti-mouse IgG (1:300; Accurate) or fluorescein isothiocyanate-
conjugated goat anti-rabbit IgG (1:300; Jackson ImmunoResearch). Slides were
examined with a Nikon TE300 fluorescence microscope equipped with a Bio-Rad MRC
1024 scanning laser confocal system (version 3.2).
Total RNA from cells was prepared with the RNeasy system (Qiagen). The cDNA
was obtained using 1 µg of total RNA, oligo(dT) as the priming oligonucleotide, and
Super Script II reverse transcriptase (Invitrogen). For HEK293 and HeLa cells , cDNA
was obtained with the Super Script III cell Direct cDNA synthesis system (Invitrogen).
The polymerase chain reaction was performed in the presence of 3 mM MgCl2 with either
Taq polymerase (Fisher) or Platinum Taq polymerase (Invitrogen). PCR products were
separated by electrophoresis in 2% agarose gels. Primers were chosen according to
Page 10 of 60
sequences of FXYD genes from rat, mouse and human (Table 1) using software provided
FXYD1 is associated with Na,K-ATPase in NRK-52E cells.
Expression of proteins in cultured cells often parallels the expression pattern in
the tissues they originate from. However, expression of regulatory subunits of Na,K-
ATPase (FXYDs) does not entirely follow the same paradigm. FXYD1,
phospholemman, is abundant in muscle, brain, and heart, where it represents the major
substrate for PKA and PKC. In kidney, though, its expression is limited to afferent
arteriole and lacis cells in the juxtaglomerular apparatus (68). Nevertheless, as shown in
Fig. 1A, the FXYD1 protein was readily detectable in crude membrane preparations (P2)
from renal NRK-52E cells, which possess characteristics of proximal tubule cells.
Moreover, FXYD1 remained tightly bound to Na,K-ATPase from NRK-52E cells during
a purification procedure that involves mild SDS treatment of crude membranes and
extraction of the majority of membrane proteins other than Na,K-ATPase (5). The blots
were stained with the C2 antibody that is specific for non-phosphorylated FXYD1 and
exhibits a very weak cross-reactivity with phosphorylated FXYD1 (55). No signal was
observed when identical blots were stained with the CP68 antibody (raised against the
peptide phosphorylated at the Ser68 site) (55) (not shown). Since no protein phosphatase
inhibitors were employed here, the assumption was that any phosphorylation of FXYD1
at Ser68 site was labile and was lost during membrane preparation. Therefore, unless
otherwise specified, we used staining by C2 as a measure of the total level of FXYD1
Page 11 of 60
Interaction of the α subunit of Na,K-ATPase and FXYD1 was further
corroborated by their co-immunoprecipitation from crude membranes solubilized with
non-ionic detergent C12E8 (Fig. 1B). Antibody against FXYD1 brought down the α
subunit of Na,K-ATPase, and anti-α antibodies precipitated FXYD1 as well (not shown).
[The doublets of FXYD1 seen in some figures but not in others may have something to
do with the different gel systems employed (SDS-Tricine vs 4-12% gradient SDS-MES
gels from Invitrogen) and were not investigated further]. All together the data indicate
that FXYD1 is an authentic component of Na,K-ATPase in renal NRK-52E cells.
Interestingly, the ratio between FXYD1 and the alpha subunit was significantly
lower in the purified preparation of Na,K-ATPase compared to crude membranes (Fig.
1A) implying the existence of a pool of FXYD1 not tightly bound to Na,K-ATPase.
We used immunocytochemistry to monitor the location of FXYD1 in NRK-52E
cells. Cells were fixed with 2% paraformaldehyde and stained for FXYD1 and the α
subunit of Na,K-ATPase. Two different antibodies were employed here to distinguish
between phosphorylated (Ser68) and non-phosphorylated forms of FXYD1. As shown in
Fig. 2, subcellular location of FXYD1 was apparently affected by the phosphorylation
status of the protein. Good co-localization of FXYD1 phosphorylated at Ser68 site and α
was observed at plasma membrane (Fig. 2, A-C), while a majority of unphosphorylated
FXYD1 was seen intracellularly (Fig. 2, D-F). The data are in line with prior data (39)
suggesting that phosphorylation of FXYD1 may be prerequisite for trafficking to plasma
Thus, based on immunofluorescence data, we speculate that a “free pool” of
FXYD1 may consist of an intracellularly located non-phosphorylated FXYD1, whereas a
Page 12 of 60
major fraction of FXYD1 associated with the pump at plasma membrane consists of a
phosphorylated form of the protein.
We also detected FXYD1 in other renal cell lines originating from proximal
tubules: porcine LLC-PK1 and opossum OK cells (Fig. 3). Meanwhile no FXYD1 was
detected in canine MDCK (Fig. 3) or mouse mIMCD3 cells (not shown), both originating
from distal nephron segments, or in embryonic HEK293 cells. The antibody’s epitope is
conserved. This implies cell (tubule)-specific control of FXYD1 expression in culture.
Interestingly, in kidney, proximal tubules are positive for FXYD2a, while more distal
segments express either FXYD2b (distal convoluted tubules) or a combination of
FXYD2b and FXYD2a (inner medullary collecting duct). Although abundant in tissue,
FXYD2 is normally absent from any mammalian renal cell line (5,62). The attractive
hypothesis is that FXYD1 substitutes for FXYD2a within the Na,K-ATPase complex in
cells in culture in order to adjust intrinsic properties of the pump properly for the
Transfection with FXYD2 affects the expression of endogenous FXYD1.
We have shown previously that expression of the FXYD2 splice variants can be
achieved in NRK-52E cells by transfection (2,5), although the level of expression of
FXYD2a or FXYD2b was never as high as in kidney membranes. With both FXYD2a
and FXYD2b there was a reduction in the activity of Na,K-ATPase and a reduction in the
rate of cell growth. Since FXYD1 is endogenously expressed in NRK-52E cells as an
authentic component of Na,K-ATPase, we tested whether expression of FXYD2 by
transfection would cause a replacement of one FXYD protein with another. Several
clones expressing splice variants of γ (FXYD2) have been tested, and Fig. 4 shows a
Page 13 of 60
representative Western blot analysis of crude membranes from γa-, γb-, and mock-
transfected cells. Transfection with γa correlated with only slight (if any) reduction of the
total level of FXYD1 expressed in NRK-52E: 92 +/- 13% (n = 3) expressed as the ratio
between the staining of PLM (C2) and the α subunit (K3) in transfected vs control cells.
In contrast, the FXYD1/α ratio was significantly lower in γb-transfectants compared to
γa- (18.1 +/- 4.5 %, n = 4) and mock-transfected cells (11.2 +/- 2.3 %, n = 4), even
though the amount of γ was lower in γb- compared to γa transfectants (Fig. 4). The data
imply a cross-talk in regulation of FXYD protein expression and a potential competition
between γb and phospholemman for binding to Na,K-ATPase. Since both γa- and γb-
transfectants exhibited similar slow growth compared to mock-transfected cells, the
conclusion is that introduction of FXYD2 rather than relative changes in expression of
FXYD1 had the impact on the rate of cell proliferation, which is in agreement with our
recent data on knock-down of FXYD2 in NRK-52E cells (67), where silencing restored
Na,K-ATPase activity and the normal rate of growth in the continued presence of
Reciprocal regulation of FXYD1/FXYD2a expression under stress-related conditions
As we demonstrated previously (67), FXYD2a is not expressed in renal cells in
culture under normal conditions, but can be induced by several kinds of cellular stress,
such as hypertonicity (with NaCl and sucrose, but not with urea), heat shock, exogenous
oxidants, or heavy metal treatment. The newly synthesized FXYD2a associates with the
Na,K-ATPase and inhibits its activity. Here we tested what happens with the
constitutively expressed FXYD1 during stress-mediated induction of FXYD2a.
Page 14 of 60
Fig. 5 shows a representative Western blot of crude membranes from either NRK-
52E cells (Fig. 5A) or C6 glioma cells (Fig. 5B) exposed to isotonic (ctr) or hypertonic
conditions (500 mOsm with NaCl) in the presence of 10% FBS. In both cases induction
of FXYD2a correlated with a reduction of the total level of FXYD1. (No protein
phosphatase inhibitors were added during membrane preparation). Based on densitometry
analysis, the FXYD1/α ratio was decreased by 31.8 +/- 3.5 % (n = 5) in membranes from
NRK-52E cells acutely treated with hypertonicity compared to control. Down regulation
of FXYD1 with hypertonicity was greater (at least 50% or more) in C6 cells suggesting
specificity of cellular response.
The changes in the reciprocal expression of FXYD1/FXYD2a were even more
dramatic in NRK-52E cells slowly adapted to hypertonicity. Although these cells
originate from proximal tubules (40), which do not experience high hypertonicity in vivo,
NRK-52E can be adapted to live under high salt similar to what was described for
mIMCD3 cells (15). Adapted cells (700 mOsm with NaCl in the presence of 10% FBS))
are viable, but proliferate much slower than control cells grown in 300 mOsm medium
(Fig. 6A). Western blot analysis of crude membranes showed that adaptation to high salt
caused a) an increase in protein expression of the α subunit ( 1.7 +/- 0.5 fold, n = 4) ; and
b) a reciprocal switch between FXYD proteins: induction of FXYD2a was accompanied
by a complete loss of FXYD1 from cells slowly adapted to high salt (Fig. 6B).
Functionally, there was a significant 1.5 +/- 0.4 (n = 3) fold increase in Na,K-ATPase
activity in membranes from adapted cells that correlated nicely with an increase in the
amount of α, implying no significant changes in activity per unit of protein measured
under optimal conditions. The data are in qualitative agreement with a report by Capasso
Page 15 of 60
et al. (16) that showed a 5-fold up-regulation of α and β subunits in mIMCD3 cells
adapted to live under hypertonic conditions (600 mOsm with NaCl) as well as a 10-fold
or larger increase in Na,K-ATPase activity in cell lysates.
The complete substitution of one FXYD for the other had a dramatic effect on
Na,K-ATPase kinetic properties. As shown in Fig. 6C, the apparent affinity for Na+was
significantly decreased in preparations of Na,K-ATPase isolated from adapted cells
where FXYD1 was completely replaced with FXYD2a. Based on nonlinear regression
analysis of data fit to the Hill equation, K0.5for Na+ was shifted from 4.9 +/- 0.6 mM (n =
6) in wild-type NRK-52E cells (expressing “pure” FXYD1) to 12.6 +/- 0.8 mM (n = 5) in
adapted cells (expressing “pure” FXYD2a). The data show an even greater K0.5 Na+ than
measured in partially purified enzyme from rat renal medulla (9.5 +/- 0.4 mM) (2) or
whole mouse kidney (7.6 mM) (36), where there is a mixture of γa and γb. The measured
affinity is within the physiological range of intracellular concentration of Na+ (14.9 +/-
0.6 mM) determined in proximal tubules (9). Thus the data highlight the physiological
significance of FXYD2a as a modulator of apparent affinity for Na+ of Na,K-ATPase.
Another example of reciprocal changes in FXYD1/FXYD2 expression under
stress conditions is shown in Fig. 7. NRK-52E cells were treated with 1mM ouabain for
24 or 96 hours. Although this concentration of ouabain should inhibit the rat Na,K-
ATPase about 85-90%, cells were not dying and only slowed down in the rate of
proliferation (not shown). This is very different from HeLa, where ouabain induces
apoptosis (53). Based on Western blot analysis, there was induction of FXYD2a in a
time-dependent manner. Staining of an identical blot with the C2 antibody revealed a
Page 16 of 60
significant reduction in the level of non-phosphorylated FXYD1, while no signal was
detected with the CP68 antibody (not shown). Since no precautions were taken to keep
phosphorylation at Ser68 intact, the data suggest a reduction in the total level of FXYD1
during ouabain treatment while gaining FXYD2a. Reduction in FXYD1/α ratio was
already significant after 24 hours treatment (66.8 +/- 7.0 %, n = 4) and was even more
pronounced with longer exposure (96 hours) (81.7 +/- 4.3 %, n = 3). At this point it is
not clear why ouabain-treated cells induce expression of FXYD2a (which acts as an
inhibitor of Na,K-ATPase) when the pump is already inhibited by the ouabain. One of the
possibilities is that ouabain activates cell signaling pathways convergent with those
employed for up-regulation of FXYD2a by stress.
All together the data suggest that interchangeability of FXYDs, regulatory
subunits of Na,K-ATPase, represents a part of a general mechanism to provide a proper
cellular response to physiological or pathological stimuli.
Dual response of FXYD1 to hypertonicity and trophic factor-dependent pathways
Hypertonicity activates multiple signaling pathways entailing protein kinases and
protein phosphatases. To monitor whether phosphorylation of FXYD1 is changed in
response to this kind of stress, NRK-52E cells were subjected to hypertonicity (500
mOsm with NaCl for 48 hours) in the presence of 10% or 0.5% FBS, and cell lysates
were tested on Western blots (Fig. 7). The reduced serum condition is a method often
used experimentally to amplify the effects of hypertonicity on signaling pathways.
Protein phosphatase inhibitor cocktail was added to the lysis buffer in order to preserve
phosphorylation of FXYD1. In both serum concentrations there was induction of
FXYD2a with no substantial changes in the level of α1 of Na,K-ATPase (Fig. 8A).
Page 17 of 60
However the response of FXYD1 was different depending on serum concentration. In
normal osmolarity, there was a higher proportion of phosphorylation of FXYD1 in the
presence of 10% serum than 0.5% serum. A hypertonic challenge of 24-48 hours in the
presence of 10% fetal bovine serum caused a reduction in the level of the non-
phosphorylated form of FXYD1: the FXYD1/α ratio was decreased in the stressed cells
by 31.4+/5.6% (n = 3). Simultaneously there was an even more significant drop (>60%)
in the level of the phosphorylated form of FXYD1. The data imply a decrease of the total
level of the FXYD1 protein as well as lability of phosphorylation under stress conditions
Interestingly, reduction in the phosphorylation level seen in 10% serum was
observed even after short-term (up to 5 hours) exposure to hypertonicity (Fig 8B). To test
whether dephosphorylation of FXYD1 is functionally significant, NRK-52E cells were
treated with hypertonicity (500 mOsm with NaCl) for 5 hours, and crude membranes
were isolated and analyzed in the presence of protein phosphatase inhibitor. There was a
reduction of Na,K-ATPase activity by 23.3 +/- 7.1 % (n = 3) in membranes from stressed
cells compared to control. Less than 5% of the activity was lost in cells exposed to
similar conditions for just 1 hour. We previously showed that induction of FXYD2a by
48 hour exposure to hypertonicity correlated with a significant reduction of Na,K-ATPase
activity. Since no FXYD2a was synthesized yet after 5 hours in hypertonic medium (67),
we suggest that dephosphorylation of FXYD1 led to inhibition of the Na-pump activity to
survive an apoptotic insult under stress-related conditions.
The incubation of NRK-52E cells in reduced serum isotonic medium resulted in a
significant loss of phosphorylation of FXYD1 at Ser68 (>50% as compared to
Page 18 of 60
phosphorylation of FXYD1 in 10% serum-supplemented isotonic conditions), which was
not significantly affected further by application of hypertonicity (Fig. 8A). However, the
reduction in the level of non-phosphorylated FXYD1 under hypertonic conditions in the
presence of 0.5% FBS was more dramatic than in serum-supplemented medium:
FXYD1/α ratio was reduced by more than 85% (observed in three independent
Serum growth factors apparently enhance pathways that result in higher basal
phosphorylation of FXYD1. Dephosphorylation of FXYD1 is the major event
accompanying hypertonic stress in NRK-52E cells grown in 10% serum, whereas
significant down regulation of non-phosphorylated FXYD1 occurred under hypertonic
stress in the reduced serum conditions. Interestingly, cells grown in low serum
proliferated significantly slower than control cells grown in the presence of 10% FBS and
at a rate similar to those grown in serum-supplemented conditions but treated with
hypertonicity (Fig. 8C). Thus there was an apparent correlation between the rate of
proliferation and the phosphorylation of FXYD1. Since phosphorylation of FXYD1 has
been implicated in activation (or release of inhibition) of Na,K-ATPase, this is further
supporting evidence that Na-pump activity is critical in supporting cell growth.
Stress-induced changes in FXYD1/FXYD2 entail regulation of gene expression
Reduction in the level of FXYD1 protein may indicate that a transcriptional or
translational block occurred in response to hypertonicity, or that mRNA levels are normal
and degradation was increased. To detect any change in mRNA level, we performed RT-
PCR analysis for FXYD1 and FXYD2a from NRK-52E cells grown under normal
conditions or in hypertonic medium (acute treatment for 48 hours at 500 mOsm with
Page 19 of 60
NaCl or chronic treatment with 700 mOsm). To our surprise, we detected the PCR
product corresponding to the FXYD2a mRNA under all experimental conditions,
including the controls, although the intensity of the band was higher with acute
hypertonicity and most pronounced under chronic hypertonicity (Fig. 9). In contrast, the
intensity of the FXYD1 PCR product bands declined with acute hypertonic treatment and
was completely lost in the adapted cells. The data suggest that 1) there is a transcriptional
and a translational block in expression of FXYD2a in NRK-52E under normal tissue
culture conditions, in line with related observations of Capasso et al. (17) on inner
medullar collecting duct cells; and 2) that FXYD1 mRNA levels are regulated by
hypertonicity. Whether the latter is due to the rate of transcription of the FXYD1 gene or
to changes in stability of the FXYD1 mRNA, awaits further investigation.
Expression of FXYDs in different cell lines
The finding of FXYD1 as an authentic component of Na,K-ATPase in renal cells
was quite unexpected. Therefore we performed a systematic RT-PCR analysis of FXYD
expression in several cell lines. Positive controls for the specific primers were included
for each species. Table 2 summarizes the data from NRK-52E, mIMCD-3, and the Caco-
2 intestinal epithelial cells lines grown under normal conditions or exposed to
hypertonicity (500 mOsm with NaCl for 48 hours). Interestingly, all of these cell lines
express FXYD3 and FXYD5 mRNAs under basal conditions, the level of which was
unaffected by hypertonicity in NRK-52E and mIMCD3 cells but reduced significantly in
Caco-2 cells. In contrast, all cells demonstrated an increase in the expression of FXYD2a
mRNA in response to high salt treatment. Only mIMCD3 cells showed an increase in the
Page 20 of 60
expression level of FXYD2b mRNA. NRK-52E cells were the only ones that expressed
FXYD1 mRNA, and it showed down regulation under hypertonic conditions.
Similar PCR analysis was performed on human HEK-293, HeLa, and retinoic
acid-differentiated SH-SY5Y cells grown under isotonic conditions (300 mOsm). As can
be seen from Table 2, FXYD5 was detected in all three cell lines, whereas FXYD3 was
identified in HeLa and FXYD6 was found in HEK-293. Surprisingly human
neuroblastoma cells SH-SY5Y displayed almost a complete set of FXYDs except
FXYD2a and FXYD1. Expression of FXYD6 mRNA was the most pronounced, although
“kidney specific” FXYD2b and FXYD4 were also found in these “neuronal”cells. The
data further support the hypothesis of plasticity and complexity of FXYD expression in
different settings such as tissues or cultured cells.
Since HEK293 and HeLa cells have been used in the past as expression systems
for different FXYDs, we have tested whether FXYD5 was expressed as a protein in these
cells. Fig. 10 demonstrates the results of Western blot with both crude membranes and
partially purified preparations of Na,K-ATPase from HEK293 and HeLa cells. The major
bands recognized by anti-dysadherin antibody were about 17-20 kDa (marked with a
single arrow), which very likely represented the core protein (with a calculated molecular
mass about 17 kDa), and a fuzzy band within a range of 28-35 kDa that is presumably
glycosylated (marked with a double arrow). We detected more highly glycosylated
FXYD5 (50 kDa) in membranes from LLC-PK1 cells (not shown). FXYD5 is a heavily
O-glycosylated protein (65), and multiple patterns of glycosylation of FXYD5 are
expected that may vary in different cell types. At least some of the FXYD5 remained
bound to Na,K-ATPase during the SDS-extraction purification procedure, consistent with
Page 21 of 60
the idea that that FXYD5 represents an endogenous regulatory subunit of Na,K-ATPase
expressed in these cells.
It is been several years since the discovery of the FXYD family members (61) as
tissue-specific regulators of Na,K-ATPase [reviewed in (21)]. FXYD2, the gamma
subunit, has been studied the most extensively, and the fact that this protein is complexed
with the pump in several nephron segments in the kidney but not in renal cells in culture
(5,62) supported the hypothesis that FXYD2 (and FXYDs in general) are not obligatory
but rather auxiliary subunits required for fine tuning the pump properties.
The novelty of this work is a demonstration of FXYDs as essential subunits of
Na,K-ATPase complex, and a demonstration of their interchangeability. There are at
least two lines of evidence in support. First, every cell line we have checked apparently
contains one or more FXYD(s) mRNA. Remarkably, there is no direct correlation
between FXYDs expressed in a tissue of origin and a cell line. For instance, expression of
phospholemman (FXYD1) in kidney is limited to afferent arterioles and lacis cells in the
juxtaglomerular apparatus (68). Nevertheless it is highly expressed in cultured renal cells
of proximal origin: NRK-52E, LLC-PK1, and OK cells (Fig. 1), but not in MDCK and
mIMCD3 cells derived from more distal tubules. On the other hand, kidney is almost the
only tissue expressing FXYD2 under normal conditions (45), and yet no protein can be
detected in renal cells in culture (5,62,64). Expression of FXYD5 in kidney is restricted
to distal nephron: connecting tubules, collecting tubules, and intercalated cells of the
collecting duct, although labeling of the apical membrane in long thin limb of Henle's
loop was also observed (43). Here we found a message for FXYD5 in renal cell lines of
Page 22 of 60
different origin: NRK-52E (proximal tubules), IMCD3 (collecting duct), and HEK-293
cells. Moreover we found FXYD5 protein in HEK293 and HeLa cells. This may be
related to cell transformation, though, since FXYD5 was identified in different kinds of
tumors [reviewed in (33)]. A similar argument may explain expression of FXYD3 mRNA
in a majority of cell lines tested in this work (NRK-52E, mIMCD3, Caco-2, HeLa cells).
FXYD3 was originally identified in breast and prostate tumors, and expression of
FXYD3 may be induced in culture by ras- and neu- oncogenes. Whether expression of
FXYD3 and FXYD5 proteins parallel the expression of FXYD3 and FXYD5 mRNAs in
cultured cells remains to be investigated.
Second, reciprocal exchange of FXYD1/FXYD2 proteins has been observed with
stimuli such as hypertonicity or ouabain treatment. Interchangeability is apparently
required for proper adaptation of cells to any given physiological or pathological
The major implication of this work is that Na,K-ATPase in cells in culture is
always paired with a FXYD protein(s), expression of which does not necessarily parallel
the expression pattern in the tissue(s) the cells originate from. One of the outcomes is a
call for rigorous systematic analysis of kinetic influences of the FXYD proteins on Na,K-
ATPase properties isolated from the dynamic background of host cell FXYDs. For
instance, the functional significance of FXYD2 for the Na,K-ATPase complex was
earlier assessed in different heterologous expression systems. There was a general
agreement that a major functional consequence of association with FXYD2 is a
modulation (reduction) of apparent affinity for Na+. Indeed, introduction of FXYD2 into
Na,K-ATPase complex resulted in a ~1.5 fold decrease in the affinity for Na+, from 5
Page 23 of 60
mM to 7.5 mM [reviewed in (4)]. However, K0.5 Na+ was still lower than in purified
enzyme from kidney (8.4-9.5 mM), and therefore skepticism was raised concerning the
physiological role of FXYD2. Here we showed that apparent affinity for Na+ can be
indeed modulated by association with FXYD2a and reduced to values (12.5 mM) even
exceeding those found in purified kidney enzyme, but only in homogenous preparations
of Na,K-ATPase containing FXYD2a and no FXYD1 as regulatory subunit. Such a
change would be large in a physiological context, bringing the affinity close to the
intracellular concentration of 14.9 mM.
Besides modulation of the affinity for Na+, other functional effects of FXYD2
were observed in heterologous expression systems including modulation of apparent
affinity for K+ (2,12) , affinity for ATP (52,63), and changes in K+ antagonism of
apparent Na+ affinity (2,63). Results in different studies were sometimes at odds.
Although such discrepancies may yet depend on which splice form of FXYD2 was
expressed and whether post-translational modification occurred, it is also likely that there
was an interplay between the FXYD2 introduced by transfection and the different
endogenous FXYDs (Tables 2 and 3) in the Na,K-ATPase complex.
Xenopus oocytes have been used as an expression system for different FXYDs
[reviewed in (31)] and FXYD2 in particular (12). We have searched the EST database
from Xenopus with tBLASTn analysis using the sequence of mouse FXYD3. Previously
unidentified orthologs were found in four different libraries constructed from unfertilized
egg of Xenopus tropicalis (7 entries in 3 libraries) and Xenopus laevis (4 entries from 1
library). Protein sequences are quite similar to each other (88% identity) and probably are
Page 24 of 60
the products of the orthologous gene. They also possess a similar degree of homology to
both FXYD3 (53 and 54% for X. tropicalis and X. laevis, respectively) and FXYD4 (46
% for both) and thus could represent the orthologs of either FXYD3 or FXYD4 expressed
in Xenopus egg, or could be independent FXYD proteins. If expressed as proteins, these
endogenous FXYDs may have been in competition with the products of introduced
FXYD cRNAs in oocyte expression studies. FXYD3 and FXYD4 from mouse show
55% identity with each other. Fig. 11 demonstrates the sequence alignment (A) and a
cladogram tree (B), showing phylogeny without indication of the amount of evolutionary
“time”. Interestingly, the FXYD orthologs found in Xenopus egg libraries are only
expressed in reproductive tissues (egg, testis) or tissues from early developmental stages
of Xenopus embryo (gastrula, early neurula, anterior neuroectoderm, etc.) but nowhere
We also showed that cells of different origin may express more than one mRNA
at a time, suggesting that more than one regulatory subunit of Na,K-ATPase may be
expressed. Since only one molecule of any kind of FXYD can apparently be bound to
alpha-beta monomer (32), this could lead to the co-existence of different multisubunit
complexes of Na,K-ATPase. This kind of phenomenon was already observed in the
middle segment of inner medullary collecting duct in rats (50): all principal cells
exhibited basolateral staining for FXYD2a, FXYD2b, and FXYD4. Another known
example is co-expression of FXYD2a and FXYD2b in medullary thick ascending limb,
demonstrated by immunocytochemistry and co-immunoprecipitation of mixed oligomeric
Page 25 of 60
Phosphorylation of FXYD1 as a mode of regulation of Na,K-ATPase
Different functional effects of FXYD1 on Na,K-ATPase have been reported.
Expression in oocytes led to a reduction of the apparent affinity for Na+, i.e. inhibition of
Na,K-ATPase activity at physiological Na+ concentrations (20). Overexpression of
FXYD1 in normal adult rat myocytes resulted in a significant reduction of Vmax without
appreciable changes in K0.5 for Na+ and K+(72). On the other hand, a stimulatory role
for FXYD1 was proposed based on reduction of total Na,K-ATPase activity in FXYD1-/-
mouse relative to littermate controls (35). Recent findings suggest that not only
expression but phosphorylation of FXYD1 is essential for modulation of Na,K-ATPase
(28,58). Substantial activation of Na,K-ATPase has been observed in isolated
sarcolemma under ischemic conditions that correlated with activation of PKA and
phosphorylation of FXYD1 but not of the α subunit (28). Treatment of isolated guinea
pig cardiomyocytes with forskolin led to activation of Na,K-ATPase correlated with
phosphorylation of FXYD1 at Ser68 (58). Increase in hydrolytic activity of Na,K-ATPase
and phosphorylation of FXYD10 (PLMS) was reported for shark rectal gland membranes
phosphorylated by PKC (44). The opposite functional effects of FXYD1 on modulation
of apparent affinity for Na+ when expressed with α/β complex in P. pastoris (decrease in
K0.5 Na+) and HeLa cells (increase in K0.5 Na+) were attributed to the presence or absence
of endogenous phosphorylation at the Ser68 site, respectively (41).
Here using phospho-specific antibodies we showed a dramatic reduction in
phosphorylation of FXYD1 per unit of protein in response to acute hypertonicity in
serum-supplemented conditions. Dephosphorylation occurred within the first hours of
treatment and correlated with a reduction of Na,K-ATPase activity, thus serving as an
Page 26 of 60
immediate response to reduce the activity of the pump while a “true repressor”, the
FXYD2a subunit, is synthesized (67). On the other hand, we observed a reduction in the
amount of FXYD1 along with an increase in phosphorylation of FXYD1 per unit of
protein under hypertonic stress in reduced serum. In this case, as we reported previously
(67) and in agreement with reports from other laboratories (14,25,48,70), the activity of
Na,K-ATPase is augmented. While the reduced levels of FXYD1 would imply less pump
activity, the enhanced phosphorylation of FXYD1 would counteract the expected
decrease in pump activity by stimulating the remaining pumps. The data suggest a
consistent underlying molecular mechanism, but experimental conditions (serum levels)
influence the cellular response. Interestingly, a significant drop in the expression level of
FXYD1 was observed in failing rabbit and human heart samples, whereas the fraction of
FXYD1 phosphorylated at Ser68 was increased dramatically (13).
Based on immunofluorescence, the intracellular distribution of FXYD1 depends
on the phosphorylation status of the protein. While phosphorylated FXYD1 is co-
localized with the α subunit of Na,K-ATPase at the plasma membrane, the non-
phosphorylated form of FXYD1 is largely retained in intracellular compartments. An
intracellular location of FXYD1 may indicate a role distinct from regulation of Na,K-
ATPase, a hypothesis that needs further investigation. It should be mentioned that at
least FXYD1 and FXYD4 have been already implicated in modulation of two other ion
transporting systems, Na+/Ca2+ exchanger in cardiac myocytes (1,71), and depolarization-
activated K+-channels, KCNQ1 (Kv7.1) (34), respectively. On the other hand,
phosphorylation of FXYD1 was required for plasma membrane delivery in MDCK cells
transfected with FXYD1 constructs (39). This was consistent with an earlier report by
Page 27 of 60
Mounsey et al. (47) that co-expression of PKA and FXYD1 in Xenopus oocytes
increased the amplitude of an ion current and the amount of FXYD1 at the plasma
In summary, we demonstrated for the first time that the cellular response to stress
entails an exchange of FXYD proteins, apparently to make a smooth transition of
properties of Na,K-ATPase, and this phenomenon represents a part of the adaptive
mechanism. Whether an exchange in regulatory subunits is essential only for modulation
of enzymatic activity or is also crucial for signal transduction pathways that involve the
Na,K-ATPase (8,56,69) awaits further investigation.
The authors wish to thank Ms. J. Pascoa and Ms. N. Asinovski for superb
The work was supported by research grants HL036271 and NS45083 (KJS), DK
443351 (CSIBD for EA), G12RR03051 and G11HD046326 (SC).
Page 28 of 60
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Fig. 1. FXYD1 is endogenously expressed and associated with Na,K-ATPase in NRK-
52E cells. A. Crude membranes (P2) and purified preparation of Na,K-ATPase (SDS)
were tested on Western blots with antibodies against α1, β1 and FXYD1 (C2-PLM). SL,
positive control (dog sarcolemma). B. C1-PLM antibodies (PLM) precipitated FXYD1
and the α subunit of Na,K-ATPase from crude membranes of NRK-52E cells. The blots
were stained with C2-PLM and K3 (α1) antibodies. Notably K3 antiserum does not
recognize β1 from dog membranes. St, starting material; IP, immunoprecipitated
material; Ctr, non-immune IgG experimental control. The figure is representative of three
Fig. 2. Subcellular location of FXYD1 is affected by phosphorylation at Ser68. NRK-
52E cells were fixed as described in Materials and Methods, and stained with McK1 (A
and D), CP68 (B), and PLM-1 (E) antibodies. Sections C and E represent the merged
images AB and DF, respectively. Bar, 20 µm.
Fig. 3. FXYD1 is expressed in renal cell lines of proximal, but not distal origin. Equal
amounts of crude membranes from MDCK, LLC-PK1, and OK cells were tested on
Western blots with anti-PLM antibody (PLM-C2). DSL, dog cardiac sarcolemma.
Fig. 4. FXYD1 is partially replaced in FXYD2 transfectants. Crude membranes from
wild type, mock-, γa- (FXYD2a), and γb- (FXYD2b) transfected NRK-52E were tested
on Western blots with antibodies against FXYD1 (C2-PLM), FXYD2 (RCT-G1) (γ), and
the α subunit (K3). Noticeably, the ratio between FXYD1 and α staining was higher in
mock-transfectants, lower in γa-transfectants, and the lowest in γb-transfectants. Several
γa- and γb-clones were tested, and a representative blot is shown.
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Fig. 5. FXYD1 is down-regulated with hypertonicity. A. Western blot analysis of crude
membranes from NRK-52E grown in the presence of 10% FBS either in control (300
mOsm) or in hypertonic medium (500 mOsm, supplemented with NaCl) for 48 hours.
Detection was with K3 (α1), RCT-G1 (γ) and C2-PLM (FXYD1) antibodies. B. C6
glioma cells were grown in the presence of 10% FBS in control (300) or hypertonic (500)
medium (with NaCl) for 48 hours. Crude membranes were tested on Western blots with
the antibodies against α1 (K1), γ (RCT-G1), and FXYD1 (C2-PLM). Hypertonicity
caused an induction of γ and down regulation of PLM.
Fig. 6. Complete replacement of FXYD1 with FXYD2a in adapted NRK-52E cells.
A. NRK-52E cells were adapted to high hypertonicity (700 mOsm with NaCl), and cell
proliferation assay was performed with WST reagent. Initial plating for control NRK-52E
cells (300 mOsm) and adapted cells (700 mOsm) was at 5x103 cells/well. B. Crude
membranes from the adapted cells were compared with those from control (300 mOsm).
(+) indicates membranes from either rat kidney or dog sarcolemma used as positive
controls to detect α1/FXYD2 or FXYD1, respectively. Induction of γ (FXYD2a)
correlated with the complete disappearance of PLM (FXYD1). C. Na,K-ATPase activity
was measured as a function of Na+ in purified preparations from control (300 mOsm) and
adapted cells (700 mOsm). The data are the average of at least four independent
experiments and are expressed as percentage of maximal activity recovered in each
Fig. 7. The loss of FXYD1 during induction of FXYD2a by ouabain treatment. NRK-
52E cells were exposed to 1 mM ouabain in a medium supplemented with 10% FBS for
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24 or 96 hours. Crude membranes were tested on Western blots with antibodies against
α1 (K1), α1 dephosphorylated at Ser18 (α1*) (McK1), γ (RCT-G1), and FXYD1 (C2-
PLM). Induction of FXYD2a (γ) correlated with down regulation of FXYD1 (PLM).
Fig. 8. Dual response of FXYD1 to hypertonicity and trophic factor-activated programs.
NRK-52E cells were grown in the presence of either 10% or 0.5 % FBS. Hypertonicity
treatment (with NaCl, 500 mOsm) was for 48 hours, and afterwards the cells were lysed
with the NP-40 lysis buffer containing protein phosphatase inhibitor cocktail. The lysates
were tested on Western blots with antibodies against α1 (K3), γ (RCT-G1), non-
phosphorylated form of FXYD1 (C2-PLM) and phosphorylated FXYD1 (CP-68).
Induction of γ correlated with down regulation of FXYD1 and a reduction of
phosphorylation at the Ser68 site. Down regulation of FXYD1 was more prominent under
reduced serum conditions. B. NRK-52E cells were treated with hypertonicity (500
mOsm) for 1 or 5 hours. Cell lysates were tested on Western blots with antibodies against
α1 and the phosphorylated form of FXYD1. Dephosphorylation of FXYD1 at the Ser 68
site occurred as early as 5 hours after initiation of treatment. C. NRK-52E cells were
grown either in control medium supplemented with 10% FBS (solid line, filled circles) or
0.5% FBS (solid line, filled triangles), or in hypertonic medium with 10% FBS (dashed
line, open circles). Cell proliferation assay was performed with WST reagent. Initial
plating was at 5x103 cells/well.
Fig. 9. Exchange of regulatory subunits entails changes in gene expression. 1µg of total
RNA from control NRK-52E cells (1, 4), cells acutely treated with hypertonicity (500
mOsm, 48 hours) (2, 5), or cells adapted to hypertonicity (700 mOsm) (3, 6) were taken
for cDNA synthesis followed by PCR analysis with primers specific for rat FXYD2a (γa)
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(1-3) or FXYD1 (PLM) (4-6). Arrows indicate the positions of specific products of 226
and 96 bps for FXYD2a and FXYD1, respectively.
Fig. 10. FXYD5 protein is expressed in HEK293 and HeLa cells. Crude membranes and
partially purified preparations of Na,K-ATPase from HEK293 and HeLa cells were tested
on 4-12% Novex NuPAGE MES gels. Blots were stained with anti-dysadherin antibody.
Single arrows indicate presumed positions for the core protein (17-20 kDa), whereas
double arrows indicate positions for partially glycosylated forms (28-35 kDa). A broad
range of molecular masses was defined with molecular weight markers: SeeBlue Plus2
(Invitrogen) for HeLa and Low Range pre-stained (Bio-Rad) for HEK293.
Fig. 11. Xenopus oocytes may have an endogenous FXYD.
A. Sequence alignment of several FXYD family members from mouse (FXYD1-
FXYD4), Xenopus laevis (FXYD1X and FXYD2X) and two new FXYDs found in ESTs
from Xenopus tropicalis (FXYDXt) and Xenopus laevis (FXYDXl) egg libraries. The
alignment was done with Clustal W. The stars indicate the positions of the conserved
residues. B. Cladogram tree of the FXYDs listed in A as defined by Clustal W algorithm.
Table 1. Nucleotide sequences of PCR primers specific for FXYDs from rat, mouse and
Table 2. Expression of FXYD mRNA is dynamic in response to hypertonicity. 1 µg of
total RNA from NRK-52E, mIMCD3 and Caco-2 cells grown under control conditions
(300 mOms) or treated with hypertonicity (500 mOsm with NaCl, 48 hours) were taken
for cDNA synthesis followed by PCR analysis with the FXYD-specific primers (Table
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Table 3. Expression of FXYD mRNA in human cell lines. HEK-293 (human
embryonic kidney cells) , HeLa (human cervical carcinoma) and retinoic acid-
differentiated SH- SY5Y (human neuroblastoma) were grown in the presence of 10%
fetal bovine serum. cDNA was obtained either with SuperScript II First DNA synthesis
kit or SuperScript III Cell Direct . PCR reactions were with Platinum Taq polymerase and
specific primers (Table 1).
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