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3 Subunit
3 IN LUNG AND LIVER ADDRESSES THE PROBLEM OF
THE MISSING SUBUNIT*
(Received for publication, June 12, 1997)

From the Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129
The Na,K-ATPase belongs to a family of P-type
ion-translocating ATPases sharing homologous catalytic subunits (
)
that traverse the membrane several times and contain the binding sites
for ATP and cations. In this family, only Na,K- and H,K-ATPases have
been shown to have a second subunit, a single-span glycoprotein called
. Recently a new isoform (
3) has been identified in mammals. Here
we describe structural features and tissue distribution of the
3
protein, utilizing an antiserum specific for its N terminus.
3 was
the only
detected in Na,K-ATPase purified from C6 glioma. Treatment
with N-glycosidase F confirmed that
3 is a glycoprotein containing N-linked carbohydrate chains. Molecular masses
of the glycosylated protein and core protein were estimated to be 42 and 35 kDa, respectively, which are different from those of the
1
and
2 subunits. Detection of
subunits has historically been difficult in certain tissues. Sensitivity was improved by
deglycosylating, and expression was evaluated by obtaining estimates of
3/
ratio. The proportion of
3 protein in the rat was highest
in lung and testis. It was also present in liver and skeletal muscle,
whereas kidney, heart, and brain contained it only as a minor component of the Na,K-ATPase. In P7 rat, we found skeletal muscle and lung Na,K-ATPase to be the most enriched in
3 subunit, whereas expression in liver was very low, illustrating developmentally regulated changes
in expression. The substantial expression in lung and adult liver very
likely explains long-standing puzzles about an apparent paucity of
subunit in membranes or in discrete cellular or subcellular
structures.
The Na,K-ATPase1
catalyzes the active uptake of K+ and efflux of
Na+ ions, thus controlling ionic gradients through the
enzymatic hydrolysis of ATP. It is a heterodimer of two different kinds of subunit, a large
subunit with multiple membrane spans and a
smaller glycoprotein subunit,
, with just one span and the bulk of
its mass in the extracellular space. Both subunits of Na,K-ATPase are
encoded by multigene families. In the rat, three isoforms of
subunit and two isoforms of
subunit have been identified and
substantially characterized (1, 2). Although the major ATPase
characteristics are assigned to the
subunit, the
subunit is
required for folding and transport of
,
-heterodimers to the
plasma membrane (2). Nonetheless, there have been a number of reports
in which little or no
1 or
2 subunit or mRNA was detected
despite significant levels of
or its mRNA (3-6), or
alternatively, in which
and
mRNA or protein did not appear to colocalize in tissue sections (7-9).
Recently several cDNA fragments having homology with Na,K-ATPase
subunits were identified in the GenBankTM expressed sequence tag
library. The full-length human clone revealed 59% sequence identity
with the
3 subunit of Xenopus laevis and 38 and 48% identity with human
1 and
2 subunits, respectively, and was named
3 (10). Full-length clones were independently isolated from cDNA
libraries from C6 rat glioma cells (GenBankTM D84450) and mouse retina
(11). Northern blot analysis and representation in the expressed
sequence tag data bank has revealed
3 message in a wide variety of
tissues. Expression of the
3 subunit at the protein level and its
association with the Na,K-ATPase has not been demonstrated yet however.
Here we accomplish these aims with an antibody, specific for the rat
3 subunit, that will be a useful addition to an existing panel of
isoform-specific antibodies to the Na,K-ATPase.
Antipeptide-directed antiserum RNT
3 (rat N
terminus of
3) was raised at Quality Control Biochemicals, Inc.
(Hopkinton, MA), against the 13-mer peptide
TKTEKKSFHQSLAC-(NH2), corresponding to the N-terminal
sequence of the rat
3 isoform (GenBankTM D84450) plus a terminal
cysteine residue. The peptide was conjugated to keyhole limpet
hemocyanin as a carrier, and two rabbits were immunized. The immune
response was tested with an enzyme-linked immunosorbent assay with
bovine serum albumin-coupled peptide on solid phase. The titer of crude
sera against the peptide was >200,000 for the better rabbit. Other
antibodies were generous gifts of other investigators: antiserum
against the KETYY peptide representing the C terminus of all
subunits of Na,K-ATPase from R. Bayer and J. Kyte (University of
California, San Diego); antipeptide antiserum 757 against sheep
1
from W. J. Ball, Jr. (University of Cincinnati); anti-fusion protein antiserum FP
1 against rat
1 from A. McDonough (University of Southern California); antiserum SpET
2 against human
2 from P. Martin-Vasallo (University de la Laguna, Tenerife, Spain); and
monoclonal antibody SM-GP50 for
2 from J. Gurd (University of
Toronto) and P. Beesley (Royal Holloway and Bedford New College, Egham,
United Kingdom).
The C6 glioma cell line was obtained from the American Type Culture Collection. Cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum and penicillin/streptomycin.
Membrane Isolation and Gel ElectrophoresisMembrane preparations from rat tissues were made as follows. Brain, lung, liver, kidney, and testis were minced and homogenized with a motor-driven Teflon homogenizer in 10 volumes of buffer containing 250 mM sucrose, 30 mM imidazole, pH 7.2, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. Unhomogenized material and nuclei were removed by low-speed centrifugation at 7,000 rpm for 15 min, and membranes were sedimented at 33,000 rpm for 30 min. For cardiac and skeletal muscle preparations, the tissues were minced and homogenized in 40 volumes of buffer containing 250 mM sucrose, 20 mM Tris-HCl, pH 7.2, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, followed by further disruption with an Omni homogenizer for 30 s at the maximal setting. The homogenates were centrifuged at 14,000 rpm for 30 min. Sarcolemma-enriched fractions were obtained by centrifugation of the supernatant at 33,000 rpm for 30 min. Protein concentration was determined according to Lowry. Partial purification of the Na,K-ATPase from rat brain microsomes and C6 crude membranes by extraction with SDS was performed as described elsewhere (12).
Gel electrophoresis and Western blots were performed as described earlier (13), except that detection was by chemiluminescence using luminol-based reagents (Pierce). Blots were scanned with a Molecular Dynamics laser densitometer.
Enzyme DigestionDeglycosylation was performed as follows.
Membranes were resuspended at 1-2 mg/ml in 0.25 M sodium
phosphate buffer, pH 7.4, denatured with 0.1% SDS and 1%
-mercaptoethanol for 30 min at 37 °C, followed by addition of
1-2 units of N-glycosidase F (Boehringer Mannheim) and up
to 0.5% Triton X-100. The reaction was performed overnight at room
temperature or at 37 °C.
3 Protein Detection and Gel Mobility
To detect
3
protein, we generated an antiserum based on the N-terminal sequence
deduced from the rat cDNA sequence. To test its reactivity with
3, rat C6 glioma cells were used, since the clone had been isolated
from a C6 cDNA library. The antiserum stained C6 cell plasma
membranes by immunofluorescence (data not shown). To determine whether
3 was associated with the Na,K-ATPase, the enzyme was partially
purified from C6 crude membranes using SDS treatment followed by
equilibrium centrifugation on a sucrose density gradient. During such
treatment most other membrane proteins are solubilized, while
Na,K-ATPase remains associated with plasma membrane. As illustrated in
Fig. 1A, the RNT
3 antibody
stained a fuzzy band with a molecular mass of about 42-45 kDa in
preparations that had a Na,K-ATPase specific activity of 100-150
µmol of Pi/mg/h (equivalent to 30-50-fold purification).
Since both
1 and
2 subunits have been shown to be glycoproteins
containing several N-linked oligosaccharide chains (14, 15),
N-glycosidase F treatment was performed to determine whether
3 was also glycosylated. As seen in Fig. 1A, this
resulted in a decrease in apparent molecular mass to 35 kDa. Fig.
1B demonstrates that
3 is the only known
isoform of
the Na,K-ATPase purified from C6 glioma cells, contrasting with the
1 and
2 isoforms found in Na,K-ATPase purified from rat brain.
Different dilutions of sample were added to facilitate comparisons. The
content of
(
1 only for C6; all three
subunits for rat brain)
was determined by staining the blots with the KETYY antibody against
the C-terminal peptide of all
subunits. Equal amounts of the sodium
pump
subunits were loaded for rat brain and C6 samples, but the C6
membranes had no
1 or
2 and were more than four times more
enriched in
3 than rat brain enzyme.
3 isoform of Na,K-ATPase is expressed in
C6 rat glioma cells. A, 7.5 µg of purified Na,K-ATPase
from C6 rat glioma cells were pretreated (+) or not (
) with
N-glycosidase F. The Western blot was stained with RNT
3
antibody. Positions for mature and core
3 protein are marked
3m,
3c, respectively. Molecular weight markers (Bio-Rad) are indicated. B, four
identical blots containing 10 µg (lanes 1 and
4), 5 µg (lanes 2 and 5), and 2.5 µg (lanes 3 and 6) of purified Na,K-ATPase from
C6 rat glioma cells (lanes 1-3) or rat brain (lanes
4-6) were stained with anti-KETYY ("
"), 757 antiserum (
1), monoclonal antibody SM-GP50
(
2), and RNT
3 (
3).
Removal of oligosaccharide chains often facilitates glycoprotein
detection on blots. As seen in Fig. 2,
fully glycosylated
3 could be detected in rat brain microsomes only
when 25-50 µg of protein were loaded. In deglycosylated samples, the
binding was almost linear within a 5-50 µg range, however.
Consequently, in further experiments we routinely used glycosidase
treatment.
3 subunit facilitates
its detection on blots. Rat brain microsomes, 50 µg (lanes
1, 2, 9, and 10), 25 µg (lanes 3 and
4), 10 µg (lanes 5 and 6), or 5 µg
(lanes 7 and 8) were deglycosylated (lanes
2, 4, 6, 8, and 10) or not (lanes 1, 3, 5, 7, and 9), and blots were stained with anti-KETYY
(lanes 1-8), RNT
3 (lanes 1-8), or with
secondary antibody alone (lanes 9 and 10).
3m and
3c indicate the
positions of mature and core
3 protein, respectively.
3 stain
from control and N-glycosidase F-treated samples was
quantified by scanning densitometry. The stain increased linearly in
both cases, though with much greater sensitivity after
deglycosylation.
Molecular masses of the mature and core
3 protein calculated from
their mobilities on SDS gels were different from those of
1 and
2
subunits. As seen in Fig. 3A,
the mature
3 protein migrated faster than the mature
2 subunit.
This was not surprising since rat
3 has only two potential
N-linked glycosylation sites in the extracellular domain,
whereas rat
2 has seven. As shown in Fig. 3B (in an
experiment where no compensation was made for the smaller amount of
3 than
1 or
2), the core
3 protein migrated significantly
slower than the core
2 protein and slightly slower than the core
1 subunit. Average values for glycosylated proteins were 46-50 kDa
for
1 and
2 and 42-45 kDa for
3. Average values for core
proteins were 34-37 kDa for
1, 32-34 kDa for
2, and 35-38 kDa
for
3. The apparent size of core
3 is larger than predicted from
the cDNA clone (30 kDa); the biochemical basis for the discrepancy
remains to be determined.
subunit have
different mobilities on SDS gels. A, 50 µg (
3) or 5 µg (
2) of rat brain microsomes, deglycosylated (+) or not (
),
were stained with RNT
3 (
3) and SM-GP50 (
2) antibodies. The
positions of mature and core
subunits are indicated. B,
10 µg of deglycosylated (+) and control (
) rat brain microsomes
were stained with polyclonal antiserum FP
1 (1), peptide-directed
antibodies 757 (2) and RNT
3 (3), monoclonal antibody SM-GP50 (4),
and polyclonal antiserum SpET
2 (5). The core proteins had different
electrophoretic mobilities.
Fig. 3B also illustrates an incidental peculiarity of
glycoprotein staining.
1 and
2 were each stained with two
different antibodies, and certain antibodies were substantially less
sensitive to the glycosylated protein than to the core. This is likely
to reflect differences in the proximity of their epitopes to
glycosylation sites.
The species specificity
of the RNT
3 antibody was examined with blots of 50 µg of
deglycosylated brain microsomes from several species.
3 protein was
detected readily in rat and mouse brain samples and more weakly in
guinea pig samples (data not shown). All other samples, including
human, chicken, rabbit, dog, cat, sheep, calf, monkey
(Macaca), frog (Rana), and goldfish brain membranes, were negative. There is only 69% identity within the first
13 amino acids among mammalian
3 subunits, and even less for other
vertebrates.2
|
3 is
weakly permissive, but substitution of asparagine in human
3
prevents RNT
3 binding. RNT
3 has also been observed to be negative
on human
3 expressed in insect
cells.3
Expression of the
3 Isoform in Rat Tissues
A panel of
adult rat tissues was examined for the presence of the Na,K-ATPase
3
subunit on blots. In Fig. 4A,
concentrations of samples were adjusted to ensure reasonable levels of
the
subunit, as detected with anti-KETYY.
3 staining was strong
in testis, lung, and liver when as much as 150 µg of microsomes were analyzed. A strong signal was also obtained with kidney outer medulla
microsomes, whereas much less was detected in rat brain microsomes.
Only a little
3 was seen in heart and skeletal muscle samples. Blots
were scanned, and Fig. 4B shows the relative abundance of
the
3 protein within the Na,K-ATPase complex estimated by taking the
ratios between
3 and
subunit staining. We found lung and testis
to have the highest proportion of
3 isoform expression per
Na,K-ATPase unit among the adult rat tissues tested.
3 content was
lower in skeletal muscle, liver, and kidney outer medulla, and in heart
and brain Na,K-ATPase it comprised only a minor fraction. It should be
stressed that the data are expressed relative to the
3/
ratio in
lung. It is known that lung also expresses
1 protein (18), and the
proportion of the two isoforms has not yet been determined.
3 protein in rat tissues.
A, microsomes from adult rat tissues, including brain (20 µg, lane 1), heart (20 µg, lane 2), lung (150 µg, lane 3), liver (150 µg, lane 4), skeletal
muscle (150 µg, lane 5), kidney outer medulla (20 µg, lane 6), and testis (150 µg, lane 7), were
deglycosylated, and blots were stained with the peptide-directed
antibodies anti-KETYY ("
") and RNT
3
(
3). B, different exposures of blots were
scanned to ensure that the signal was in a linear range, and the ratio of
3 to
staining was calculated. The data are expressed as arbitrary units relative to the
3/
ratio found in rat lung. C, microsomes from 7-day-old rat tissues, including brain
(20 µg, lane 1), heart (20 µg, lane 2), lung
(150 µg, lane 3), liver (150 µg, lane 4),
skeletal muscle (150 µg, lane 5), and whole kidney (20 µg, lane 6), were treated and stained as in A. D,
3/
ratios for tissues from P7 animals.
The same tissues (except testis) were analyzed at day 7 after birth.
Fig. 4C illustrates that as in the adult, the expression of
3 protein in lung was high, but it was almost undetectable in the
liver. Surprisingly, the strongest signal for
3 was in sarcolemma-enriched membranes from hind limb skeletal muscle. Since
there was almost no difference in the amount of
subunit between
lung and skeletal muscle samples as judged by anti-KETYY staining, this
indicates that skeletal muscle has the highest proportion of
3 in
the P7 rat of the tissues studied (Fig. 4D). Comparison of
Fig. 4, B and D, illustrates that brain and heart from newborn rat, like skeletal muscle, were more enriched in the
3
protein per Na,K-ATPase unit than those tissues from adult rat. Thus
expression of the
3 subunit of the Na,K-ATPase is subject to
developmental regulation in several tissues.
In several experimental systems it has been possible to form
active combinations of Na,K-ATPase or H,K-ATPase
subunits with different
subunits, even ostensibly mismatched pairs
(i.e. NaK
/HK
). The outcome is that exchange of
subunits has effects on the enzymatic properties of the complex, most
notably on the affinities for K+ or Na+
(19-25). This implies that tissue- or developmentally controlled differences in
-
combination may have direct physiological
consequences mediated by alteration of substrate affinity.
2 has
also been implicated in intercellular adhesion in the developing
nervous system (26). Since the
isoforms differ in the number and
structure of their carbohydrate groups (14, 15), such differences
(reflected here in differences in gel mobility) may be important in
early development.
The observation that
3 is expressed at the highest level (relative
to total
subunit) in liver and lung may resolve some long-standing
controversies. In the liver,
1 subunit has been detected only after
density gradient purification (18); in prior studies it was not seen
(6, 27). Even mRNA for
1 has been frequently undetectable or low
(3, 4, 28, 29), although it was high in liver from young animals and
was specifically increased by thyroid hormone and by resection-induced
proliferation (4, 30, 31). Most of the negative results could in
principle be attributed to insufficient sensitivity, but an analysis of
the distribution of Na,K-ATPase in liver suggests a more important problem. In early work there were discrepancies between the subcellular distribution of histochemical reaction for Na,K-ATPase activity and
immunoreactivity (reviewed in Ref. 32). The controversy centered on
whether the pump was located on sinusoidal or canalicular subcellular
membranes, which is relevant to understanding the mechanism of salt and
water transport during the secretion of bile. Histochemistry indicated
a sinusoidal (basolateral) distribution, while initial antibody
staining suggested a canalicular (apical) distribution. The
interpretation was complicated by evidence that membrane lipid fluidity
affected Na,K-ATPase activity and that fluidization uncovered cryptic
activity in the canalicular domain (33). Later immunocytochemistry by
the same investigators documented that Na,K-ATPase
1 subunits were
found in both domains, but the
1 subunit was found predominantly in
the sinusoidal domain (7). Oddly, a less well characterized
anti-Na,K-ATPase antibody selectively stained the canalicular domain.
The polarized distribution of reactivity for
1 correlated with the
prior histochemistry, suggesting that the presence of
1 makes the
enzyme active. Most recently, this group reported that the increased
1 seen during hepatic regeneration was specifically localized to the
canalicular domain and lateral surfaces of the hepatocyte (8).
Fluidization of the membrane increased Na,K-ATPase activity in the
canalicular fraction in control rats (where
1 was absent), but not
in that from rats undergoing regeneration (where
1 was present).
Thus there appear to be functional differences in canalicular
Na,K-ATPase depending on whether or not
1 is expressed there (8). We
can now speculate that the missing
subunit in normal adult rats is
3 and that it is responsible for the difference. Little or no
mRNA for
3 has been detected on Northern blots of adult mouse or
rat liver RNA (10, 11), but this is likely to be due to the low level
of expression of any Na,K-ATPase subunit, requiring greater
sensitivity.
Another puzzle that may be explained by the presence of
3 in rat
liver is a report of tissue-specific kinetic differences in
Na,K-ATPases from lamb kidney and liver (18). Although both of these
enzyme preparations contained
1 and
1, the liver preparation had
significantly higher affinities for ATP, Na+, and
K+. The presence of
3, as well as
1, in the liver
enzyme may account for the difference, although
3 conferred lower
affinity for K+ than
1 with Bufo or
Xenopus subunits expressed in Xenopus oocytes (20, 21).
The literature on Na,K-ATPase expression in lung also has some relevant
peculiarities. Na,K-ATPase activity increases sharply during the
perinatal period because of its role in fluid reabsorption immediately
after birth. In whole lung,
1 and
1 mRNA levels peaked at
birth and then fell to a lower level maintained in the adult (9, 34,
35). Both type II pneumocytes in alveoli and bronchiolar epithelial
cells have high levels of Na,K-ATPase (36, 37). The most intriguing
results were obtained by examining mRNA levels with in
situ hybridization (9, 37). During the perinatal period,
1
mRNA increased dramatically in bronchiolar epithelium, but not so
much in alveoli, while
1 mRNA increased dramatically in both
loci. Beginning at day 8 postnatally,
1 signal developed a punctate
pattern in alveoli, while
1 signal remained broadly distributed. We
can speculate that the some of the
1 may be associated with
3,
since
1 appeared to be distributed in specific cells of the adult
alveolar epithelium. mRNA for
3 has been detected in adult rat
and mouse lung (10, 11).
In conclusion, the presence of
3 associated with Na,K-ATPase in
various tissues presents a more complete picture of this enzyme's
isoform composition and points to possible functional differences
important for the physiology of several tissues. It has not been ruled
out that
3 may also associate with H,K-ATPases, a possibility
supported by the interchangeability of
subunits in expression
systems. The effect of
subunits on
substrate affinities, and
now potentially on the presence or absence of measurable activity,
supports the idea that
has a regulatory role.
To whom correspondence should be addressed: 149-6118, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-8579; Fax: 617-726-7526.
2, this isoform of
from
the chicken is grouped with
3 sequences by dendrogram analysis of
evolutionary relationships (2).
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