Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
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ABSTRACT |
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Specific
functions served by the various Na+-K+-ATPase
-isoforms are likely to originate in regions of structural
divergence within their primary structures. The isoforms are nearly
identical, with the exception of the NH2 terminus
and a 10-residue region near the center of each molecule
(isoform-specific region; ISR). Although the NH2 terminus
has been clearly identified as a source of isoform functional
diversity, other regions seem to be involved. We investigated whether
the central ISR could also contribute to isoform variability. We
constructed chimeric molecules in which the central ISRs of rat
1- and
2-isoforms were exchanged. After stable transfection into opossum kidney cells, the chimeras were characterized for two properties known to differ dramatically among the
isoforms: their K+ deocclusion pattern and their response
to PKC activation. Comparisons with rat full-length
1-
and
2-isoforms expressed under the same conditions
suggest an involvement of the central ISR in the response to PKC but
not in K+ deocclusion.
-subunit; rat;
1- and
2-isoforms; chimeras; potassium deocclusion
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INTRODUCTION |
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NA+-k+-atpase
is critical for maintaining the ionic gradients across the plasma
membranes of animal cells. This enzyme complex extrudes Na+
from the cell and accumulates K+, using metabolic energy
derived from ATP hydrolysis. The enzyme complex consists of two
dissimilar subunits, and
, that exist in multiple forms. A
fundamental question surrounding the pump is the physiological
relevance of this subunit diversity. The
-polypeptide is the
catalytic subunit and contains the binding sites for ions and
substrates. Four distinct isoforms (
1,
2,
3, and
4) of the
Na+-K+-ATPase
-subunit have been described
so far, with differences in enzyme kinetics and response to second
messengers. The primary structures of these isoforms are nearly
identical, with the exception of the NH2 terminus and a
10-residue region near the center of the molecule (19, 24)
[isoform-specific region (ISR);
K489-L499; Fig.
1]. It seems likely that the specific
functions served by the various Na+-K+-ATPase
-isoforms must originate within these regions of structural divergence.
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The NH2-terminal region has been extensively studied.
Heterologous expression of NH2-terminal deletions and
chimeric constructs has shown that the mutant -subunits display
changes in kinetics and regulation of ion transport properties.
Although the NH2 terminus of
is not required for
Na+-K+-ATPase activity (21), a
truncated enzyme lacking the first 32 amino acids has distinctive
kinetics at micromolar ATP concentrations, conditions in which
K+ deocclusion becomes rate limiting in the overall
catalytic cycle. Thus Na+-ATPase activity of the truncated
enzyme is stimulated by low concentrations of K+, whereas
activity of wild-type
1 is inhibited. Interestingly, the
2-isoform resembles the truncated enzyme in this
respect. However, unexpected characteristics of chimeras resulting from exchanges between
1 and
2 NH2-terminal
domains suggested that this distinctive kinetic behavior of
Na+-K+-ATPase
-isoforms is not entirely due
to the NH2-terminal region but rather to its interaction
with other ISRs of the
-protein (8).
Another isoform-specific property that clearly involves the
NH2-terminal region is the regulation of
Na+-K+-ATPase transport activity by PKC.
Indeed, we have shown that stimulation of endogenous PKC with phorbol
esters increases pump-mediated Rb+ transport in cultured
opossum kidney (OK) cells expressing the exogenous rat
Na+-K+-ATPase 1-isoform. This
increase was abolished in cells expressing a mutant missing the first
26 amino acids of the rodent
-subunit, consistent with a role for
the NH2-terminal region in PKC regulation. Ser16 and Ser23, which are believed to be in
vivo targets of PKC phosphorylation, are found within the
NH2-terminal domain of the rat
1-isoform. We
have shown that PKC stimulation of transport is completely abolished in
S16A and S23A mutants (9). However, comparisons with other
species and experimental systems raise some doubts about the role of
these residues. For example, Ser16 is well conserved among
mammalian
1-isoforms, but it is only weakly
phosphorylated. Ser23, on the other hand, is missing in
many species. Neither residue is contained within a PKC consensus
sequence, and neither residue is conserved in
2 or
3. Nevertheless, there are other serines and threonines
in the NH2 termini of these isoforms that may serve as
phosphorylation sites. Given the variability of the response and the
differences among species and isoforms, it is therefore tempting to
speculate that additional regions of the
-subunit are contributing
to the effect of PKC.
We hypothesized that the central ISR may be one of these additional
regions. To test this hypothesis, chimeric molecules were constructed
in which the central ISRs of the rat 1- and
2-isoforms were exchanged. After stable transfection
into OK cells, the chimeras were characterized for their enzymatic
properties. Comparisons of the chimeras with rat full-length
1- and
2-isoforms expressed under the
same conditions suggest an involvement of the central ISR in the PKC
response but not in K+ deocclusion.
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METHODS |
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Preparation of full-length 1 and ouabain-resistant
2 sequences.
Wild-type
1 and
2 cDNAs, a gift from Dr.
Jerry B. Lingrel and colleagues (22), were subcloned by
our laboratory into pGEM-3Z (Promega, Madison, WI) as described
(21). A ouabain-resistant form of the rat
2-isoform (designated
1 and
Preparation of chimeras.
The 1 and
1 and
1 and
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Expression vectors, gene transfer, and selection.
cDNAs encoding 1,
construct downstream of the enhancer-promoter
regions of the immediate early gene of human cytomegalovirus, followed
by polyadenylation signals from the bovine growth hormone gene.
Membrane preparations. Crude plasma membranes were isolated from control and transfected OK cells. Confluent monolayers from twelve 100-mm dishes were washed twice with phosphate-buffered saline, and cells were then harvested by scraping with a rubber policeman. Crude membranes were isolated from the resulting cell suspension after hypotonic lysis and differential centrifugation, followed by treatment with sodium iodide as described (21).
Gel electrophoresis and immunoblotting.
Expression of introduced -subunits in transfected OK cells was
confirmed by electrophoresis and subsequent immunoblotting of proteins
from the isolated membranes. Electrophoresis of samples through
SDS-polyacrylamide gels (7.5%), electroblotting, and probing of the
blots with appropriate antibodies were performed as described (19). The presence of transferred proteins on the blots
and equality of loading among the lanes were confirmed by staining with
Ponceau S. For detection and characterization of rat isoform and
chimera expression in OK cells, two site-directed rabbit polyclonal antibodies were used. Anti-HERED recognizes the rat
2
ISR (residues 494-506). Anti-NASE recognizes the rat
1 ISR (residues 494-505). However, anti-NASE also
recognizes the opossum
1 sequence (19); therefore, it could not be used for confirmation in constructs in which
its target sequence was expected.
RNA isolation, reverse transcription, and DNA amplification.
In instances where the cross-reactivity of anti-NASE precluded specific
detection of the 1 IRS, we confirmed the structure of
the expressed mRNA. Isolation of total RNA from confluent monolayers of
cells in 3.5-cm culture dishes was accomplish using a commercial preparation, Ultraspec (Biotecx Laboratories, Houston, TX), a modification of the guanidium thiocyanate-phenol-chloroform protocol of
Chomczynski and Sacchi (6). The recovered RNA was
dissolved in diethylpyrocarbonate-treated water, and only preparations
with an A260/A280 amplification ratio >1.5
were analyzed further.
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Enzymatic activity. The activity of Na+-K+-ATPase in isolated membranes from transfected cells was determined from the hydrolysis of radiolabeled ATP as described (17). Briefly, membranes (1 mg/ml) were treated with 0.4-0.7 mg/ml deoxycholate for 30 min at room temperature to activate latent Na+-K+-ATPase activity. Detergent-treated membranes were then diluted 1:20 and incubated at 37°C for 30 min in a total volume of 0.2 ml containing 20 mM NaCl, 25 mM histidine, 3 mM MgCl2, 0.2 mM EGTA, and 1 µM ATP (pH 7.5 at room temperature). To minimize the contribution of endogenous Na+-K+-ATPase, 3 µM ouabain was also included. The Na+-ATPase activity of the introduced enzyme was estimated by the decrement in hydrolysis in the presence of 3 mM ouabain. Various concentrations of KCl, ranging from 0.05 to 5 mM, were also added, and the resulting activity was standardized to the Na+-ATPase estimate measured in the absence of K+.
Active transport. Na+-/K+ pump-mediated transport was assessed by measuring the ouabain-sensitive uptake of the K+ congener 86Rb+ as described (20). Briefly, the difference in accumulation of the radioisotope over 5 min at 37°C was determined in cells exposed to standard culture medium plus 3 µM or 1 mM ouabain. As with the enzymatic analyses, a dose of 3 µM was used to inhibit the ouabain-sensitive endogenous opossum enzyme, whereas the 1 mM ouabain dose was necessary to inhibit the ouabain-resistant enzyme introduced. The effect of PKC activation was determined by treating confluent monolayers for 5 min with the DMSO vehicle or phorbol ester agonist PMA before the addition of radiolabeled Rb+. This was achieved by adding 5 µl of a 10 mM solution of PMA (diluted in DMSO) or 5 µl of DMSO alone (control condition) directly to the 5 ml of culture medium.
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RESULTS |
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Expression of full-length 1- and
1,
1
2
1, and
2
1
2 produced
ouabain-resistant colonies, indicating that all four exogenous
sequences were capable of producing functional
Na+-K+-ATPase. To confirm the structure of the
introduced subunit, membranes from transfected cells were evaluated by
immunoblotting (Fig. 2). As a probe, we
used anti-HERED, a polyclonal antibody directed against the
2 ISR (19). A band corresponding to 116 kDa
was detected in
1
2
1, as
well as rat brain membranes or membranes containing the full-length
1-isoform),
1-transfected OK membranes, or nontransfected OK
membranes that were included as negative controls. No signal was
detected in membranes from
2
1
2-transfected OK cells
(Fig. 2B), consistent with an absence of the
2 ISR in the chimera. Taken together, these data suggest
that the structure of the
1
2
1 chimera was as
intended, with substitution of the
2 ISR into the
1-isoform. Conversely, the
2 ISR was not
detected in membranes expressing
2
1
2. The same immunoblots
were probed with anti-NASE, a polyclonal antibody directed against the
1 ISR. Unfortunately, given the cross-reactivity of this
antibody with opossum-derived
1, a band was detected in
nontransfected OK cells, precluding any conclusion about exogenous
expression in transfected cells (data not shown). Indeed, a previous
study has shown this same cross-reactivity with opossum-derived samples
(19).
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Effect of ISR exchange on enzymatic properties.
In micromolar concentrations of ATP sufficient to saturate the
high-affinity phosphorylation site, the response of
Na+-dependant ATP hydrolysis to varying concentrations of
K+ is a convenient and sensitive indication of
isoform-specific differences in the E2(K)E1 pathway of the
Na+-K+-ATPase reaction (8). This
part of the reaction becomes rate limiting at low ATP concentration,
and K+ inhibits Na+-ATPase activity of the
1 enzyme. In contrast,
2 is
stimulated. Previous work using chimeric enzymes obtained by exchanges
between
1 and
2 NH2 termini
has suggested that the distinctive kinetic behavior of
1
and
2 was not due to the NH2-terminal domain
alone but rather to its interaction with other ISRs of the protein
(8). Accordingly, a series of experiments was
designed to determine whether ISR sequence diversity was involved in
the kinetic difference between
1 and
2.
In Fig. 4, results are presented relative
to ouabain-sensitive Na+-ATPase activity, which was
34.8 ± 6.1, 25.2 ± 2.9, 24.0 ± 7.4, and 29.4 ± 1.4 nmol Pi · mg
protein
1 · h
1 for
1,
1
2
1,
*2, and
2
1
2, respectively.
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Effect of ISR exchange on PKC-dependent activation of cellular
Na+-K+-ATPase-mediated
Rb+ transport.
In basal conditions, Na+-K+-ATPase-mediated
Rb+ transport was in the same range for nontransfected and
transfected cells, regardless of the construct introduced. Indeed,
before PMA-stimulation,
Na+-K+- ATPase-mediated Rb+
transport was 8.14 ± 0.6, (n = 7) in
nontransfected OK cells, 6.44 ± 0.38 (n = 7) for
1, 6.99 ± 0.76 (n = 7) for
1
2
1, 6.41 ± 1.56 (n = 11) for
2, and 7.99 ± 0.52 nmol · mg
protein
1 · h
1 (n = 6) for
2
1
2.This strongly
suggests that Na+-K+-ATPase was not
overexpressed in the transfected cells and that the overall activity
was not compromised by the mutations.
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DISCUSSION |
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The primary structures of -isoforms of the
Na+-K+-ATPase are nearly identical, except for
the NH2 terminus and an approximately 10-residue region
near the center of the molecule, the central ISR (Ref. 19,
Fig. 1). We and others have established the influence of
the NH2-terminal region on isoform-specific enzyme kinetics and regulation (8, 9, 17, 26). However, this work has also
raised suspicions that another site on the catalytic subunit may
contribute to these differences. We thought it likely that this
putative site is also a region of structural divergence between the
isoforms, and we considered the ISR a prime candidate. To address this
question, we produced a pair of chimeric molecules in which the ISRs of
1 and
2 were switched. These chimeras
were then expressed in opossum renal cells in culture after
DNA-mediated gene transfer.
A difficulty with heterologous expression of the
Na+-K+-ATPase in mammalian cells is the nearly
ubiquitous distribution of this enzyme complex. To distinguish an
endogenous from introduced enzyme in the opossum cells, we took
advantage of the varied sensitivity of the
Na+-K+-ATPase to digitalis glycosides
(10, 25). Selection of transfected cells was achieved by
growth in a concentration of ouabain sufficient to kill nontransfected
cells but not cells that express the more resistant, introduced form.
Clearly, this strategy is dependent on the ability of the introduced
subunit to sustain active transport despite the presence of digitalis
glycoside. Mutations severe enough to inhibit catalytic turnover or to
interfere with targeting to the plasmalemma would not produce
ouabain-resistant cells and could not be studied with this procedure.
For this reason, the successful production of ouabain-resistant
colonies after transfection with
1
2
1 or
2
1
2 suggests that overall
enzymatic function was not compromised, despite the switch in ISRs.
Of course, there was always the possibility that the observed ouabain
resistance was not a consequence of the transfected -subunit.
Resistance to sublethal concentrations of ouabain is occasionally
achieved, for instance, by significant overexpression of pumps rather
than the introduction of a resistant enzyme (15). We
ensured against this eventuality by selecting cells in ouabain at
concentrations well above the K1/2 for inhibition
of the endogenous enzyme. Moreover, we confirmed the expression and
structure of the introduced forms by direct detection of the exogenous
polypeptides and mRNAs with specific probes (Figs. 2 and 3).
Having confirmed the successful expression of the introduced isoforms
and chimeras, we next evaluated the effect of ISR exchange between
1 and
2 on their K+
activation/inhibition profile, a kinetic parameter known to differ dramatically between these two isoforms. Indeed, the distinctive behavior of the isoforms is apparent when the reaction is carried out
under micromolar ATP concentrations because the K+
deocclusion pathway of the reaction cycle becomes rate limiting (18). Under such conditions, the activity of exogenous
1 expressed in HeLa and COS-1 cells is inhibited by
K+, whereas
2 is stimulated (8,
17). The present observations have confirmed these results in OK
cells. Daly et al. (8) have argued that it is not solely
the difference in amino acid sequences at the NH2 termini
of
1 and
2 that is responsible for this
kinetic difference. Rather, it is likely to be an interaction of the
segment between 24 and 32 of
1 with some other region of
the
1 protein that determines the
K+-sensitive pattern displayed by this isoform. We
hypothesized that the ISR could be this region. However, this seems
unlikely because exchange of the
1 and
2
sequences did not result in a modification of the K+
activation/inhibition profile compared with the appropriate unaltered isoform (Fig. 4). Taken together, the results of Daly and co-workers and the present data suggest that it is the interaction of the NH2-terminal segment with a region of
1
other than the ISR that is a determinant of the observed K+
inhibition profile.
Another possible role for the ISR region could be in the
isoform-specific response to second messengers. Numerous laboratories have cataloged differences in isoform regulation after stimulation of
both protein kinase A and C (reviewed in Refs. 2,
3, and 14). For the present set of experiments, we
chose to focus on PKC activation of the
Na+-K+-ATPase induced by the phobol ester
agonist PMA. We observed a 15% stimulation of pump-mediated
Rb+ transport for 1 and
1
with the
2 sequence, this activation was increased
twofold (Fig. 5). Conversely, substituting the
2 ISR with
1 in the
2-isoform resulted in a
nonresponding phenotype. These data clearly argue in favor of a role
for the ISR in PKC regulation. Moreover, the paradoxical response to
the agonist seems to imply an inhibitory effect of the
1
ISR. When it was removed from
1, the PMA response was
increased (
1
2
1), but when
it was substituted into
2
(
2
1
2), the previously
observed response was blocked.
It is tempting to speculate about what mechanism may underlie
this inhibitory effect of the 1 ISR. Efendiev et al.
(9) have shown that PMA-induced activation of
Na+-K+-ATPase in OK cells is the result of pump
translocation from intracellular pools via clathrin-coated vesicles,
resulting in an increased abundance in the plasma membrane
(9). This process requires the phosphorylation of
Ser11 and Ser18 (not present in
2-isoform) of the
1-isoform by the PKC
-isoform and involves the adaptor activator protein (AP)-1
(9). Adaptors mediate the incorporation of cargo onto
transport vesicles by interacting with sorting signals present in the
cytosolic domain of transmembrane proteins. Four adaptors (AP-1, AP-2,
AP-3, and AP-4) have been described so far. AP-1 and AP-3 mediate
sorting events at the level of the trans-Golgi network
and/or endosomes, whereas AP-2 functions in endocytic clathrin-coated
vesicle formation (reviewed in Ref. 4). Recent evidence
has shown that AP-4 participates in basolateral sorting in epithelial
cells (23). Using the same transfection strategy into OK
cells as was used in the present study, investigators have shown that,
unlike PMA, dopamine inhibits Na+-K+-ATPase
activity. Interestingly, this inhibition also involves membrane
trafficking, in this case by internalization via clathrin-coated vesicles. This internalization requires activation of the atypical PKC
and the adaptor AP-2, as well as the binding of
phosphoinositide-3 kinase to a proline-rich motif of the
-subunit
(5, 26). Little is known about AP-3 and AP-4, but AP-1 and
AP-2 are known to recognize their target by consensus signals in the
cytoplasmic domain of proteins. These consensus sequences are either
di-leucine motifs or tyrosine-based signals, specifically Y-X-X-Ø,
where Ø is a bulky hydrophobic amino acid. Recent work by Cotta
Doné et al. (7) has identified Tyr537 as
an essential element for AP-2 binding and the clathrin-dependent endocytosis of Na+-K+-ATPase that mediates
dopamine-induced inhibition.
In addition to this tyrosine-based signal, the
1-isoform also displays a di-leucine motif, which
appears to be in the ISR but is not present in
2. The
di-leucine motif might represent the molecular basis of the inhibitory
effect that we have attributed to the
1 ISR. It could
act as a dynamic retention signal that favors
1
internalization, even during PMA stimulation. In apparent contradiction, it has been clearly shown that mutating the second leucine of this motif (i.e., Leu500) is not sufficient to
alter PMA-induced activation (7). However, this finding
may not be inconsistent with an involvement of the first leucine of the
motif (i.e., Leu499), which is the last amino acid of the
1 ISR and is absent from the
2 sequence.
Moreover, dynamic retention signaling can involve an entire region,
including several, sometimes redundant, consensus motifs. For instance,
insulin-regulated aminopeptidase dynamic retention within the endosomal
compartment requires 2 of 3 distinct motifs present in a 30-amino-acid
region of its cytoplasmic tail (13). It may be that
Leu499 is the more important residue of the signaling
motif, but other residues might contribute to a more extensive domain
that includes the
1 ISR sequence but is missing in the
2 sequence.
Along the same lines, it should be kept in mind that although the ISR represents one the most striking sequence variabilities among the isoforms, other regions of the intracellular domain also display various degrees of diversity, especially in the so-called large cytoplasmic loop (between TM4 and TM5; see Fig. 1 in Ref. 2). As mentioned previously, the NH2-terminal region is also clearly an ISR. By swapping ISRs, we might have disrupted an important interaction with another part of the molecule, thereby interfering with a dynamic retention signal composed of several motifs that may be far apart in the primary structure. In short, it is not clear at this point if ISR swapping is disrupting a signal contained in the sequence itself or rather an important interaction with one or several other motifs within the intracellular domains of the protein.
With previous studies, the present data, and the above-stated
hypothesis taken into account, the stimulation of 1 that
we are observing under PMA treatment likely results from the
contribution of at least two facilitating components and one inhibitory
component. Accordingly, their presence in or absence from the different
isoforms would determine the amplitude of the individual response to
the phorbol ester. First, an isoform-specific effect is very likely to
be involved in the PMA-induced increase observed with
1
It may involve Ser11 and Ser18 phosphorylation
(which are not present in
2) and AP-1. The effect is an
increase in the number of Na+-K+-ATPase
complexes expressed at the membrane (9). It is also possible that tyrosine-based motifs such as Tyr469,
expressed by
1 but not
2, could mediate
the interaction. Second, a mechanism of internalization mediated by the
di-leucine motif and the surrounding area in the ISR (present only in
1) may contribute to the response. This effect would be
a dynamic retention, taking place even in basal conditions. Third, an
activator mechanism seems to be shared by
1 and
2. Accordingly, removing the inhibitory ISR of the
1 sequence and replacing it by the "neutral"
2 ISR results in an increased response to PMA as shown
for
1
2
1. On the same
basis, the PMA-induced stimulation of
2 could be
compromised by the addition of the inhibitory sequence of the
1 ISR, as shown by the absence of PMA-induced
stimulation in
2
1
2.
Clearly, the chimeras used in the present study are just the first step
in a systematic evaluation of isoform-specific structure and its
influence on function. Future experiments will be needed to determine
the individual amino acid residues contributing to the PKC response, as
well as the role played by the ISRs of other -isoforms.
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ACKNOWLEDGEMENTS |
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This work has been supported by American Heart Association, Texas Affiliate, Grant 98G-385 and National Center for Research Resources Grant RR-19799.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. V. Pierre, Dept. of Physiology, Texas Tech Univ. Health Sciences Ctr., 3601 4th St., Lubbock, TX 79430 (E-mail: Sandrine.Pierre{at}ttuhsc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 4, 2002;10.1152/ajprenal.00153.2002
Received 19 April 2002; accepted in final form 28 May 2002.
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