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1 Prassis Research Institute
Sigma-Tau, Adducin point
mutations are associated with genetic hypertension in Milan
hypertensive strain (MHS) rats and in humans. In transfected cells,
adducin affects actin cytoskeleton organization and increases the
Na+-K+-pump
rate. The present study has investigated whether rat and human adducin
polymorphisms differently modulate rat renal
Na+-K+-ATPase
in vitro. We report the following.
1) Both rat and human adducins
stimulate
Na+-K+-ATPase
activity, with apparent affinity in tens of nanomolar concentrations.
2) MHS and Milan normotensive strain
(MNS) adducins raise the apparent ATP affinity for
Na+-K+-ATPase.
3) The mechanism of action of
adducin appears to involve a selective acceleration of the rate of the
conformational change E2 (K)
cytoskeleton; blood pressure; genetics
INTERACTIONS BETWEEN cytoskeletal and integral membrane
proteins are fundamental for the maintenance of polarity in epithelial or neuronal cells (25, 39) and the regulation of ion transport (4,
10). The actin-based cytoskeleton has been demonstrated to
interact with ion transport proteins such as the band 3-anion exchanger
(17), epithelial Na+ channels (4),
the
Na+-K+-Cl The best characterized functions of adducin are to promote
spectrin-actin association and to bind actin and bundle actin filaments (3). The Animals.
Inbred MHS/Gib and MNS/Gib rats derived from the original stock colony
(Prassis Research Institute, Settimo Milanese, Milan, Italy) were used
for erythrocyte adducin, ankyrin, and kidney Na+-K+-ATPase
purifications. Rats were fed on a standard diet (Altromin MT, Rieper,
Vandois, Italy) containing 2.5 g/kg NaCl. Indirect systolic blood
pressure was recorded by the tail-cuff method (BP Recorder, Ugo Basile,
Comerio, Italy) in 3-mo-old rats and averaged 167 ± 2 and 132 ± 1.8 mmHg in MHS and MNS, respectively.
Purification of erythrocyte adducin and ankyrin.
Rat and human erythrocyte adducins were purified from the whole blood
of MHS and MNS rats (400 ml) or human volunteers (200 ml). The
purification procedure was basically as reported by Hughes and Bennett
(26). Briefly, washed erythrocytes were lysed in hypotonic buffer, and
membranes were recovered by Pellicon cassette filtration (Millipore).
Membranes were extracted with low-ionic strength buffer, and the
extract was absorbed on SP-Sepharose (Pharmacia) and eluted with 500 mM
NaBr. Final purification was performed on a Resource-Q fast-performance
liquid chromatography column (Pharmacia). The fractions were analyzed
by SDS-PAGE, and those containing adducin were dialyzed twice versus
20% sucrose, 10 mM Tris · HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 40 mg/l 4-(2-aminoethyl)benzenesulfonyl fluoride
hydrochloride (Pefabloc, Sigma). The yield of purified adducin from rat
and human erythrocytes was ~0.5 mg of protein for each purification.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E1 (Na) or
E2(K) · ATP
E1Na · ATP.
4) Apparent affinities for mutant
rat and human adducins are significantly higher than those for wild
types. 5) Recombinant human 
- and
-adducins stimulate Na+-K+-ATPase
activity, as do the COOH-terminal tails, and the mutant proteins
display higher affinities than the wild types.
6) The cytoskeletal protein ankyrin,
which is known to bind to
Na+-K+-ATPase,
also stimulates enzyme activity, whereas BSA is without effect; the
effects of adducin and ankyrin when acting together are not additive.
7) Pig kidney medulla microsomes
appear to contain endogenous adducin; in contrast with purified pig
kidney
Na+-K+-ATPase,
which does not contain adducin, added adducin stimulates the
Na+-K+-ATPase
activity of microsomes only about one-half as much as that of purified
Na+-K+-ATPase.
Our findings strongly imply the existence of a direct and specific
interaction between adducin and
Na+-K+-ATPase
in vitro and also suggest the possibility of such an interaction in
intact renal membranes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cotransporter (49), and
Na+-K+-ATPase
(41, 42). In polarized renal tubular cells, the confinement of
Na+-K+-ATPase
to the basolateral surface involves the direct anchorage of the enzyme
to ankyrin (28, 43, 50) and, thence, to fodrin (41). The mechanisms of
actin polymerization (21) and the formation of the
spectrin/fodrin-actin network (2) are regulated by a variety of
proteins, including adducin (34). Adducin is a heterodimeric
cytoskeletal protein composed of related but nonidentical subunits
(, , or 
). Adducin is involved in signal transduction mechanisms through the modulation of the actin cytoskeleton at cell-cell contact sites (2, 34). Much experimental evidence indicates
that adducin may be a candidate protein to explain genetic alterations
in ion transport associated with primary or essential hypertension (8).
In rats of the Milan hypertensive strain (MHS), a primary increase of
renal tubular Na+ reabsorption (1)
is involved in the development of hypertension. In particular, the MHS
rat shows an increased activity and expression of
Na+-K+-pump
units per cell compared with their Milan normotensive strain (MNS)
controls (18). In MHS and MNS rats, two missense point mutations in the
- (F316Y) and -subunits (Q529R) of adducin have been demonstrated
to be genetically associated with hypertension (8). Moreover, rat renal
cells (NRK-52E) transfected with MHS adducin cDNA show a significant
increase of
Na+-K+-pump
activity and a higher immunohistochemical reactivity for the
Na+-K+
pump compared with cells transfected with MNS adducin (47). Finally,
adducin polymorphisms in the human -subunit (G460W, S586C) have been
found to be genetically associated (12, 13, 38) and linked (13) with
essential hypertension in humans and to affect the relationship between
renal Na+ excretion and blood
pressure (13, 27). Despite the different mutations in hypertensive rat
and human adducins, the evidence so far suggests that these mutations
produce similar alterations in renal
Na+ handling of hypertensive rats
and humans (1, 38). To our knowledge, this is the first demonstration
showing that a spontaneous polymorphism in the -adducin gene affects
blood pressure in two species (rat and human) that diverged ~40
million years ago.
-/-dimer of adducin is arranged so that pairs of head
domains form a globular core that caps the end of actin filaments, whereas tails of the - and -subunits participate in the lateral contacts between several actin filaments and the -subunit of spectrin, recruiting additional spectrin units to the actin filaments (26, 35). The increase in
Na+-K+-pump
activity at maximal stimulation
(Vmax) and the
promotion of actin bundling on transfection of cells with adducin may
therefore lead to a reasonable assumption that an increase in
Na+-K+-pump
density is the result of a change in cellular turnover due to
adducin's property of organizing the cytoskeleton. A direct cytoskeleton-Na+-K+-pump
interaction is known to be mediated by ankyrin, which binds both
Na+-K+-ATPase
and spectrin (3, 43). On the other hand, observations that adducin
accumulates at cell-cell contacts (32) have led to a proposal that
adducin may itself recognize a specific membrane receptor, in addition
to spectrin and actin. Therefore, we were interested in examining
whether a direct functional interaction exists between adducin and
Na+-K+-ATPase
in a cell-free system. The present study has been designed to evaluate
this possibility. After preliminary experiments that demonstrated in
vitro effects of adducin on renal
Na+-K+-ATPase,
we looked at differential effects of wild-type and mutant adducins from
Milan rats and humans. We have also made some initial observations to
examine the possibility that
adducin-Na+-K+-ATPase
interactions exist in native renal membranes.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Expression and purification of full-length and COOH-terminal tails
of - and -adducins.
A pGEMEX plasmid containing cDNA coding for human -adducin and the
COOH-terminal domain of - (residues 430-737 and 530-737) and -adducin (residues 530-726) were provided by Dr. V. Bennett (Dept. of Cell Biology and Biochemistry, Duke University, Durham, NC).
The original human -adducin construct encodes the amino acids
Gly-460 and Cys-586. Gly-460 was replaced by Trp via site-directed mutagenesis on the original recombinant vector with a U.S.E.
Mutagenesis Kit (Pharmacia) and the target mutagenic primer AHW
(5'-GCTTCCATTCTGCCATTCCTCGGAAGCTTC) to obtain the hypertensive
variant cDNA. Similarly, Cys-586 was replaced by Ser with the target
mutagenic primer AHS (5'-CCAGATTCTCTTCAGAGCCCTTCTCTTCC) to
obtain the normotensive variant cDNA. The pGEMEX recombinant expression
vectors were transformed into the Escherichia
coli BL21pLysS strain (26).
-D-galactopyranoside,
and bacteria (1 liter) were incubated for 4 h at 37°C after
induction and then harvested and lysed with DNase I in 1% Triton
X-100, 20 mM Na-phosphate, pH 7.4, 200 mM NaCl, 1 mM dithiothreitol,
and 100 mg/l Pefabloc (lysis buffer). The full-length protein was
purified from inclusion bodies, which were dissolved in
buffer A (10 mM Na-phosphate, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.05% Tween 20, and 40 mg
of Pefabloc) containing 7 M urea and 1 M NaBr and centrifuged for 1 h
at 30,000 g. The supernatant was
dialyzed versus buffer A containing 4 M urea and was loaded on a 6-ml Resource-Q column that was eluted with
a NaBr gradient (0-300 mM) in buffer A containing 4 M urea.
The expressed COOH-terminal domains were obtained after lysis of the
bacteria and centrifugation for 20 min at 2,700 g. The supernatant was heated for 20 min at 70°C and centrifuged for 1 h at 30,000 g. The supernatant was diluted with
buffer A and purified on SP-Sepharose
and a Resource-Q column as described above. The expressed recombinant
proteins accounted for roughly 25% of the total protein. The yield was
~15-20 milligrams of protein per liter of bacteria. Protein
contents of the expressed constructs were estimated from SDS-PAGE by
comparing the intensities of their Coomassie blue-stained bands with
known amounts of BSA. Reflectance densitometry was carried out with a
Bio-Rad model 620 densitometer.
A purified 31-amino acid synthetic peptide, corresponding to the
COOH-terminal residues 696-726 of human -adducin tail
(GSPSKSPSKKKKKFRTPSFLKKSKKKEKVES), was a kind gift of Dr. V. Bennett
and Y. Matsuoka (40). The purity of the synthetic peptide was >95%
as determined by C18 reversed-phase column HPLC. The concentration of
the synthetic peptide was determined by the amino acid composition
analysis that confirmed the sequence (40).
Purification and assay of
Na+-K+-ATPase.
Microsomal membranes and purified
Na+-K+-ATPase
were prepared from the renal outer medulla of pigs or 3-mo-old MHS rats
(29). Na+-K+-ATPase
activity was assayed by the release of
32Pi
from 32P-labeled ATP
([32P]ATP) as
described previously (18, 29). Whenever the reaction medium is
unspecified, it consisted of 100 mM NaCl, 3 mM
MgCl2, 5 mM KCl, 50 mM HEPES-Tris,
1 mM EGTA, 3 mM Tris-ATP, and 20 nCi of
[32P]ATP (0.5-3
Ci/mmol, Amersham), pH 7.4. The specific activities of the purified MHS
rat or pig
Na+-K+-ATPases
used in this study were 30-35 and 15-20 µmol
Pi · min1 · mg
protein1, respectively. The
activity was inhibited 99% by 5 mM ouabain. For the study of kinetic
effects of adducin, concentrations of Na+,
K+, and ATP were varied at
saturating concentrations of the other pump ligands. Where necessary,
the ionic strength of the medium was maintained constant by the
addition of choline chloride. For the study of effects of adducin, 10 µl of purified adducin (0.08-8 µg of protein), or its medium,
were incubated in duplicate for 30 min at 37°C with 10-25 ng
of purified
Na+-K+-ATPase
in 100 µl of standard assay medium. With the assumption of a relative
molecular mass
(Mr) for
adducin of 160 kDa (- plus -subunits), the final concentration
was in the range of 3-300 nM.
Gel electrophoresis, blotting to polyvinylidene fluoride, and
immunoblotting.
Procedures for running 10% tricine
(N-tris[hydroxymethyl]methylglycine)
SDS-PAGE, electroblotting to polyvinylidene difluoride paper, and
immunoblotting have been described in detail previously (11, 24).
Immunoblots were developed by enhanced chemiluminescence (ECL;
3-5 µg protein/lane) with the use of anti-rabbit IgG-horseradish peroxidase conjugate and the protocol supplied with ECL reagents from
the 1998 Amersham-Pharmacia Life Science Products catalog. For the
quantification of bands, the X-ray films were scanned with a Bio-Rad
GS-690 imaging densitometer and analyzed with Bio-Rad Multi-Analyst
software (version 1.01). For immunoblots, we used 1) an affinity-purified polyvalent
antibody referred to as "anti-KETYY," which recognizes the five
COOH-terminal residues of the -subunit of
Na+-K+-ATPase
(1:200 dilution), and 2) a
monoclonal antibody raised against the human adducin -subunit at
Prassis laboratories. In brief, mice were immunized with bacterial tail
constructs in Freund's adjuvant (4 boosts every 15 days, 100 µg/boost). The spleen was fused with mice myeloma cells, and
hybridomas were grown in a selective medium and screened for a positive
reaction with antigen on ELISA plates. Positive clones were subcloned twice.
Kinetic analysis. Kinetic parameters were calculated by nonlinear regression (Enzfitter, Elsevier-Biosoft, Cambridge, UK) and expressed as means ± SE. Statistical comparisons were performed by ANOVA. P < 0.05 was regarded as significant.
Materials. All chemicals were reagent grade from Sigma. [32P]ATP (0.5-3 Ci/mmol) was purchased from Amersham.
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RESULTS |
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TOP
ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Purity of rat and human erythrocyte adducins and
Na+-K+-ATPase.
Rat adducin preparations consisted of three bands (Fig.
1, lanes
2-5). The major 105-kDa band contained both -
and -subunits, which comigrate in this species, and accounted for
~60% of the total. The 70- and 60-kDa bands always copurified with
the 105-kDa band and are either proteolytic fragments produced within
the erythrocyte or alternatively spliced forms. In preparations of human erythrocyte adducin, - and -subunits ran separately, and the 70- and 60-kDa bands were also found, although in much smaller amounts than for rat adducin (Fig. 1, lane
6), as previously shown (22). Several preparations of
MHS, MNS, and human adducin were made. Only those batches of adducin
showing an electrophoretic pattern like that in Fig. 1 were used for
the experiments. The purified MHS rat
Na+-K+-ATPase
used for most experiments in this study showed essentially only the two
bands of the - and -subunits with apparent
Mr values of 95 and 55 kDa, respectively, and minor impurities (Fig. 1, lane 1). The specific activity of
this preparation was particularly high, 30-35 µmol
Pi · min1 · mg
protein1, with an estimated
sevenfold enrichment compared with the microsomal fraction with a
specific activity of 3.5-4.5 µmol
Pi · min1 · mg
protein1 (see Ref. 29).
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Effects of adducin on
Na+-K+-ATPase
activity.
Figure 2 shows that both MHS and MNS
adducin significantly stimulated MHS rat
Na+-K+-ATPase
activity in a medium containing 0.1 mM ATP, 100 mM NaCl, 5 mM KCl, and
3 mM MgCl2. The extent of
stimulation was 78.7 ± 3.1% (n = 11) for MHS adducin and 83.4 ± 1.7%
(n = 11) for MNS adducin (see also
Table 1). Stimulation by
adducin was fitted to hyperbolic curves with an apparent affinity
(K0.5) of 14.2 ± 1.7 nM (n = 11) for MHS and 60.9 ± 11.7 nM (n = 11) for MNS (see
Table 1). The stimulation of
Na+-K+-ATPase
activity by adducin was 100% inhibited by ouabain, and no hydrolysis
of [32P]ATP by
purified adducin was detected (data not shown). The effect of adducin
was lost if it was subjected to extensive proteolysis by proteinase K
(56°C for 24 h), followed by heating at 95°C for 1 h.
Stimulation of
Na+-K+-ATPase
by adducin was largely prevented by preincubation of the adducin (75 nM) with an anti-adducin antibody (1:100 dilution) (Table
2). This control excluded the possibility
that minor protein contaminants in the adducin preparation were
responsible for the stimulation of
Na+-K+-ATPase
activity.
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Tests of the mechanism of stimulation of
Na+-K+-ATPase
activity.
A large stimulation of
Na+-K+-ATPase
activity by adducin at 1 µM ATP implies that the adducin accelerates
the rate of the conformational change
E2(K) E1Na, which is the
rate-limiting step of the
Na+-K+-ATPase
cycle in this condition (44). This inference can be tested
independently by looking at the effect of
K+ on
Na+-ATPase activity at 1 µM ATP
(14, 48). Normally, the addition of
K+ to a reaction medium containing
Na+,
Mg2+, and 1 µM ATP inhibits the
rate of ATP hydrolysis because the rate-limiting step
E2 E1 for
Na+-K+-ATPase
activity is slower than that for
Na+-ATPase activity in the absence
of K+
(E2-P E2). Thus, in Fig.
4, we compared the effects of
K+ on ATP hydrolysis at 1 µM ATP
in the absence or presence of adducin. In the absence of adducin,
K+ inhibited the ATP hydrolysis as
expected. In contrast, in the presence of adducin, the addition of
K+ produced a small stimulation
first, followed by a reduction of the rate but, overall, no inhibition
of the ATPase activity. In the absence of
K+ adducin had little or no effect
on the rate of Na+-ATPase
activity.
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E1Na at pH 6, more so by
E1-P E2-P at neutral pH, and by the
rate of phosphorylation E1
E1-P at pH > 8 (20). Data in Table 4 show that the degree of
stimulation by adducin is greatest at pH 6, somewhat less at pH 7, and
altogether nonexistent at pH 9.
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E1Na at the low ATP
concentration (see DISCUSSION).
Effects of ankyrin and BSA on
Na+-K+-ATPase
activity.
An indication of the specificity of the
adducin-Na+-K+-ATPase
interaction was obtained by comparing the effects with those of another
cytoskeletal protein, ankyrin, which is known to bind to the
Na+-K+-ATPase
(28, 41-43, 50), and with BSA, which should not bind. Na+-K+-ATPase
activity was measured at 0.1 mM ATP, with
Na+,
K+ and
Mg2+ at saturating concentrations
and increasing concentrations of adducin, ankyrin, or BSA (Fig.
5). As expected, adducin induced a
substantial stimulation of activity
(K0.5 = 9.5 nM,
73%
Na+-K+-ATPase
stimulation). Purified ankyrin also increased the
Na+-K+-ATPase
activity, although to a lower degree and with lower affinity than
adducin (K0.5 = 110 nM, 47%
Na+-K+-ATPase
stimulation). BSA did not affect the
Na+-K+-ATPase
activity up to 1 µM. The experiment shown in Fig.
6 looked at the combined effects of adducin
and ankyrin on
Na+-K+-ATPase
using both adducin and ankyrin at saturating or near-saturating concentrations. Evidently the stimulation of
Na+-K+-ATPase
activity by adducin and ankyrin when acting together is much lower than
could be expected for the additive effects of the two proteins acting
independently (see DISCUSSION).
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Effects of human recombinant adducin on
Na+-K+-ATPase
activity.
Further evidence of the specificity of the
adducin-Na+-K+-ATPase
interaction was obtained by comparing the effects on
Na+-K+-ATPase
of full-length wild-type or mutant human adducins or truncated forms
purified from extracts of E. coli
(Fig. 7). Erythrocyte adducin is formed by
- and -subunits, each composed of three distinct domains (40): a
39-kDa NH2-terminal globular
protease-resistant head domain connected by a 9-kDa neck domain to a
COOH-terminal protease-sensitive tail domain containing the calmodulin
binding site and the regions involved in the adducin-spectrin-actin
binding (16, 26, 30, 31, 35, 40). The constructs used in this study
code for recombinant human full-length -adducin containing the
wild-type G460/S586 and mutant W460/C586 substitutions, wild-type and
mutant tail fragments of the -subunit (residues 430-737 and 530-737), or wild-type full-length -subunit and tail fragments (residues 530-726 and 696-726). The human - and
-adducins, added alone or as mixtures, and tail fragments stimulated
the
Na+-K+-ATPase
activity, assayed at 0.1 mM ATP and saturating concentrations of
Na+,
K+ and
Mg2+ in a dose-dependent fashion
(concentration range: 3-1,000 nM) (Fig. 7). The degree of
Na+-K+-ATPase
stimulation of wild-type and mutant full-length -subunit or -
plus -subunit mixtures was ~75%. The
K0.5 values for
the activation of
Na+-K+-ATPase
show that mutant W460/C586 full-length -subunit or the W460/C586
- plus -subunit mixture had a significantly higher affinity than
the wild-type full-length G460/S586 -subunit or wild-type G460/S586
- plus -subunit mixture. Both wild-type and mutant tail fragments
(residues 430-737) of the -subunit retained the ability to
stimulate the
Na+-K+-ATPase,
although to a lower extent (60%) than the full-length proteins (75%).
The apparent affinities of wild-type and mutant -subunit tail
fragments were much lower than those of the full-length -adducins or
- plus -subunit mixtures (25 ± 3.9 vs. 408 ± 67 nM and
9.8 ± 1.3 vs. 157 ± 7 nM for full-length -subunit compared with tail fragments, respectively). It is of interest that the K0.5 values of
the -subunit tail fragments (residues 430-737) containing both
point mutations were significantly reduced compared with those of
wild-type -subunit tail fragments (residues 430-737) (157 ± 7 vs. 408 ± 67 nM). The shorter mutant -subunit tail fragment (residues 530-737) still retained the ability to stimulate the Na+-K+-ATPase
activity by ~30% with a
K0.5 value
comparable to that of the -subunit tail fragment (residues
430-737).
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-subunit stimulated the enzyme by ~70%, with a
K0.5 of 30 nM, as
did the tail fragment (residues 530-726), although to a lower
extent (30%). Again, the affinity of the -subunit tail fragment was
much lower than that of the full-length -subunit (109.7 mM vs. 30 nM). The 31-mer synthetic peptide corresponding to the tail of the
-subunit, with a positively charged COOH-terminal region, was inactive.
For comparison with the recombinant proteins, ,-adducin purified
from human erythrocytes was also tested. Human -adducin genotypes
were established (13), and two subjects with the wild-type G460/S586
form and three subjects with the mutant W460/C586 form of adducin were
studied. As was found for the recombinant proteins, the human adducins
stimulated
Na+-K+-ATPase
by 75%, and the mutant erythrocyte adducin showed a higher affinity
than the wild-type protein (10.2 ± 1.8 vs. 25.7 ± 2.6 nM,
n = 3). These findings provide a
strong indication that the properties of the recombinant proteins are
similar to those of the native proteins and are not artifactual effects
of denatured protein.
An
adducin-Na+-K+-ATPase
interaction in intact renal membranes?
If adducin and
Na+-K+-ATPase
interact in kidney in vivo, one could expect that membranes isolated
from renal medulla would contain adducin. With the use of the
anti-adducin antibody to screen immunoblots, preliminary experiments
showed that there is very little intact adducin in rat kidney medulla
microsomes, although there appear to be adducin fragments. In contrast,
in pig kidney medulla microsomes, we detected a band that runs in the
same position as intact human adducin -subunit, as well as bands
that could be fragments of adducin. The immunoblot in Fig.
8 attempted to quantify the amount of this
band in pig kidney medulla microsomes and the pig kidney enzyme with
the use of known amounts of human adducin to calibrate the immunoblot.
In 30 µg of microsomal protein, the amount of the band corresponding
to intact adducin is comparable to ~1 µg of human adducin
-subunit, and there is also a substantial amount of smaller bands
that recognize the antibody. In contrast, in 40 µg of the pig kidney
Na+-K+-ATPase
preparation, anti-adducin detected no proteins. For comparison, the
immunoblot in which anti-KETYY was used shows that there were approximately equal amounts of -subunit of
Na+-K+-ATPase
in 1 µg of microsomes and 0.2 µg of purified enzyme, consistent with the fivefold lower specific activity of microsomes (3.5 µmol Pi · min1 · mg1)
compared with that of purified
Na+-K+-ATPase
(16.5 µmol
Pi · min1 · mg1).
The absolute amount of -subunit of
Na+-K+-ATPase
in microsomes is estimated readily from the known site concentration
(~0.2-0.3 nmol/mg protein) (18, 24) and
Mr value of 153 kDa ( + + -subunits) as 30-45 µg/mg microsomal
protein. This value is similar to that of the band tentatively assigned as intact adducin (~30 µg/mg microsomes). If indeed intact
endogenous adducin is present in the microsomes and a significant
fraction of the
Na+-K+-ATPase
is bound, one could predict that added adducin would stimulate the
Na+-K+-ATPase
less than the purified enzyme, which contains no endogenous adducin.
This prediction was tested in the experiment shown in Fig.
9. The percentage of control
Na+-K+-ATPase
activity in each case was plotted as a function of the added adducin
concentration. The curves represent best fits to hyperbolas with the
following parameters: enzyme,
Vmax = 387 ± 32.5% of control and
K0.5 = 30.5 ± 6.7 nM adducin; and microsomes, Vmax = 149.5 ± 12.8% of control and
K0.5 = 33.5 ± 7.4 nM adducin. Thus added human adducin stimulated the
Na+-K+-ATPase
activity of the microsomes only one-half as much as purified pig
Na+-K+-ATPase,
whereas the apparent affinity for adducin was the same on enzyme and
microsomes. These two findings indicate that, in microsomes, part of
the
Na+-K+-ATPase
is unavailable for interaction with exogenous adducin, whereas the
available fraction of
Na+-K+-ATPase
interacts with exogenous adducin in the same way as does the purified
enzyme preparation.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study we have provided evidence for a novel interaction between the cytoskeleton and the Na+-K+-ATPase in vitro. The experiments raise questions as to the mechanism of stimulation of Na+-K+-ATPase, the specificity of the adducin-Na+-K+-ATPase interaction, and, in particular, the relation of the in vitro effects to the physiological or pathophysiological roles of adducin polymorphism in modulating Na+-K+-pump activity, renal Na+ transport, and blood pressure in rats and humans that have been inferred from previous work.
Mechanism of stimulation of
Na+-K+-ATPase
by adducin.
The key to an understanding of the mechanism of stimulation of
Na+-K+-ATPase
by adducin is the finding that the degree of stimulation increased
greatly when ATP concentration was reduced from 3 mM to 0.1 mM and then
to 1 µM (Table 1, Fig. 3). At the lowest ATP concentration (1 µM),
the rate of ATP hydrolysis is limited almost exclusively by a slow rate
of the conformational transition
E2(K) E1Na, in which occluded
K+ is deoccluded and released to
the cytoplasmic surface, where Na+
binds. ATP binds with low affinity to
E2(K) and greatly accelerates the
rate of the conformational transition from
E2(K) · ATP to E1Na · ATP, the
form to which ATP is bound with a high affinity and phosphorylates the
enzyme. In an ATP hydrolysis experiment, the apparent affinity for ATP
reflects its binding affinity to E2(K) and the relative proportion
of the E2(K) form among the other
states of the enzyme (E1,
E1-P,
E2-P), which itself is determined by the rate constants of the transitions between the different states.
With these kinetic considerations in mind, it is clear that the major
effect of both MHS and MNS adducin is to accelerate the conformational
transition E2(K) E1Na. The effect on ATP hydrolysis
would be to decrease the relative proportion of
E2(K) relative to
E1Na and the other forms
(E1-P,
E2-P) and thereby increase the
apparent affinity for ATP. At saturating concentrations of ATP, the
rate of ATP hydrolysis is only partially limited by the rate of
E2(K) · ATP
E1Na · ATP, thus
explaining the much lower degree of stimulation by adducin. A necessary
corollary of the proposed action of adducin, to shift the
conformational equilibrium between
E2(K) · ATP and
E1 · ATP toward
the latter form, is that adducin should itself bind more tightly to the
E1 · ATP form.
This prediction is confirmed by the finding that the apparent affinity
for both MHS and MNS adducins is raised when the ATP concentration is
raised (Table 1). Independent confirmation that adducin accelerates the
rate of the conformational change E2(K) E1Na at low ATP concentrations (1 µM) was obtained in the experiment showing no inhibition or even
slight stimulation of Na+-ATPase
by K+ in the presence of adducin
but showing the expected inhibition by
K+ in the absence of adducin. The
biphasic effect of K+ observed in
Fig. 4 is a reproducible phenomenon. It can be explained by assuming
that the binding of only one potassium ion is required to catalyze
dephosphorylation of E2P and that
the rate of the conformational change depends on whether one or two
potassium ions are bound in E2.
However, this does not affect the conclusion concerning acceleration of
the conformational change by adducin. The pH dependence of the effect
of adducin as shown in Table 4 (strongest at pH 6, somewhat less at pH
7, and absent at pH 9) is completely in accordance with the stimulation
of E2(K) E1Na or
E2(K) · ATP
E1Na · ATP.
E1Na at low ATP, by
either interacting with the ATP binding site and raising the binding
affinity of ATP to E2(K), thus
accelerating E2(K) · ATP E1Na · ATP, or accelerating the
E2(K) E1Na
conformational transition directly, with equivalent results on the
kinetics. Stimulation by adducin of E2(K) E1Na or E2(K) ·ATP E1Na · ATP but a lack of effects of
adducin on ATP hydrolysis in conditions in which other steps are rate
limiting (phosphorylation at pH 9 for
Na+-K+-ATPase
experiments or E2-P E2 for
Na+-ATPase) indicates selectivity
of the functional effect. This suggests the presence of a specific
structural interaction of adducin with the enzyme.
By assuming that adducin accelerates the rate of
E2(K) · ATP
E1Na · ATP, one
could predict secondary effects on the apparent K+ and
Na+ affinities for activating ATP
hydrolysis due to the changes in the distribution of enzyme forms. The
direction of these effects should be to lower apparent
K+ affinity and raise apparent
Na+ affinity, and the size of such
changes should be smaller than the change in apparent ATP affinity. A
moderate reduction of apparent K+
affinity was indeed observed (Table 3). No significant effect on
apparent Na+ affinity was
observed. In view of the lower overall affinity of
Na+, the expected change might be
too small to be detected.
Specificity of the interaction between adducin and
Na+-K+-ATPase.
The results of this study strongly imply that we are dealing with a
direct interaction between adducin and
Na+-K+-ATPase.
Apart from the inference of a specific structural interaction on the
basis of the functional effects, the following features imply that the
interaction requires a specific structural domain, or conformation, of
adducin. 1) The
apparent affinities of MHS and MNS rat and human adducins for
stimulating
Na+-K+-ATPase
are in the tens of nanomolar concentration range.
2) The apparent affinities are
associated with the genetic variants found in both hypertensive (MHS)
and normotensive (MNS) rats [- (F316Y) and -adducin
(Q529R)] (8) and in humans (-adducins G460W and S586C) (Fig.
7) (12, 13, 38). In particular, the mutant MHS rat adducin stimulates
rat renal
Na+-K+-ATPase
activity with a higher affinity than does the wild-type MNS rat adducin
(Table 1), and a similar phenomenon is observed for wild-type and
mutant (G460W, S586C) human adducins (Fig. 7; see also below).
3) The stimulation of
Na+-K+-ATPase
is retained by restricted portions of both the - and -adducin
COOH-terminal tails, even though it is lower than that of the
full-length protein (60 or 30%, according to the length of the
fragment, vs. 75%), whereas it is lost when the tail fragment is
reduced to 31 amino acids (see Fig. 7 for domain organization). The
finding that the affinity of tail fragments is lower than that of
full-length adducin (Fig. 7) could indicate that both head/neck and
tail regions are required for binding or that removal of the head/neck
region affects the structure of the tail region, making it suboptimal
for binding. In addition, the mutant tails show a higher affinity than
wild-type tails (Fig. 7). 4) The
cytoskeletal protein ankyrin, which is known to bind directly to the
Na+-K+-ATPase
(28, 41, 42, 43, 50), is also able to stimulate the enzyme activity,
although to a lower extent (47 vs. 73%) and with lower affinity than
adducin (110 vs. 10 nM), whereas a noncytoskeletal protein (BSA) has no
effect (Fig. 5). The finding that the stimulatory effects of adducin
and ankyrin are not additive (Fig. 6) implies that these two proteins
may interfere with each other on the pump. Ankyrin is known to bind to
the -subunit of
Na+-K+-ATPase
at two distinct cytoplasmic domains (15). These domains have been
identified within residues 140-166 of the minor cytoplasmic loop
between transmembrane segments M2 and M3 (50) and also to a motif ALLK
in the central cytoplasmic loop between transmembrane segments M4 and
M5 (28). Therefore, it is possible that adducin also binds at or near
one or both of these cytoplasmic ankyrin binding regions.
Physiological and pathophysiological role of adducin in modulating
Na+-K+-pump
activity.
How are the present findings relevant to the functional and
pathophysiological roles of adducin polymorphism known to be involved in the mechanisms responsible for genetic hypertension (8, 12, 13, 27,
38, 47)? In genetically hypertensive MHS rats, the development of
hypertension is linked to a primary renal defect,
"transplantable" with the kidney (6, 7), that consists of an
increased tubular Na+ reabsorption
(5, 9, 19) associated with higher basolateral Na+-K+-pump
activity (18). The increase in
Na+-K+-pump
activity in MHS kidney is already present before the development of
hypertension, is accounted for by a higher number of functionally active
Na+-K+-pump
sites on the cell membrane surface (18), and is also associated with an
increased expression of the - and -subunit mRNA of the Na+-K+-pump
(18).
-subunit, between transmembrane segments M2 and M3 (minor loop) and
between M4 and M5 (major loop), interact in the
E2(K) state and move apart in the
E1Na state. A variety of
structural modifications, such as proteolytic cleavages or mutations in
the interacting sequences, appear to interfere with the loop
interactions and thereby stabilize the
E1 state. It is easy to imagine
that extrinsic proteins that bind to the cytoplasmic loops could also hinder their interactions, thus poising the
E2/E1
equilibrium toward E1 and raising
the apparent ATP affinity. This mechanism could apply to both adducin
and ankyrin, which is known to recognize both minor and major
cytoplasmic loops (Fig. 5) (15, 28, 50). In other words, the in vitro
functional effect of adducin or ankyrin on
Na+-K+-ATPase
may be incidental to the physiological role. The principal significance
of the functional effect could be that it indicates the presence of a
specific and direct interaction between adducin and the
Na+-K+
pump. In the physiological context this interaction may regulate the
cellular cycling of
Na+-K+
pumps and their density. Indeed, the higher affinity for mutant MHS
compared with that for wild-type MNS adducin (Table 1) may serve to
anchor the cytoskeleton to the
Na+-K+
pump more tightly and reduce the rate of internalization.
The evidence presented here, which suggests the existence of a novel
adducin-Na+-K+-ATPase
interaction, supports previous evidence showing that adducin polymorphisms are involved in genetic alterations of cell
Na+ transport and the pathogenesis
of primary hypertension in rats and humans. In particular, despite the
differences in the mutation positions between rat and human adducins,
the "hypertensive" adducin variants of both species affect the
Na+-K+-ATPase
activity similarly. These findings strongly support the notion that rat
genetic studies provide relevant information for understanding the
genetic and molecular mechanisms of human primary hypertension.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Vann Bennett, Yoichiro Matsuoka, and Xiaolin Li for
the kind gift of the human - and -adducin expression constructs and the 31-mer peptide mimicking the polybasic domain. We are grateful
to Dr. J. Kyte, University of California, San Diego, for providing the
anti-KETYY antibody. The excellent technical assistance of Sabrina
Pastore is also acknowledged.
| |
FOOTNOTES |
|---|
Part of this work was supported by the European Community Biomed Program (EURHYPGEN Concerted Action).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. J. D. Karlish, Biochemistry Dept., Weizmann Institute of Science, Rehovot, Israel 76100 (E-mail: bckarlis{at}weizmann.weizmann.ac.il).
Received 7 December 1998; accepted in final form 10 May 1999.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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|
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) and MNS (
)
adducin.
Na+-K+-ATPase
activity was measured at 0.1 mM ATP in presence of 100 mM NaCl, 5 mM
KCl, and 3 mM MgCl2 using 25 ng of
rat enzyme. Percentages of
Na+-K+-ATPase
activation were calculated over a control sample run in absence of
adducin. Curves represent means ± SE of 11 experiments run in
duplicate. Specific activity of purified
Na+-K+-ATPase
at 0.1 mM ATP was 7.1 µmol · min
) curve was run in parallel in absence
of adducin.
Na+-K+-ATPase
was measured in presence of 100 mM NaCl, 5 mM KCl, and 3 mM
MgCl2 at varying ATP
concentrations (0.05-3 mM) using 25 ng of rat enzyme. Curves
represent means of 3 experiments performed in duplicate with both
adducins.
) or presence (+) of adducin. Rat
Na+-K+-ATPase
(7.5 ng) was incubated without (
), and BSA
(
) were incubated with 25 ng rat
Na+-K+-ATPase
in presence of 100 mM NaCl, 5 mM KCl, 3 mM
MgCl2, and 0.1 mM ATP.
