(Received for publication, July 22, 1994; and in revised form, December 27, 1994)
From the
Rat brain sodium channels are phosphorylated at multiple serine
residues by cAMP-dependent protein kinase. We have identified soluble
rat brain phosphatases that dephosphorylate purified sodium channels.
Five separable forms of sodium channel phosphatase activity were
observed. Three forms (two, approximately 234 kDa and one, 192 kDa) are
identical or related to phosphatase 2A, since they were 85-100%
inhibited by 10 nM okadaic acid and contained a 36-kDa
polypeptide recognized by a monoclonal antibody directed against the
catalytic subunit of phosphatase 2A. Immunoblots performed using
antibodies specific for isoforms of the B subunit of phosphatase 2A
indicate that the two major peaks of phosphatase 2A-like activity, A1
and B1, are enriched in either B` or B. The remaining two
activities (approximately 100 kDa each) probably represent calcineurin.
Each was relatively insensitive to okadaic acid, was active only in the
presence of CaCl
and calmodulin, and contained a 19-kDa
polypeptide recognized by a monoclonal antibody raised against the B
subunit of calcineurin. Treatment of synaptosomes with okadaic acid to
inhibit phosphatase 2A or cyclosporin A to inhibit calcineurin
increased apparent phosphorylation of sodium channels at cAMP-dependent
phosphorylation sites, as assayed by back phosphorylation. These
results indicate that phosphatase 2A and calcineurin dephosphorylate
sodium channels in brain, and thus may counteract the effect of
cAMP-dependent phosphorylation on sodium channel activity.
Voltage-sensitive sodium channels mediate sodium influx during
the initial phase of the action potential in excitable cells. Sodium
channels from rat brain contain three glycosylated subunits: (260
kDa), which contains both the ion pore and voltage sensor;
1 (36
kDa), which is associated with
by ionic interactions and
modulates channel inactivation;
2 (33 kDa), which is covalently
attached to
by disulfide bonds (reviewed in (1) ). The
subunit contains four homologous domains, termed I-IV, each
of which has multiple membrane spanning segments. The
electrophysiological responses of brain sodium channels are modified by
cAMP-dependent protein kinase (PKA) (
)and protein kinase C.
Cyclic AMP-dependent phosphorylation decreases sodium current during
depolarization(2, 3) . Protein kinase C-dependent
phosphorylation slows channel inactivation (4) and also
decreases sodium current during
depolarization(4, 5, 6) . The
subunit
of brain sodium channels is phosphorylated in synaptosomes and in
cultured cells by cAMP-dependent protein kinase on Ser-573, Ser-610,
Ser-623, and Ser-687, which are clustered in a single intracellular
loop of the
subunit between homologous domains I and II of the
subunit(7, 8, 9, 10, 11) .
Protein kinase C phosphorylates the
subunit of purified
reconstituted sodium channels on two sites, Ser-610, which is also
phosphorylated by PKA, and Ser-1506 which is not phosphorylated by PKA
and is located within an intracellular loop connecting homologous
domains III and IV of the
subunit(12) . Phosphorylation
of Ser-1506 mediates the effect of protein kinase C on channel
inactivation (13) and enhances the effect of cAMP on channel
function(14) .
Purified soluble sodium channels were
dephosphorylated by purified calcineurin, a calcium- and
calmodulin-dependent protein phosphatase, and the catalytic subunit of
phosphatase 2A, but not the catalytic subunit of phosphatase
1(11) . A mixture of the purified catalytic subunits of
phosphatases 1 and 2A reversed the electrophysiological response of
sodium channels to cAMP in Chinese hamster ovary cells tranfected with
the brain sodium channel subunit(3) . When brain sodium
channels were expressed in Xenopus oocytes, the catalytic
subunit of phosphatase 2A, but not that of phosphatase 1, could reverse
the effect of PKA on sodium current(2) . It is not known which
of these serine/threonine phosphatases dephosphorylate sodium channels in vivo. To fully understand how neuronal sodium channels are
regulated by reversible phosphorylation, it is necessary to identify
the phosphatases that dephosphorylate sodium channels in brain and to
learn how their activity is controlled. In this study we demonstrate
that calcineurin and phosphatase 2A in rat brain extracts
dephosphorylate sodium channels that have been phosphorylated at
cAMP-dependent phosphorylation sites. We also provide evidence that
both of these phosphatases dephosphorylate sodium channels in
synaptosomes. These observations establish that calcineurin and
phosphatase 2A participate in controlling the phosphorylation state of
sodium channels in brain.
For all
other studies, one to three fresh rat brains were homogenized in
homogenization buffer, 23 ml/brain, and centrifuged at 100,000 g for 1 h. The resulting supernatant was applied to a
DEAE-Sephadex ion exchange column (2.4
20 cm) equilibrated in
the same buffer. The column was washed until the effluent absorbance at
280 nm was less than 0.1. Bound material was then eluted at 1 ml/min
with a 600-ml gradient of 0-1 M NaCl. Three-ml fractions
were collected and assayed for sodium channel phosphatase activity.
DEAE fractions containing sodium channel phosphatase activity were
pooled, and saturated (NH)
SO
(pH
7.4) was added to a final concentration of 55% at 4 °C. The mixture
was stirred for 30 min, then centrifuged at 10,000
g for 30 min. The pellet was resuspended in 1 ml of homogenization
buffer, applied to a Sephacryl S-300 gel filtration column (1.7
96 cm) equilibrated in homogenization buffer containing 0.1 M NaCl, and eluted at 0.2 ml/min. One-ml fractions were collected
and assayed for phosphatase activity.
When P-labeled phosphorylase a was used as
substrate, it was present at a final concentration of 10 µM and 5 mM caffeine was included in the assay mixture.
Sodium channel phosphatase activity was not affected by caffeine, and
phosphorylase a phosphatase activity was not affected by the presence
of 0.2% Triton X-100. For samples treated with okadaic acid, a
concentrated stock (124 µM) of okadaic acid in N,N-dimethylformamide was diluted to a final
concentration of 10 nM or 500 nM in assay samples.
Control samples received an equal amount of N,N-dimethylformamide, which did not alter
phosphatase activity. When testing the sensitivity of phosphatase
activity to CaCl
and calmodulin, samples were assayed in
the presence of 2 mM EGTA and the absence of CaCl
and calmodulin.
Okadaic
acid inhibits serine/threonine protein phosphatases to varying
degrees(31) . The effect of okadaic acid or CaCl and calmodulin on phosphatase activity in the soluble extract was
determined using two substrates, sodium channel and phosphorylase a, a substrate for phosphatases 1 and 2A (Fig. 1). Two
concentrations of okadaic acid were tested: 10 nM to
preferentially inhibit phosphatase 2A, and 500 nM to inhibit
both phosphatases 2A and 1. Phosphorylase a phosphatase activity was
40% inhibited by 10 nM okadaic acid and completely inhibited
by 500 nM okadaic acid, consistent with the presence of both
phosphatase 1 and 2A. Sodium channel dephosphorylation was 75%
inhibited by 10 nM okadaic acid, however little or no further
inhibition was achieved by 500 nM okadaic acid. This suggests
that phosphatase 2A or a phosphatase with similar sensitivity to
okadaic acid in the brain extract dephosphorylates sodium channels. In
addition, those form(s) of phosphatase 1 present in the soluble extract
and active toward phosphorylase a do not dephosphorylate sodium
channels. Removal of CaCl
and calmodulin further reduced
sodium channel dephosphorylation to 6% of maximal activity, suggesting
that calcineurin in the crude soluble extract may also dephosphorylate
sodium channels.
Figure 1:
Dephosphorylation of sodium channels or
phosphorylase a by soluble rat brain extract. Dephosphorylation of P-labeled sodium channels (
) or
P-labeled phosphorylase a (&cjs2112;) by soluble rat brain
extract was measured as described under ``Experimental
Procedures.'' Dephosphorylation in the presence of 10 nM or 500 nM okadaic acid, or in the presence of 10 nM okadaic acid and 2 mM EGTA is expressed as a percent of
control dephosphorylation (100%) in the presence of 1 mM CaCl
and 50 nM calmodulin. Average values
± S.E. from three separate experiments are shown. (O.A., okadaic acid; *, no detectable dephosphorylation; N.D., not determined).
Figure 2:
DEAE chromatogram of soluble rat brain
sodium channel phosphatase activity. Soluble rat brain extract was
subjected to DEAE ion exchange chromatography as described under
``Experimental Procedures.'' Phosphatase activity was
measured by the release of phosphate from P-labeled sodium
channels. Results from a single experiment are shown and are
representative of five separate studies.
Figure 3: Gel filtration chromatograms of DEAE peaks A, B, and C. DEAE peaks A, B, and C were each pooled, concentrated, and subjected to gel filtration through Sephacryl S-300. Sodium channel phosphatase activity was measured as described under ``Experimental Procedures.'' The elution of gel filtration standards are indicated: a, ferritin, 440,000; b, catalase, 232,000; c, aldolase, 158,000; d, bovine serum albumin, 66,000; e, carbonic anhydrase, 24,000; f, cytochrome c, 12,000. Results from a single experiment are shown and are representative of four separate studies.
Fractions A1, A2, B1, and B2 obtained from gel filtration
and pooled concentrated fractions from DEAE peak C were assayed for
sensitivity to calcium and calmodulin or okadaic acid (Fig. 4).
A1, B1, and C were unaffected by the removal of CaCl and
calmodulin, but were 80-100% inhibited by 10 nM okadaic
acid. In contrast, A2 and B2 were insensitive to 10 nM okadaic
acid, but were reduced in activity by 85-100% in the absence of
CaCl
and calmodulin. These results suggest that A1, B1, and
C are similar to phosphatase 2A with regard to okadaic acid
sensitivity, whereas A2 and B2 may represent calcineurin.
Figure 4:
Sensitivity of sodium channel phosphatases
to okadaic acid or CaCl and calmodulin. Gel filtration
peaks A1, A2, B1, B2, and DEAE peak C were assayed for sodium channel
phosphatase activity in: control buffer containing 1 mM CaCl
and 50 nM calmodulin (&cjs2108;); buffer
containing 2 mM EGTA with no added CaCl
or
calmodulin (
); buffer containing 10 nM okadaic acid
(&cjs2113;). Dephosphorylation is expressed as a percent of control
dephosphorylation (100%) in the presence of CaCl
and
calmodulin. Average values ± S.E. from three separate
experiments are shown, except for B2, which was assayed once in
triplicate. (*, no detectable
dephosphorylation).
To test
whether the okadaic acid-sensitive sodium channel phosphatase fractions
contained phosphatase 2A, immunoblots were performed using antibodies
specific for the catalytic (C) subunit of phosphatase 2A. Fractions of
A1 and B1 from gel filtration chromatography and C from DEAE
chromatography were immunoreactive with a monoclonal antibody that
specifically recognizes the catalytic subunit of phosphatase 2A (Fig. 5A). Mobility of the immunoreactive species in
these samples coincided with that of the 36-kDa catalytic subunit of
phosphatase 2A purified from rabbit skeletal muscle. In contrast, A2,
which was active only in the presence of calcium and calmodulin, did
not contain an immunoreactive polypeptide of 36 kDa. Recognition of the
36-kDa polypeptide in soluble brain extract was blocked by
preincubation of antibody with the purified catalytic subunit of
phosphatase 2A (data not shown), indicating that recognition was
specific. Western (21, 55) and Northern (38, 39, 55) blot analyses indicate that as
many as five forms of the phosphatase 2A B subunits may be present in
brain. Immunoblots using antibodies specific for two isoforms of the B
subunit, B and B`, demonstrated that A1 was enriched in B` (Fig. 5B), whereas B1 was enriched in B
(Fig. 5C). Peak C stained weakly with antisera for both
types of B subunits. The doublet visualized with the B` subunit
antiserum has also been reported in cardiac tissue(20) , where
the B`-containing isoform is the major species of phosphatase 2A. These
data indicate that A1 contains phosphatase 2A
, and B1
contains phosphatase 2A
. Fraction C may contain proteolyzed
forms of both phosphatase 2A
and 2A
, as has
been observed by others(17) .
Figure 5:
Immunoblot analysis of sodium channel
phosphatases with antibodies to the catalytic or B subunit of
phosphatase 2A. Peak fractions from DEAE or gel filtration
chromatography were subjected to SDS-PAGE, then transferred to membrane
and probed as described under ``Experimental Procedures''
with antibodies to either the catalytic subunit (A) or the B` (B) or B (C) subunits of phosphatase 2A (B). A gel filtration peak fractions A1, A2, B1, and
DEAE peak C, and the catalytic subunit of phosphatase 2A purified from
rabbit skeletal muscle (PP-2Ac). B and C,
gel filtration peak fractions A1, B1, and C. The migration of
prestained standards (bovine serum albumin (76 kDa), carbonic anhydrase
(28 kDa), biotinylated standards (phosphorylase b` (97 kDa),
bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase B
(31 kDa), and the catalytic subunit (PP2A-C), the B` subunit (PP2A-B`) or the B
subunit (PP2A-B
) of
phosphatase 2A are indicated.
Immunoblots were also performed to determine whether the calcium and calmodulin-sensitive fractions contained calcineurin. A polypeptide of approximately 19 kDa in peaks A2 and B2 was detected using an antibody directed against the B subunit of calcineurin (Fig. 6). The migration of this polypeptide coincided with that of the B subunit of purified calcineurin. A1, which was insensitive to calcium and calmodulin, was not recognized by this antibody. Recognition of the 19-kDa polypeptide in soluble brain extract was blocked by preincubation of antibody with the purified calcineurin (data not shown), again indicating that recognition was specific.
Figure 6: Immunoblot analysis of sodium channel phosphatases with an antibody to the B subunit of calcineurin. Peak fractions A1, A2, and B2 from DEAE chromatography and purified calcineurin (CN) were subjected to SDS-PAGE, transferred, and probed as described under ``Experimental Procedures.'' The migration of prestained standards (carbonic anhydrase (28 kDa), and lysozyme (16 kDa)) and the B subunit of calcineurin (CNB) are indicated.
To determine whether A2 and B2 represent
two separate peaks of CaCl/calmodulin-sensitive phosphatase
activity, soluble brain extract was subjected to DEAE chromatography as
before. Fractions were then assayed in the presence or absence of 10
nM okadaic acid (Fig. 7). In the presence of okadaic
acid, two distinct peaks of activity were observed. In the presence of
10 nM okadaic acid and the absence of CaCl
and
calmodulin, all sodium channel phosphatase activity was lost. This
indicates that A2 and B2 are separate peaks containing calcineurin, but
it is unclear whether they represent distinct isoforms of calcineurin,
or if one is derived from the other via proteolysis.
Figure 7:
Two separable peaks of
CaCl/calmodulin-sensitive sodium channel phosphatase
activity. DEAE ion exchange chromatography was performed as in Fig. 2, with phosphatase activity measured in the presence of
CaCl
and calmodulin (
) as usual, in the presence of
CaCl
, calmodulin and 10 nM okadaic (
), or in
the presence of 10 nM okadaic acid and 2 mM EGTA with
no added CaCl
or calmodulin
(
).
Figure 8:
Back phosphorylation of sodium channels
from synaptosomes treated with okadaic acid or cyclosporin A. A, synaptosomes were incubated at 37 °C with 10 µM forskolin for 10 min, 300 nM okadaic acid for 15 min, or
1 µM cyclosporin A for 30 min, and then sodium channels
were isolated and back phosphorylated with the catalytic subunit of PKA
and []P-labeled ATP as described under
``Experimental Procedures.'' B, dose dependence of
the effect of okadaic acid on synaptosomes. Results are expressed as
the percent decrease in back phosphorylation compared to control in the
absence of drug, with appropriate vehicle controls. Results are the
average ± S.E. of three or more
experiments.
Previous studies have shown that PKA
phosphorylates four serine residues in sodium channel subunits in vitro and in situ(11) . These sites
incorporated different levels of
P during back
phosphorylation or metabolic labeling, suggesting that they may be
differentially phosphorylated by PKA. In back phosphorylation studies,
Ser-623 incorporated nearly twice as much
P as other
residues, whereas in metabolic labeling studies, Ser-687 incorporated
twice as much
P as any of the other sites. To determine
whether calcineurin or phosphatase 2A selectively dephosphorylated any
of the four identified PKA phosphorylation sites, phosphopeptide maps
were generated from back phosphorylated sodium channels isolated from
synaptosomes treated with phosphatase inhibitors or forskolin, and
phosphate incorporated into each phosphorylation site quantified
separately as described previously (11) (Fig. 9). This
allows changes in phosphorylation of each site to be examined,
regardless of the total amount of
P incorporated.
Treatment of synaptosomes with forskolin, okadaic acid or cyclosporin A
led to a moderate decrease in back phosphorylation of each Ser residue
phosphorylated by PKA. Ser-573 was less affected by treatment with
forskolin or okadaic acid than were the other phosphorylation sites,
although the effect of treatments on back phosphorylation of this site
was variable. Since Ser-573 accounts for 20-25% of the total
phosphate incorporated into all four sites (11) and is the
least affected of any site by all treatments, differences in back
phosphorylation at this site contribute modestly to the overall changes
in back phosphorylation seen when all four sites are considered
together (Fig. 8). Ser-687 was more sensitive to treatment with
okadaic acid than forskolin or cyclosporin A, indicating that
phosphatase 2A exerted greater influence on the phosphorylation status
of this site than did PKA or calcineurin.
Figure 9: Phosphopeptide analysis of sodium channels after treatment of synaptosomes with okadaic acid or cyclosporin A. Synaptosomes were treated as in Fig. 7with A, forskolin; B, okadaic acid; or C, cyclosporin A, then sodium channels were isolated, back phosphorylated, and phosphopeptide maps generated as described under ``Experimental Procedures.'' Phosphopeptides corresponding to each serine residue were scraped and counted. Results are expressed as the percent decrease in back phosphorylation compared to control in the absence of drug, with appropriate vehicle controls. Results are the average ± S.E. of three or more experiments.
At least two different types of serine/threonine phosphatases with properties similar to those of phosphatase 2A and calcineurin dephosphorylate sodium channels in brain. These two phosphatases appear to account for all the sodium channel phosphatase activity detected in a soluble rat brain extract.
Activity present in gel filtration
peaks A1 and B1, and in DEAE peak C resembles phosphatase 2A in its
sensitivity to low concentrations of okadaic acid, and each peak
contains a 36-kDa polypeptide recognized by an antibody directed
against the catalytic subunit of phosphatase 2A. The best characterized
forms of phosphatase 2A contain a catalytic subunit complexed with one
or more regulatory subunits, A (61 kDa) and B (54-74 kDa), which
can influence the activity and substrate specificity of the catalytic
subunit (summarized in (36) ). Distinct B subunits have been
observed in brain(21, 55) , and multiple forms of all
three subunits in brain are predicted from cloning
studies(37, 38, 39, 55) .
Immunoblots indicate that peaks A1 and B1 contain phosphatase 2A (AC-B`), and phosphatase 2A
(AC-B
), respectively,
suggesting that both of these isoforms of phosphatase 2A may be active
toward sodium channels. However, further work will be required to
determine the contribution of these and other brain forms of
phosphatase 2A to sodium channel dephosphorylation. The activity and
substrate specificity of phosphatase 2A is modulated by the type of B
subunit present(21) . Recent studies suggest that phosphatase
2A may also be regulated by
phosphorylation(40, 41, 42, 43) ,
the binding of ceramide (44, 45) , and possibly by
carboxymethylation(54) , but it is not yet clear how these
events are controlled in brain. It will be important to determine
whether post-translational modifications or B subunit isoforms modulate
the activity of phosphatase 2A toward sodium channels. In addition, it
will be interesting to determine whether any of the brain forms of
phosphatase 2A are colocalized with sodium channels in neurons.
Two separable calcineurin-like phosphatase activities, A2 and B2, were also detected using sodium channel as substrate. Each was dependent on calcium and calmodulin for activity, was insensitive to 10 nM okadaic acid, and contained a 19-kDa polypeptide recognized by antibodies directed against the B subunit of bovine brain calcineurin. Calcineurin is a heterodimer containing a catalytic subunit, termed A (60 kDa) and a regulatory B subunit (19 kDa). Molecular biological, immunological, and biochemical studies indicate that multiple isoforms of the A subunit are expressed in brain (46, 47, 48, 49, 50, 51, 52, 53) . Further studies will be required to determine whether the two peaks of calcineurin-like activity are distinct forms of the enzyme or if the minor peak, B2, is derived by proteolysis. If A2 and B2 represent distinct forms of calcineurin, it will be of interest to determine whether they play distinct roles in sodium channel dephosphorylation.
The observation that phosphatase 2A and calcineurin can dephosphorylate sodium channels in brain extracts and in synaptosomes suggests a role for these phosphatases in controlling the state of sodium channel phosphorylation. This conclusion is consistent with previous in vitro studies showing that purified calcineurin and the catalytic subunit of phosphatase 2A, but not that of phosphatase 1, can dephosphorylate sodium channels(11) , and with electrophysiological studies showing that the catalytic subunit of phosphatase 2A, but not phosphatase 1, can reverse the effects of PKA on sodium channel function(2) . Despite these observations, it remains possible that a latent form of phosphatase 1 may dephosphorylate sodium channels when appropriately activated.
Inhibition of phosphatase 2A or calcineurin in synaptosomes led to
an apparent increase in phosphorylation of all four cAMP-dependent
phosphorylation sites of the sodium channel subunit. This result
differs from earlier findings that purified calcineurin or the
catalytic subunit of phosphatase 2A selectively dephosphorylated
Ser-623 or Ser-610 in purified sodium channels,
respectively(11) . These differences may arise from the
presence of additional subunits or post-translational modifications of
enzymes or substrate, or may reflect the competing basal activity of
phosphatases and PKA within synaptosomes.
The identification of phosphatase 2A and calcineurin as enzymes that dephosphorylate sodium channels in brain is an important step in understanding how the state of sodium channel phosphorylation is regulated. The physiological circumstances controlling the activity of calcineurin and phosphatase 2A toward sodium channels remain to be determined. Since the sodium channel contains multiple phosphorylation sites and is a target for multiple protein kinases and phosphatases, control of its state of phosphorylation is expected to be complex.