1Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454; and 2Department of Molecular Physiology and Biophysics and Center for Molecular Neuroscience, Vanderbilt Medical Center, Nashville, Tennessee 37232
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chen, Huan-Xin, Nikolai Otmakhov, Stefan Strack, Roger J. Colbran, and John E. Lisman. Is Persistent Activity of Calcium/Calmodulin-Dependent Kinase Required for the Maintenance of LTP?. J. Neurophysiol. 85: 1368-1376, 2001. Calcium/calmodulin-dependent protein kinase II (CaMKII) is concentrated in the postsynaptic density (PSD) and plays an important role in the induction of long-term potentiation (LTP). Because this kinase is persistently activated after the induction, its activity could also be important for LTP maintenance. Experimental tests of this hypothesis, however, have given conflicting results. In this paper we further explore the role of postsynaptic CaMKII in induction and maintenance of LTP. Postsynaptic application of a CaMKII inhibitor [autocamtide-3 derived peptide inhibitor (AC3-I), 2 mM] blocked LTP induction but had no detectable affect on N-methyl-D-aspartate (NMDA)-mediated synaptic transmission, indicating that the primary function of CaMKII in LTP is downstream from NMDA channel function. We next explored various methodological factors that could account for conflicting results on the effect of CaMKII inhibitors on LTP maintenance. In contrast to our previous work, we now carried out experiments at higher temperature (33°C), used slices from adult animals, and induced LTP using a tetanic stimulation. However, we still found that LTP maintenance was not affected by postsynaptic application of AC3-I. Furthermore the inhibitor did not block LTP maintenance under conditions designed to enhance the Ca2+-dependent activity of protein phosphatases 1 and 2B (elevated Ca2+, calmodulin, and an inhibitor of protein kinase A). We also tested the possibility that CaMKII inhibitor might not be able to affect CaMKII once it was inserted into the PSD. In whole-brain extracts, AC3-I blocked autophosphorylation of both soluble and particulate/PSD CaMKII with similar potencies although the potency of the inhibitor toward other CaMKII substrates varied. Thus we were unable to demonstrate a functional role of persistent Ca2+-independent CaMKII activity in LTP maintenance. Possible explanations of the data are discussed.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Long-term potentiation (LTP)
has been widely studied as a cellular model of learning and memory.
Induction of LTP is triggered by a transient increase in intracellular
Ca2+ concentration, which activates the
biochemical cascade leading to enhanced synaptic transmission (reviewed
in Bliss and Collingridge 1993; Nicoll and
Malenka 1999
). There is now substantial evidence for a key role
of postsynaptic calcium/ calmodulin-dependent protein kinase II
(CaMKII) in this induction process. LTP induction produced by a
tetanus can be blocked by postsynaptic application of peptide inhibitors of the kinase (Feng 1995
; Hvalby et
al. 1994
; Malinow et al. 1989
). Furthermore it
was shown that LTP produced by a pairing protocol is also blocked by
CaMKII inhibitors (Otmakhov et al. 1997
). Since use of
this protocol ensures the level of postsynaptic depolarization required
for LTP induction, it is clear that CaMKII is involved in the core
processes of synaptic plasticity rather than simply in the
depolarization processes. More recently, elevation of CaMKII activity
by direct introduction of the kinase into a cell or by expression of a
constitutively active form was shown to enhance synaptic transmission
in a manner that occludes with tetanus-induced LTP (Lledo et al.
1995
; Pettit et al. 1994
; Shirke and
Malinow 1997
). This action appears to involve direct
phosphorylation of glutamate receptor subunit 1 (GluR1) and the
resulting enhancement of AMPA channel conductance (Benke et al.
1998
; Derkach et al. 1999
) as well as insertion of new GluR1 subunits into the synapse (Hayashi et al.
2000
).
CaMKII may also have a role in the maintenance of LTP. Since
autophosphorylation of Thr286 can promote further autophosphorylation of the kinase, CaMKII activity might be maintained constitutively by a
positive feedback process (Lisman 1985, 1994
;
Miller and Kennedy 1986
; Okamoto and Ichikawa
2000
; Saitoh and Schwartz 1985
; Schworer
et al. 1988
; Zhabotinsky 2000
). Consistent with
this hypothesis, biochemical work has shown that LTP induction triggers a long-lasting increase in the autophosphorylated form of CaMKII and in
its Ca2+-independent activity (Barria et
al. 1997
; Fukunaga et al. 1993
, 1995
; Lee
et al. 2000
; Ouyang et al. 1997
, 1999
).
Furthermore a mutation of Thr286 prevents LTP induction (Giese
et al. 1998
).
If persistent CaMKII activity is responsible for LTP maintenance, LTP
should be reversed if CaMKII activity is inhibited after induction.
Early work tested this prediction in mature rats by infusing CaMKII
inhibitors into the postsynaptic neuron through microelectrodes. These
studies gave conflicting results (Feng 1995;
Malgaroli et al. 1992
; Malinow et al.
1989
). One difficulty with microelectrode studies is that cells
have to be impaled after induction of LTP, and there was, therefore, no
direct evidence that LTP had occurred in the neuron that was being
recorded from. To address this problem, we developed a method for
controlled inhibitor application using a perfused patch pipette. With
this method, CaMKII inhibitor could be applied postsynaptically after verifiable LTP had been induced in a cell. Our results showed that LTP
maintenance was not affected by CaMKII inhibitor (Otmakhov et
al. 1997
), consistent with the results of Malinow et al.
(1989)
, but inconsistent with the work of Feng
(1995)
, showing that LTP can be reversed by this inhibitor.
In this paper, we have attempted to resolve a range of issues relevant
to the possible role of CaMKII in LTP induction and maintenance. In the
first series of experiments, we explored whether the prevailing notion
that CaMKII acts downstream from the NMDA channel might be incorrect.
The fact that CaMKII inhibitors block LTP induction has been taken as
evidence that CaMKII detects Ca2+ entry through
the NMDA channel and triggers subsequent processes that strengthen the
synapse. However, because the NMDA receptor is phosphorylated by CaMKII
(Gardoni et al. 1998; Leonard et al. 1999
; Omkumar et al. 1996
; Strack and
Colbran 1998
; Strack et al. 2000
), it is
possible that CaMKII inhibitors decrease the baseline NMDA conductance
and thereby inhibit LTP. We have tested this possibility.
In a second set of experiments, we addressed various technical
differences between the experiments of Feng (1995),
which did show an effect of CaMKII inhibitor on LTP maintenance, and
our previous experiments, which did not (Otmakhov et al.
1997
). Feng's experiments were conducted at higher temperature
(32-33°C), used older animals, and induced LTP using a tetanic
method. Our work was done at room temperature on younger animals, and
LTP was induced by pairing. We have tested whether these factors could
account for the discrepancies.
In a third set of experiments, we addressed the hypothesis that
reversal of LTP maintenance does not occur because postsynaptic phosphatase activity is too low. The main phosphatase that
dephosphorylates PSD-associated CaMKII is protein phosphatase 1 (PP1)
(Strack et al. 1997a). PP1 is upregulated by
Ca2+/calmodulin and downregulated by protein
kinase A (PKA) via an enzyme cascade (Shenolikar and Nairn
1991
). Specifically, when inhibitor-1 is phosphorylated by PKA,
it can effectively inhibit PP1; when it is dephosphorylated by the
calcium/calmodulin-dependent protein phosphatase 2B, calcineurin, PP1
becomes active. This makes PP1 dependent on the intracellular free
Ca2+ concentration and on calmodulin
concentration. We therefore attempted to boost postsynaptic PP1
activity by setting Ca2+concentration in pipette
solution above the resting level (0.3 µM) and including a PKA
inhibitor and calmodulin in this solution.
In a final set of experiments, we investigated whether CaMKII
inhibitors can affect CaMKII activity in the PSD. The PSD is an array
of scaffolding proteins and attached enzymes (Kennedy 1998), which could make some PSD proteins inaccessible from
cytosol. Thus CaMKII inhibitor might effectively inhibit the activity
of soluble CaMKII but might have restricted access to the PSD such that
PSD-associated CaMKII is not effectively inhibited. If LTP maintenance
requires activity of PSD-associated CaMKII, this could explain why we
have been unable to reverse maintenance with CaMKII inhibitor. To test
this possibility, biochemical experiments were conducted in which we
examined the effect of inhibitors on PSD CaMKII.
The work presented in this paper poses a puzzle: biochemical experiments show that CaMKII is persistently activated after LTP induction. However, all the experiments that we have reported previously and that we have extended in this paper seem to indicate that constitutive CaMKII activity does not play a role in LTP maintenance. Possible resolutions to this puzzle are presented in the DISCUSSION.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electrophysiology
Transverse hippocampal slices were prepared from male Long-Evans
rats (6-8 wk old) as described previously (Chen et al.
1999). The CA3 region of each slice was removed from the slice
by a surgical cut. Slices were incubated on cell culture inserts
(Falcon, 8 µm pore diameter) covered by a thin layer of artificial
cerebrospinal fluid (ACSF containing 2 mM Ca2+
and 6 mM Mg2+) and surrounded by a humidified
95% O2-5% CO2 atmosphere
at room temperature (~22°C). For recording, a single slice, after
2- to 6-h incubation, was transferred to a submerged recording chamber with continuous flow (1.5-2 ml/min) of ACSF. The ACSF contained (in
mM) 124 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 2 CaCl2, 2 MgSO4, 10 D-glucose, and 0.05 picrotoxin, gassed with 95%
O2-5% CO2 giving pH 7.4. All experiments were carried out at 32-33°C.
Whole-cell current-clamp recordings were performed from CA1 pyramidal
cells located 50-90 µm beneath the slice surface under visual
control using infrared dark-field illumination and a CCD TV camera. The
patch electrodes were made from borosilicate glass and filled with (in
mM) 125 K-gluconate, 10 HEPES, 8 NaCl, 0.2 EGTA, 2 MgATP, 0.3 Na3 GTP, and 10 phosphocreatine (pH 7.3 with KOH,
osmolarity 290-296 mOsm). The electrodes had resistance 3-5 M when
filled with internal solution. Whole-cell recordings were made in
current-clamp mode using an Axopatch-1D (Axon Instruments, Foster City,
CA). Only cells with membrane potential more negative than
65 mV were
used. To evoke synaptic responses, two glass electrodes filled with
ACSF (300 K
) were placed in the dendrite region 70 and 150 µm away
from the cell body layer to stimulate two separate groups of Schaffer
collaterals. Stimuli (100 µs) were delivered alternatively to each
input pathway through current output isolation units. The interval
between stimuli in each pathway was 6 s with 3-s interval between
pathways. Stimulation intensity was adjusted to produce an excitatory
postsynaptic potential (EPSP) with amplitude of ~30-50% of
threshold for an action potential. Whole- cell voltage-clamp recording
was used in experiments in which the isolated synaptic NMDA component
was studied. In these experiments, K-gluconate was replaced by
Cs-gluconate in the internal solution,
1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo-[f]quinoxaline-7-sulfonamide disodium (NBQX) (10 µM) was used to block AMPA component and
membrane potential was held at
45 mV. In all experiments, series and
input resistances during the recording were monitored every 3 s by
applying depolarizing voltage pulses. The series resistance ranged from 6 to 12 M
. The input resistance ranged from 70 to 150 M
.
LTP was induced by tetanic stimulation including five trains, each
containing 20 pulses at 100 Hz. The interval between trains was 10 s. The specific inhibitor of CaMKII used was an autocamtide-3 derived
inhibitory peptide (Ac-KKALHRQEAVDAL-NH2), which
resists proteolysis (AC3-I) (Otmakhov et al. 1997).
Peptide inhibitors of this class are 100-fold more specific for CaMKII
than for PKC (Braun and Schulman 1995
). A peptide with
the reversed amino acid sequence relatively to AC3-I served as a
control peptide (gift of Dr. Leslie Griffith). For inhibition of PKA, a
specific PKA peptide inhibitor PKI(6-22) amide was used.
Aliquots of the peptide stock solutions were thawed only once
immediately before an experiment. The pH of internal perfusion solution
was adjusted to 7.2-7.4. Internal perfusion was performed as described
elsewhere (Otmakhov et al. 1997
).
Data were acquired using a 486 PC computer, Labmaster DMA ADC, and program written in Axobasic. The amplitude of a synaptic response was calculated as the difference between the average of data points in a window before the stimulus and in a window around the peak of the synaptic response. The average of responses during a 5-min period before LTP induction was taken as the baseline, and all values were normalized to this baseline. Values were expressed as means ± SE. Two-tail paired t-test was used for calculation of the statistical significance of differences. Drugs used included D-2-amino-5-phosphonovaleric acid (D-AP5, RBI), Picrotoxin (Sigma), NBQX (RBI), AC3-I, and PKI(6-22) (QCB, Hopkinton, MA), Calmodulin (Sigma).
Biochemistry
Rat whole-forebrain extracts and PSDs were prepared
(Strack et al. 1997a), and baculovirus-expressed murine
CaMKII
was purified as described (McNeill and Colbran
1995
). Purified CaMKII and extracts were diluted to similar
kinase activities (~100 pmol · ml
1 · min-1) and assayed for autocamtide-2
phosphorylation (10 µM) in the presence of calcium/calmodulin (0.5 mM/1 µM) and varying concentrations of AC3-I or inactive control
peptide essentially as described (Colbran 1993
). Less
than 1% kinase activity was detected in the absence of
calcium/calmodulin. Controls omitting peptide substrate showed that
phosphorylation of endogenous proteins did not contribute significantly
to total 32P-incorporation at these dilutions.
Endogenous substrate assays were carried out following a two-phase
protocol. To autophosphorylate CaMKII, whole rat forebrain extracts
(0.25 mg/ml) were incubated for 15 s on ice in the presence of 0.5 mM CaCl2/3 µM calmodulin or 1 mM EGTA (as a
control) with 5 µM nonradioactive ATP, 2 mM Mg acetate, 20 mM HEPES,
pH 7.5, 0.5% (vol/vol) Triton X-100, 1 mM dithiotreitol, 20 µg/ml
leupeptin, 1 mM benzamidine, 1 µM microcystin-LR.
Calcium/calmodulin-independent activities of 20-30% (using the
autocamtide-2 assay as described in the preceding text) were typically
achieved during this incubation. After 15 s,
[-32P]ATP (5 µM, ~20 cpm/fmol) was added
without or with 1.5 mM EGTA to radiolabel proteins by
calcium/calmodulin-dependent or -independent phosphorylation,
respectively, and incubation was continued for 45 s on ice before
reactions were stopped by addition of 20 mM EDTA. Varying
concentrations of AC3-I and other kinase inhibitors were also present
during the second phase. Extracts were microcentrifuged (30 min, 14,000 g, 4°C) to separate soluble from particulate proteins, and
analyzed by SDS-PAGE and autoradiography. 32P
incorporation into individual bands was quantified by PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CaMKII inhibitor does not affect the NMDA receptor-dependent synaptic conductance
The induction of LTP depends on the activation of the NMDA
receptor (Bliss and Collingridge 1993). It has been
reported that CaMKII directly binds to NMDA receptor and phosphorylates
it, suggesting that NMDA activity can be regulated by CaMKII
(Gardoni et al. 1998
; Leonard et al.
1999
; Omkumar et al. 1996
; Strack and
Colbran 1998
; Strack et al. 2000
). If activation
of NMDA receptor is required for basal CaMKII activity, blocking CaMKII
activity may reduce the basal NMDA conductance and therefore block LTP induction. To test this possibility, we have examined the effect of the
inhibitory peptide on the NMDA component of the synaptic response
isolated by blocking the AMPA component with NBQX (20 µM). The
recordings were done under voltage-clamp mode at holding potential
45
mV. As shown in Fig. 1, perfusion of 2 mM
AC3-I had no effect on the basal NMDA excitatory postsynaptic current (EPSC). The amplitude of NMDA EPSC at 30 min after the perfusion of
AC3-I were 98 ± 5% of the baseline (n = 6;
P > 0.05)
|
Attempts to reverse LTP maintenance
Recordings were made in hippocampal slices from 6- to 7-wk-old rat
at 32°C in current-clamp mode, and LTP was induced by a tetanus to
match conditions used by Feng (1995). To determine the
effect of CaMKII inhibitory peptide, AC3-I, on the maintenance of LTP,
we used a two-pathway protocol. After 5 min of baseline recording, LTP
was induced in one pathway and then AC3-I (2 mM) was perfused into the
patch pipette. Fifteen minutes later, LTP was induced in the second
pathway (Fig. 2A). This
protocol allowed us to measure in the same experiment the inhibitor
effect on both the maintenance of LTP in the first pathway and on LTP
induction in the second pathway. We found that in the first pathway,
the level of LTP at 60 min after perfusion of AC3-I was not
significantly different (201 ± 14%, Fig. 2A,
n = 6) from the level of LTP in experiments in which
control peptide (2 mM) was perfused (249 ± 54%, Fig.
2B, n = 6, P > 0.05).
However, because the initial levels of LTP in the test and control
experiments were slightly different, it was desirable to check for an
effect of the inhibitor using a procedure that normalized these initial
levels. The level of LTP just after the tetanus can be obscured by
posttetanic potentiation, which can last several minutes. Therefore we
measured the initial LTP magnitude during the 5-min period starting 10 min after the tetanus. In this time, posttetanic potentiation is mostly
diminished, but the effect of intracellularly applied drug is not yet
revealed. Taking the levels of LTP at this time as 100%, we calculated
the percentages of LTP remaining 60 min after perfusion of AC3-I or control peptide. These remaining LTP levels were not significantly different (90 ± 2 and 83 ± 2% of the initial LTP level,
respectively, P > 0.05), indicating that the
maintenance of LTP was not affected by the CaMKII inhibitor. Induction
of LTP in the second pathway was completely blocked by the same
application of AC3-I (100 ± 8% of baseline at 45 min after the
tetanus). In control experiments in which control peptide was perfused
before the tetanus was delivered to the second pathway, robust LTP was
produced (255 ± 58%, P < 0.05).
|
In additional experiments, we found that lower concentrations of CaMKII inhibitory peptide (1 mM) only partially blocked LTP induction (Fig. 2C, n = 5); LTP in the second pathway in these experiments was still 144 ± 18% of baseline at 50 min after the tetanus, a value significantly lower than in control experiments (Fig. 2B, P < 0.05). The maintenance of LTP was not effected in these experiments; the level of LTP at 60 min after the tetanus was 207 ± 14% (or 100 ± 6% of the initial LTP level, P > 0.05).
One possible explanation of the lack of effect on maintenance is that although the CaMKII activity is required for the maintenance of LTP, inhibiting the kinase does not lead to dephosphorylation of the kinase substrates because the PP1 activity is too low (see INTRODUCTION). This might occur because we followed standard practice by including a low concentration of Ca2+ buffer (0.2 mM EGTA) in the internal solution, and this might lower Ca2+ to below normal levels. Also the concentration of calmodulin might be low because it is a small soluble protein that could wash out of the cell during prolonged whole-cell recordings.
To study the effects of CaMKII inhibitor under conditions where free
Ca2+ was not low, we clamped
Ca2+ in the patch pipette solution using a high
concentration of the Ca2+ buffer,
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA), set to maintain free Ca2+ at 0.3 µM.
Because of complexities introduced by the fact the internal solution
contained gluconate, which is itself a Ca2+
buffer (Christoffersen and Skibsted 1975), the
Ca2+ concentration in this solution was checked
by direct measurement using a Ca2+-specific
electrode. This concentration is slightly higher than thought to be the
case under resting conditions and should ensure that
Ca2+-dependent phosphatase activity is at least
at normal levels. The effect of using this solution in conjunction with
CaMKII inhibitor is shown in Fig.
3A. As in our previous work,
this internal solution was applied after the induction of LTP. It can
been seen that there was still no substantial inhibition of LTP
maintenance (199 ± 34% that correspond to 92 ± 10% of the
initial LTP level, P > 0.05). The response in the
nonpotentiated pathway was also not affected (99 ± 3% of
baseline at 1 h after perfusion, P > 0.05).
|
We next considered the possibility that PP1 might be low because of
elevated PKA activity or washout of calmodulin (see
INTRODUCTION). In this case, PP1 activity should be
elevated by PKA inhibitor and by addition of exogenous calmodulin. We
thus conducted experiments in which 20 µM calmodulin and 2 mM PKA
inhibitor [PKI (6-22) amide] were added to the high Ca
2+ internal solution (see in the preceding text).
These conditions should elevate PP1 activity. These concentrations of
calmodulin and PKI were shown to be effective in hippocampal neurons
(Otmakhova et al. 2000; Wang and Kelly
1995
). However, as demonstrated in Fig. 3B, these
experiments did not reveal any strong tendency to reverse maintenance
(87 ± 2% of the initial level in LTP pathway and 102 ± 7%
in control pathway at an hour after the perfusion, P > 0.05).
Characterization of CaMKII inhibition by AC3-I
LTP is associated with a rapid and maintained increase in
calcium/calmodulin-independent activity of CaMKII as a consequence of
autophosphorylation of Thr286/287 (Barria et al. 1997;
Fukunaga et al. 1993
, 1995
; Lee et al.
2000
; Ouyang et al. 1997
, 1999
). This
autophosphorylation also promotes translocation of CaMKII to
postsynaptic densities where it enhances the phosphorylation of several
postsynaptic substrates including the GluR1 subunit of the AMPA-type
glutamate receptor (Shen and Meyer 1999
; Shen et
al. 2000
; Strack et al. 1997b
). The failure of
AC3-I to block maintenance of LTP could conceivably be attributed to an
inability of this compound to inhibit the PSD-associated form of the
kinase. To address this issue, we first compared the inhibitory potency of AC3-I toward purified recombinant CaMKII and endogenous CaMKII in
whole-brain lysates and PSDs, using as a substrate the specific CaMKII
substrate autocamtide-2 (AC2). AC3-I inhibited AC2 phosphorylation in
the presence of calcium/calmodulin with similar
IC50s (20-80 µM) for different sources of
kinase (Fig. 4A). Thus CaMKII
in PSDs, crude brain lysates, and purified soluble CaMKII are similarly accessible to inhibition by AC3-I.
|
We next investigated the effect of AC3-I on phosphorylation of
endogenous CaMKII substrates. Whole forebrain extracts were briefly
incubated with calcium/calmodulin and nonradioactive ATP under
conditions that lead to selective autophosphorylation of CaMKII (see
METHODS). To label proteins by calcium/calmodulin-dependent and -independent phosphorylation, incubation was continued by adding
[-32P]ATP in the absence or presence of the
calcium chelator EGTA, respectively. Extracts were then separated into
a Triton X-100 soluble and an insoluble cytoskeletal fraction enriched
in PSDs and analyzed by SDS-PAGE and autoradiography. The substrate
profiles under both 32P-labeling conditions were
similar, but differed from samples in which both autophosphorylation
and 32P-labeling were carried out without
calcium/calmodulin (Fig. 4B), suggesting that
calcium/calmodulin-dependent and autonomous CaMKII phosphorylate
similar substrates. The pattern of 32P
incorporation in the particulate fraction was very similar to the
pattern seen when purified PSDs were phosphorylated by endogenous CaMKII (Chen et al. 1998
; Strack et al.
1997b
). Two major 32P-labeled bands
present in both soluble and particulate fraction correspond to the
autophosphorylated
and
isoforms of CaMKII. Additional CaMKII
substrates included a doublet of 76K and 78K molecular weight in the
soluble extract (S76/78) and 180K in the particulate fraction (P180).
Based on molecular weight, fractionation profile, and previous
characterization as CaMKII substrates, these bands were tentatively
identified as synapsin 1a/b (S76/78) (Huttner et al.
1981
) and a combination of the NR2B subunit of the NMDA receptor (Omkumar et al. 1996
) and the PSD protein,
densin-180 (Apperson et al. 1996
). Calcium-independent
autophosphorylation of CaMKII
in both fractions was potently
inhibited by AC3-I (IC50 0.6 ± 0.3 and
1.1 ± 0.4 µM, in soluble and particulate fractions, respectively, n = 3; Fig. 4C) but unaffected
by the PKA inhibitor PKI-tide and the PKC inhibitor
PKC19-32 (10 µM, not shown). Similar results
were obtained for AC3-I inhibition of 32P
incorporation into CaMKII
(IC50 1 µM) and
the soluble substrate S76/78 (IC50 0.3 ± 0.1 µM, n = 3 not shown). The phosphorylation of a
particulate CaMKII substrate, P180, was much more difficult to inhibit
with AC3-I (IC50 8 ± 1.2 µM,
n = 3) than S76/78. While the potency of inhibition of
phosphorylation of different substrates by AC3-I varied,
calcium-dependent and -independent activities toward a given substrate
were inhibited similarly by AC3-I (Fig. 4C).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This work is a continuation of the investigation into the role of
CaMKII in the maintenance of LTP. Our previous results indicated that
perfusion of CaMKII inhibitor after LTP induction did not block LTP
maintenance (Otmakhov et al. 1997), contrary to the results of Feng (1995)
, who found that the maintenance
of LTP could be blocked by a similar CaMKII peptide inhibitor. Here we have investigated whether age, temperature, or method of LTP induction might account for this discrepancy. In particular, we now have used
mature animals, induced LTP using a tetanus, and performed our
experiments at 32-33°C to make our conditions more similar to those
used by Feng. We also followed the recording for
1 h from the
start of the perfusion because Feng reported the effect occurred on
this slow time scale. Our method of drug delivery (diffusion from a
patch pipette) is more efficient than the infusion from the
microelectrode used by Feng, and our inhibitor concentration (2 mM) is
higher than used in his work (100 µM). Nevertheless, contrary to
Feng's finding, we did not see a block of LTP maintenance within the
1-h period after inhibitor application. The fact that the same
application of the inhibitor completely blocked LTP induction indicates
that the inhibitor was effective inside of these cells.
We have also addressed several other possible reasons for the
discrepancy with Feng. One specific concern is that reversal of
maintenance might depend on phosphatase activity and that phosphatases might "wash out" of the cell during whole- cell recording procedure that we have used. It seems doubtful, however, that phosphatases have
completely washed out or been irreversibly inactivated since cells
still expressed normal long-term depression (LTD). This was also true
in the presence of AC3-I (data not shown). Previous work has shown that
LTD depends on phosphatase activity (Mulkey et al. 1993,
1994
).
Another possibility is that the intracellular
Ca2+ concentration may have been higher in
Feng's experiments than in our experiments. This could potentially
affect the dominant PSD phosphatase, PP1, which is turned on by a
cascade activated by the
Ca2+/calmodulin-dependent phosphatase 2B
(calcineurin) and turned off by PKA (Shenolikar and Nairn
1991). The difference in Ca2+
concentration may be because in Feng's experiments, intracellular Ca2+ concentration was artificially elevated
because the relatively low-resistance microelectrode (50 M
) that
impaled the cell might not provide tight seal with the membrane and
thus make the membrane leaky to Ca2+.
Alternatively, the Ca2+ concentration in our
experiments may be lowered below normal as a consequence of the low
Ca2+ levels in our internal solution (due to use
of a small concentration of Ca2+ chelator in the
internal solution). To address these possibilities, we set the
Ca2+ level in the internal solution slightly
above (0.3 µM) resting concentration. We were still unable to reverse
LTP with CaMKII inhibitor. This was also true when additional steps to
activate PP1 were taken (addition of calmodulin and PKA inhibitor).
Still another possibility to be considered is that the activity of
PSD-associated CaMKII is less effectively inhibited by AC3-I than that
of soluble CaMKII. We initially demonstrated that the abilities of
soluble and PSD-associated CaMKIIs to phosphorylate an exogenous
synthetic peptide substrate (10 µM autocamtide-2) were similarly
inhibited by AC3-I (IC50 20-80 µM; Fig.
4A). Furthermore AC3-I also similarly inhibited
autophosphorylation of soluble and particulate CaMKII in whole-brain
extracts (IC50 0.6-1.1 µM). These results
indicate that the inhibitor does not have difficulty entering the PSD
and interacting with CaMKII. This conclusion is consistent with the
report that phosphorylation of GluR1 by PSD-associated CaMKII can be
potently inhibited by a related CaMKII inhibitor peptide
(Hayashi et al. 1997). However, further analysis of
CaMKII-mediated phosphorylation in whole-brain extracts revealed significant differences in the susceptibility of various substrate phosphorylations toward inhibition by AC3-I: while phosphorylation of
the soluble P76/78 proteins was potently inhibited
(IC50 0.3 µM), phosphorylation of
particulate/PSD-associated proteins P180 was much less potently
inhibited (IC50 8 µM). Therefore there is a
possibility that LTP induction in our experiments is blocked due to
inhibition of phosphorylation of a substrate with high sensitivity to
AC3-I, but LTP maintenance might not be blocked because the persistent
phosphorylation of a substrate with low sensitivity to AC3-I, like
P180, was not inhibited. Indeed, we found that just a twofold reduction
in concentration of AC3-I already caused a significant decrease in its
effect on LTP induction (Fig. 2), suggesting that the concentration of
the inhibitor in dendrites may be too low to block reactions that are
less sensitive to the inhibitor. The preceding interpretation, however,
requires that intramolecular phosphorylation of CaMKII should not be
blocked by this concentration of CaMKII inhibitor, or, if it is
blocked, that the kinase remains phosphorylated on T286, perhaps, due
to low phosphatase activity (see following text).
One remaining technical difference between our work and that of
Feng (1995) is that he used a microelectrode for
intracellular application of inhibitor whereas we used the whole-cell
patch-clamp method. However, our results are in agreement with
Malinow et al. (1989)
using microelectrodes to deliver
CaMKII inhibitory peptide; they also did not find that LTP maintenance
could be reversed.
Possible roles of CaMKII in LTP
Although the role of CaMKII in LTP induction is clear, the crucial
substrates are not known with certainty. One of CaMKII substrates in
the PSD is the NMDA channel (Gardoni et al. 1998; Leonard et al. 1999
; Omkumar et al. 1996
;
Strack and Colbran 1998
; Strack et al.
2000
), the function of which may be upregulated as a result of
this phosphorylation (Kitamura et al. 1993
; Kolaj et al. 1994
). It is therefore possible that if the basal
phosphorylation of the NMDA channel enhances its conductance that the
CaMKII inhibitor blocks LTP induction simply because the basal NMDA
conductance is reduced. We have tested this possibility by determining
whether CaMKII inhibitor reduces the baseline NMDA conductance and
found that it does not. A more complex possibility that we have not tested is that the NMDA conductance becomes enhanced by a
CaMKII-dependent process during the LTP induction protocol.
If this occurs, this enhancement might be necessary for LTP induction.
In general, little is known about the time-dependent changes in the
NMDA conductance during LTP induction, but it is suspected that it is
upregulated during induction through a PKC- and Src-dependent process
and that blocking this upregulation blocks LTP induction
(W. Y. Lu et al. 1999
; Y. M. Lu et al.
1998
; Yu and Salter 1999
). However, it is also
possible that the NMDA conductance could be upregulated during LTP
induction by a CaMKII-dependent phosphorylation of the NMDA channel or
indirectly through the cascade (Weng et al. 1999
)
initiated by the CaMKII-dependent phosphorylation of SynGap (Chen et al. 1998
).
There appear to be several CaMKII substrates crucial for LTP induction
that are downstream from Ca2+ entry through the
NMDA channel. First, during induction of LTP, CaMKII phosphorylates the
GluR1 subunit of AMPA channel (on S831) (Barria et al.
1997; Lee et al. 2000
; Mammen et al.
1997
), and this causes the AMPA channel conductance to increase
(Benke et al. 1998
; Derkach et al. 1999
).
Second, new AMPA channels appears to be inserted into the synapse
during LTP induction and this insertion is CaMKII dependent
(Hayashi et al. 2000
; Maletic-Savatic et al.
1998
). This last process, however, is not due to
CaMKII-dependent phosphorylation of S831, indicating that other targets
of CaMKII must be involved (Hayashi et al. 2000
). Third,
it appears that CaMKII is responsible for the addition of new synapses
after LTP induction (Toni et al. 1999
). This idea is
consistent with the data suggesting the role of CaMKII in restructuring
of dendritic branches, synapse shape, and synapse number (Koh et
al. 1999
; Rongo and Kaplan 1999
; Wu and
Cline 1998
).
Despite our evidence against a role for CaMKII activity in the
maintenance of LTP, there remain some reasons for suspecting that
CaMKII does play a role: biochemical experiments show a persistent activation of CaMKII and a persistent phosphorylation of GluR1 (Barria et al. 1997; Fukunaga et al. 1993
,
1995
; Lee et al. 2000
; Ouyang et al.
1997
, 1999
). Why then, do our data not provide any support for
the functional role of this persistent activity? Several hypotheses
need to be considered. One possibility is that the relevant phosphatase
activity may still be too low and that higher Ca2+ concentrations than we have tested in our
experiments (0.3 µM) are required to activate the phosphatase.
Indeed, it has been suggested that calcium elevation as much as ~0.8
µM (Yang et al. 1999
) are needed to induce LTD, which
is known to involve phosphatase activation. These considerations
emphasize that that the mechanisms maintaining low phosphatase activity
could be as important for LTP maintenance as mechanisms maintaining the
kinase activity. There are in fact indications that the basal
phosphatase activity decreases after induction of LTP and therefore can
contribute to LTP maintenance (Blitzer et al. 1998
;
Fukunaga et al. 2000
; Klann and Thiels
1999
). Another possibility is that there is a redundant kinase
(PKC) that phosphorylates the LTP target even after CaMKII is inhibited
(Feng 1995
; Wang and Feng 1992
;
Wang and Kelly 1996
, but see Malgaroli et al.
1992
). Finally, it cannot be excluded that constitutive CaMKII
activation and GluR1 phosphorylation do not occur at synapses and are
thus not directly relevant to synaptic function. More work is needed to
distinguish among these possibilities.
![]() |
ACKNOWLEDGMENTS |
---|
We thank Dr. Simon Levy for measuring Ca2+ concentration in the internal solution and Dr. Leslie Griffith for providing AC3-control peptide.
Present address of S. Strack: Dept. of Pharmacology, University of Iowa, 2-432 BSB, Iowa City, IA 52242.
![]() |
FOOTNOTES |
---|
Address for reprint requests: J. E. Lisman (E-mail: Lisman{at}brandeis.edu).
Received 29 September 2000; accepted in final form 18 December 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|