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The Journal of Neuroscience, June 15, 2000, 20(12):4452-4461
In CA1 Pyramidal Neurons of the Hippocampus Protein Kinase C
Regulates Calcium-Dependent Inactivation of NMDA Receptors
Wei-Yang
Lu,
Michael F.
Jackson,
Donglin
Bai,
Beverley
A.
Orser, and
John F.
MacDonald
Departments of Physiology and Pharmacology, University of Toronto,
Toronto, Ontario M5S 1A8 Canada
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ABSTRACT |
The NMDA subtype of the glutamate-gated channel exhibits a
high permeability to Ca2+. The influx of
Ca2+ through NMDA channels is limited by a rapid and
Ca2+/calmodulin (CaM)-dependent inactivation that
results from a competitive displacement of cytoskeleton-binding
proteins from the NR1 subunit of the receptor by
Ca2+/CaM (Zhang et al., 1998 ; Krupp et al., 1999).
The C terminal of this subunit can be phosphorylated by protein kinase
C (PKC) (Tingley et al., 1993). The present study sought to investigate whether PKC regulates Ca2+-dependent inactivation of
the NMDA channel in hippocampal neurons. Activation of endogenous PKC
by 4 -phorbol 12-myristate 13-acetate enhanced peak
(Ip) and depressed steady-state
(Iss) NMDA-evoked currents, resulting
in a reduction in the ratio of these currents (Iss/Ip).
We demonstrated previously that PKC activity enhances IP via a sequential activation of the focal
adhesion kinase cell adhesion kinase /proline-rich tyrosine kinase 2 (CAK/Pyk2) and the nonreceptor tyrosine kinase Src (Huang et
al., 1999; Lu et al., 1999). Here, we report that the PKC-induced
depression of Iss is unrelated to the
PKC/CAK/Src-signaling pathway but depends on the concentration of
extracellular Ca2+. Intracellular applications of
CaM reduced
Iss/Ip and
occluded the Ca2+-dependent effect of phorbol esters
on Iss. Moreover, increasing the
concentration of intracellular Ca2+ buffer or
intracellular application of the inhibitory CaM-binding peptide
(KY9) greatly reduced the phorbol ester-induced depression of
Iss. Taken together, these results suggest
that PKC enhances Ca2+/CaM-dependent inactivation of
the NMDA channel, most likely because of a phosphorylation-dependent
regulation of interactions between receptor subunits, CaM, and other
postsynaptic density proteins.
Key words:
NMDA receptor; desensitization; Ca2+-dependent inactivation; calmodulin; PKC; phosphorylation; hippocampal neurons
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INTRODUCTION |
NMDA receptors constitute a major
class of the glutamate-gated ion channels that play a key role in
synaptic development and plasticity in the CNS. Via their
interactions with specific anchoring proteins, NMDA channels are
clustered at appropriate positions within the postsynaptic density
(PSD) where the regulatory protein calmodulin (CaM) can interact with
the channel subunits to modulate channel gating (Ehlers et al.,
1996a,b; Zhang et al., 1998; Krupp et al., 1999). The molecular
organization of the PSD is essential for the precise localization of
NMDA channels and is also important for the efficient transduction of
intracellular signaling events that can be triggered after NMDA
receptor activation (Swope et al., 1999).
NMDA receptors are oligomeric complexes comprised of three families of
subunits: NR1, NR2A D, and the newly identified NR3 subunit (Moriyoshi
et al., 1991; Monyer et al., 1992; Yamakura and Shimoji, 1999). Most
native NMDA receptors are thought to be heteromers composed of various
combinations of NR1 and NR2 subunits (Sheng et al., 1994). Eight
different splice variants of the NR1 subunits arising from alternative
splicing of one short cassette in the N terminal (N1) and two cassettes
in the C terminal (C1 and C2) have also been identified (Hollmann and
Heinemann, 1994; Bennett and Dingledine, 1995). All of the NR1 splice
variants contain a common C0 region just proximal to the C
terminal C1 and C2 cassettes (Ehlers et al., 1998). These NR1 splice
variants exhibit differences in their spatial and developmental
expression patterns in the CNS (Laurie and Seeburg, 1994) and their
subcellular localization in heterologous expression systems (Ehlers et
al., 1995), as well as their agonist and antagonist potencies (Yamakura and Shimoji, 1999).
The NR1 subunit is directly phosphorylated by PKC (Tingley et al.,
1993), and activation of this kinase enhances NMDA-evoked currents in
isolated trigeminal neurons (Chen and Huang, 1992) and in isolated and
cultured hippocampal neurons (Xiong et al., 1998), as well as in
Xenopus oocytes injected previously with either brain mRNA
(Kelso et al., 1992) or cDNAs for recombinant NMDA receptor subunits
(Zukin and Bennett, 1995). Further analysis has shown that the
PKC-induced phosphorylation occurs primarily but not exclusively at
four serine residues located in the C1 cassette of the NR1 subunit
(Tingley et al., 1993). Interestingly, recombinant receptors lacking
the C1 cassette demonstrate a greater phorbol ester-induced
potentiation than do those containing this cassette (Durand et al.,
1993), suggesting that the potentiation by PKC is unrelated to
phosphorylation of these residues (Yamakura et al., 1993). In this
regard, we have demonstrated recently that phorbol esters enhance
NMDA-evoked currents in CA1 pyramidal neurons via a sequential
activation of PKC, the focal adhesion kinase cell adhesion kinase
/proline-rich tyrosine kinase 2 (CAK/Pyk2) (Huang et al., 1999),
and the nonreceptor tyrosine kinase Src rather than as a consequence of
the direct phosphorylation of NMDA receptors by PKC (Lu et al.,
1999).
NMDA channels exhibit a high permeability to
Ca2+ relative to most AMPA channels,
and this Ca2+ signal underlies much of the
NMDA receptor-dependent plasticity and neurotoxicity in the CNS.
However, the influx is limited by a rapid
Ca2+- and CaM-dependent channel
inactivation (Mayer and Westbrook, 1985; MacDermott et al., 1986;
Rosenmund and Westbrook, 1993; Rosenmund et al., 1995). Additionally,
the influx of Ca2+ may activate the
phosphatase calcineurin resulting in a downregulation of NMDA channels
(Lieberman and Mody, 1994) and an increased expression of
glycine-insensitive desensitization of NMDA receptors (Tong and Jahr,
1994). In turn, the potential dephosphorylation of NMDA receptors may
be counterbalanced by the serine-threonine kinases cAMP-dependent
kinase (PKA) (Raman et al., 1996) or casein kinase-II (Lieberman and
Mody, 1999).
Recent studies have shown that the Ca2+-
and CaM-dependent negative feedback requires regulation of
protein-protein interactions involving NMDA receptor subunits
themselves. Specifically, the C0 region of the NR1 subunit
competitively binds the actin-associated protein  -actinin2
(Wyszynski et al., 1997) as well as CaM (Ehlers et al., 1996b).
Inactivation may occur as a consequence of a competitive displacement
of -actinin2 by Ca2+/CaM or
alternatively by a Ca2+-dependent
reduction in the affinity of -actinin2 for this site on the receptor
(Zhang et al., 1998; Krupp et al., 1999). The phosphorylation of serine
residues in the C1 cassette is also correlated with dispersion of
surface-associated clusters of NR1 subunits expressed in fibroblasts
(Ehlers et al., 1995, 1996a). Furthermore, the binding of the
cytoskeletal protein spectrin to the C terminal of the NR1 receptor is
also inhibited by PKC-dependent phosphorylation of this subunit
(Wechsler and Teichberg, 1998). Therefore the present experiments
examine the hypothesis that PKC-dependent phosphorylation directly
regulates Ca2+-dependent inactivation of
NMDA receptors.
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MATERIALS AND METHODS |
Isolated neurons and whole-cell recordings of NMDA-evoked
currents. CA1 neurons were isolated from hippocampal slices taken from postnatal rats (Wistar; 12-20 d old) using previously described procedures (Wang and MacDonald, 1995). Briefly, the rats were anesthetized with halothane and killed by decapitation using a guillotine. The whole brain was removed and placed in cold (4°C) extracellular solution (see below for composition). Hippocampi were
then microdissected and cut by hand into 400- to 500-µm-thick slices
using a razor blade. The slices were incubated at room temperature for
30 min in extracellular solution containing 0.3-0.5 mg/ml
papain (from papaya latex; Sigma, St. Louis, MO). The slices were then
washed and kept in enzyme-free solution until used. All solutions were
bubbled with 100% O2. One slice was transferred into a 35 mm culture dish to facilitate isolation of neurons. The CA1
region was excised from the slice, and two polished glass pipettes were
used to isolate single cells mechanically. Only neurons that retained
their pyramidal shape, including a major primary and several secondary
dendritic processes, were used for recordings. Recordings from control
and drug-treated cells were always made on the same day.
The extracellular solution was composed of 140 mM
NaCl, 1.3 mM CaCl2, 5.0 mM KCl, 25 mM HEPES, 33 mM glucose,
0.0005 mM TTX, and 3-10 µM glycine, with a
pH of 7.4 and osmolarity between 325 and 335 mOsm. In some
experiments the extracellular concentration of
CaCl2 was changed as indicated. In addition, 10 µM EDTA was occasionally added in the extracellular
solution to chelate contaminating concentrations of
Zn2+. Whole-cell patch-clamp recordings
were performed at room temperature (20-22°C). Neurons were
voltage-clamped and lifted into the stream of the extracellular
solution supplied by a computer-controlled multibarreled perfusion
system (Warner). Currents were evoked by rapid application of NMDA (300 µM; unless otherwise indicated,  of exchange was ~2
msec). The resistance of patch pipettes used in the study was 3-4
M . To monitor series resistance, a voltage step of  10 mV was
applied before each application of NMDA. The series resistance in the
recordings was between 6 and 8 M. Recordings in which series
resistance varied by >10% were rejected. No electronic compensation
for series resistance was used. The intracellular solution contained
140 mM CsMeSO4 or CsF, 11 mM EGTA, 1 mM
CaCl2, 2 mM
MgCl2, 10 mM
HEPES, 2 mM TEA, and 4 mM K2ATP, with a pH of 7.3 and osmolarity between 295 and 300 mOsm. In certain experiments, 11 mM EGTA was replaced with 20 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid
(BAPTA) as indicated. Drugs used in this study were purchased from
Sigma or as follows: 4-phorbol 12-myristate 13-acetate (4-PMA), 4-PMA, and chelerythrine from Alexis; pp60c-Src from Upstate Biochemicals; and Src(40-58) and sScr(40-58) from Dr. M. W. Salter (Hospital for Sick Children, Toronto, Ontario, Canada).
CaM-binding peptide (CaM inhibitory peptide, KY9) and its control
peptide (KY8) were a gift from Dr. K.-W. Yau. PMA and chelerythrine
were dissolved in dimethylsulfoxide (DMSO), and stock solutions were made to 10 mM concentration. All stock solutions
of drugs were kept at 25 or 70°C and thawed only before the
experiment. Final concentrations of DMSO used in this study were kept
to <0.01%.
Currents were recorded using an Axopatch 1-B amplifier, and data were
filtered at 2 kHz, digitized, and acquired using the pClamp6 program
(Axon Instruments). All population data are expressed as the mean ± SEM. The Student's paired t test or the ANOVA test was
used whenever appropriate.
Nucleated patches from cultured hippocampal neurons and ultrafast
perfusion. Nucleated patches (Sather et al., 1992) taken from
cultured hippocampal neurons were used in conjunction with an ultrafast
perfusion system. This system consisted of a Burleigh piezoelectric
actuator and  -shape perfusion tubing, the use of which allowed a
more rapid delivery of the agonist. Changes of solution could be
achieved within 0.4 msec.
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RESULTS |
Activation of PKC enhances peak and depresses steady-state
NMDA-evoked currents
In acutely isolated hippocampal pyramidal neurons, rapid
applications of NMDA (300 µM; glycine, 3 µM) evoked a peak current (Ip) that rapidly desensitized or
inactivated to a steady-state value
(Iss) (Fig.
1A). The
steady-state-to-peak ratio
(Iss/Ip) of the current has been used previously as a measure of the degree of
desensitization or inactivation of NMDA channels (Sather et al., 1992;
Lu et al., 1998; Zhang et al., 1998). This ratio decreased by ~8%
over the first 15-20 min of recording (Fig. 1B),
reflecting a small and time-dependent increase in the extent of NMDA
receptor desensitization (Sather et al., 1992). Among the cells
examined this ratio ranged from 0.45 to 0.6 when the control
intracellular solution was used.
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Figure 1.
Activation of endogenous PKC in
hippocampal pyramidal neurons induces a complex modulation of
whole-cell NMDA-evoked currents. A1, In an acutely
isolated CA1 pyramidal neuron, currents were evoked by NMDA (300 µM) before and during bath application (indicated by
horizontal bar) of 4-PMA (100 nM). Note that the peak
(Ip) increased while the steady state
(Iss) decreased after PMA.
A2, The same effect is observed with applications of
PDD. Right, Currents were normalized to the peak
recorded after PDD. B, The inactive form of the phorbol
ester 4-PMA (100 nM) was ineffective
(middle) in contrast to 4-PMA (right)
applied to the same neuron. C, Intracellular application
of the PKC inhibitor chelerythrine (10 µM) did not affect
the current itself but blocked the effect of 4-PMA
(right; treated with 4-PMA for 6 min). The
dashed horizontal lines in A-C indicate
steady-state current level during control. D, Population
data summarize changes in
Iss/Ip
over the time course of the recording from four groups of cells. Under
control conditions (gray squares)
Iss/Ip
decreased slightly during the first 6-15 min of the recording and then
stabilized (n = 7). Applications of 4-PMA
(black circles) reduced
Iss/Ip
from 0.49 ± 0.08 in control to 0.26 ± 0.0751 15 min after
application of PMA (n = 12; two-way ANOVA,
p < 0.002). In contrast, 4-PMA
(gray triangles) had no effect
(n = 4). The effect of 4-PMA on
Iss/Ip was
reduced by inclusion of chelerythrine (10 µM;
white squares) in the patch pipette
(n = 6; two-way ANOVA, p < 0.01). E, Responses of nucleated patches to rapid
applications of NMDA (100 µM) and bath-applied PMA are
shown. E1, Two superimposed responses of a nucleated
patch taken from a cultured hippocampal neuron are shown.
E2, 4-PMA enhanced Ip to
131 ± 11% of control and depressed
Iss to 59 ± 4% of control
(n = 6; p < 0.05).
E3, This phorbol ester reduced
Iss/Ip
from 38 ± 0.7 in control to 17 ± 0.4 (n = 6; p < 0.05).
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The active phorbol ester 4-PMA (100 nM) was used
to activate endogenous PKC. Peak currents (Fig. 1A)
were enhanced by 8-35% (12 ± 6.6%; n = 12 cells analyzed) 5-8 min after exposure to PMA. The degree of
enhancement was relatively small because of the high concentrations of
NMDA and glycine used in these experiments (Lu et al., 1999). In
contrast, Iss was reduced by 32-71%
(51 ± 12%; n = 12; Fig. 1D)
resulting in a reduction in
Iss/Ip
(control, Iss/Ip = 0.49 ± 0.08; PMA,
Iss/Ip = 0.26 ± 0.07; n = 12 in both groups; Fig.
1A). The same effects were observed after
applications of a different phorbol ester, phorbol 12,13-didecanoate
(PDD; 1 µM; Fig. 1A2),
whereas the inactive analog of PMA, 4-PMA, was without effect on
NMDA-activated currents (Fig. 1B,D). To confirm that
the effects of PMA were caused by activation of endogenous PKC, we
performed a series of recordings with or without chelerythrine, a
selective inhibitor of PKC (Lu et al., 1999), in the patch pipette. Chelerythrine did not change the NMDA-evoked currents but prevented both the enhancement of IP and the
depression of Iss by PMA (Fig. 1C,D).
We also examined the effects of PMA on the responses of nucleated
patches to rapid applications of NMDA. This was done in case we were
underestimating the actual values of
Ip because of a substantial
desensitization of the receptors occurring during the onset of agonist
application. Confirming that this was unlikely to be the case, PMA
applications to nucleated patches modified NMDA-evoked currents
similarly to what was observed in whole-cell recordings (Fig.
1E).
The PKC-induced potentiation of Ip is
dependent on activation of Src, but the depression of
Iss is not
NMDA receptor-mediated currents
(Ip) in CA1 neurons are upregulated by
several G-protein-coupled receptors via the sequential activation of PKC and then the tyrosine kinases CAK/Pyk2 (Huang et
al., 1999) and Src (Lu et al., 1999). Instead of a Src-dependent downregulation of Iss, we hypothesized
that the depression was the result of a more direct effect of PKC on
NMDA channel function. To test this possibility we first perfused
pp60c-Src into the patch pipette while recording NMDA-activated
currents. As shown in Figure 2,
A and B, this kinase enhanced
Ip of the NMDA-evoked current to
131 ± 9.0% (n = 9) but caused no reduction in
Iss. Although, inclusion of Src in the
patch pipette occluded the ability of PMA to potentiate
Ip, it failed to prevent the
depression of Iss (Fig.
2A).
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Figure 2.
The phorbol ester-induced depression of
Iss was independent of Src and CAK/Pyk2
activity. A, An example of NMDA-evoked currents recorded
from an isolated neuron when pp60c-Src was included in the patch
pipette is shown. Times after the beginning of the whole-cell
configuration are indicated above the
traces. B, Similar recordings for a group
of such cells (n = 7) are summarized in this plot.
Inclusion of Src enhanced the peak (black
circles) but did not depress steady-state
(gray squares) currents. Currents
in each cell were normalized to the first response 1-2 min after
establishment of the whole-cell configuration. C,
4-PMA depressed Iss with
(gray squares) or without
(black circles) inclusion of the Src
inhibitory peptide Src(40-58) in the patch pipette (25 µg/ml).
Fifteen minutes after exposure to 4-PMA,
Iss with Src(40-58) was 42 ± 5%
(n = 7), whereas Iss
without Src(40-58) was 39 ± 7% (n = 5;
p > 0.05). D, Intracellular
application of CAK/Pyk2 (0.5 µg/ml) did not reduce
Iss, nor did it prevent the
depression of Iss by 4-PMA.
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We demonstrated previously that intracellular applications of the Src
inhibitory peptide Src(40-58) prevented both the PMA- and Src-induced
potentiation of Ip in these neurons
(Lu et al., 1999). However, inclusion of this peptide in the patch
pipette (25 µg/ml) failed to alter the PMA-induced depression of
Iss (Fig. 2C), nor did it
prevent the decrease in
Iss/Ip
(data not shown). Similarly, intracellular applications of CAK/Pyk2
failed to alter the PMA-induced depression of
Iss (Fig. 2D). Taken
together, these results demonstrate that the PMA-induced depression of
Iss is independent of the activity of
either CAK/Pyk2 or Src.
Application of PMA enhanced the apparent desensitization of
NMDA responses
The PMA-induced potentiation of
Ip is relatively small in saturating
concentrations of NMDA and glycine whereas the depression of
Iss is most prominent (Lu et al.,
1999) (Fig. 3A), suggesting a
potentiation of desensitization (Sather et al., 1992; Lu et al., 1998).
NMDA-evoked responses demonstrate at least three different forms of
desensitization or inactivation: (1) a
Ca2+-dependent inactivation, (2) a
glycine-sensitive desensitization, and (3) a glycine-independent
desensitization (McBain and Mayer, 1994). Initially, we investigated
whether or not PMA had simply increased
Ip, driving more of the receptors into
the desensitized state and causing a relative reduction in the
amplitude of Iss. Using a fixed
concentration of NMDA, we examined the relationship between the
PMA-induced depression of Iss and
the potentiation of Ip. With
increasing concentrations of glycine, the degree of potentiation of
Ip decreased (Fig. 3A,B),
and the depression of Iss increased
(Fig. 3A,C). The greatest depression of
Iss was observed in saturating
concentrations of glycine [also seen in saturating concentrations of
NMDA (Lu et al., 1999)]. To assess quantitatively the relationship
between the degree of depression and the size of the peak currents, we
plotted Iss against
Ip (Fig. 3D) both before
and after applications of PMA. Peak and steady-state components of the
evoked currents could be distinguished with glycine concentrations
ranging from 300 to 3000 µM. As shown in Figure
3D the relationship between
Iss and
Ip was linear, and the slope of this
relationship (ratio of Iss to
Ip) was reduced by treatment of the
cells with PMA (control, 0.7; PMA, 0.3; n = 7). This
analysis suggests that the PMA-induced depression of Iss was unrelated to the absolute
amplitude of Ip and indicates that the
reduction of Iss is unlikely to have
been simply a consequence of an enhanced glycine-sensitive
desensitization. Nevertheless, to minimize any potential effects of PMA
on glycine-sensitive desensitization, we used saturating
concentrations of glycine in most of the subsequent recordings.
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Figure 3.
The PMA-induced depression of
Iss was greatest in the presence of
saturating concentrations of glycine. A, Responses of an
isolated neuron to applications of various concentrations of glycine in
the presence of a fixed concentration (50 µM) of NMDA are
shown before (top) and after (bottom)
bath application of PMA. B, The normalized
IP is plotted against glycine concentration
before (black squares) and after
(gray squares) applications of PMA
(n = 7). Currents were normalized to the response
to 10,000 nM glycine. C, A similar plot of
Iss is shown with currents normalized to
that evoked by 30,000 nM glycine. Note the greater
depression of the Iss in the presence of
higher concentrations of glycine. D, For the same group
of recordings shown in B and C,
Iss was plotted against
Ip before (Control,
black circles) and after
(gray circles) PMA. The
relationship between Iss and
Ip was linear. The slope of the
Iss-Ip
relationship was reduced from 0.7 to 0.3 after treatment of the cells
with PMA.
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Depression by PKC of Iss is dependent on
extracellular calcium
An alternative explanation for the PMA-induced depression of
Iss is an enhancement of
Ca2+ inactivation. We therefore tested the
effect of PMA on Ip and Iss in the presence of three different
concentrations of extracellular Ca2+ (0.5, 1.3, and 5.0 mM
[Ca2+]e) with the
objective of increasing the influx of Ca2+
through NMDA channels. At first we examined responses to relatively low
concentrations of NMDA, mimicking previous protocols used in cultured
cells (Zhang et al., 1998). Extracellular
Ca2+ depressed the amplitude of the
currents as reported previously (Ascher and Nowak, 1988; Lieberman and
Mody, 1994). However, unlike cultured cells (Zhang et al., 1998)
minimal time- and Ca2+-dependent
inactivation was observed in isolated CA1 neurons until PMA was
applied. Furthermore, applications of PMA enhanced the entire response
to applied NMDA (Fig. 1A1), and the steady-state current recorded in the presence of PMA was actually larger than the
entire corresponding control response. The
Ca2+ dependency of the PMA-induced
inactivation was quantified by plotting
Iss/Ip
against [Ca2+]e
(Fig. 4A2) and using
the slope of this relationship as a measure of its
Ca2+ sensitivity (control
k = 0.012 mM 1;
n = 5; PMA k = 0.045
mM1; n = 6; two-way ANOVA,
p < 0.05). We therefore continued to examine responses
to near-saturating concentrations of NMDA in which there are smaller
PMA-induced increases in Ip and large
decreases in Iss.
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Figure 4.
The PMA-induced depression of
Iss was dependent on extracellular calcium.
A1, B1, In the presence of three
different concentrations of extracellular Ca2+
(indicated above the traces), currents
were evoked by a low concentration (10 µM;
A1) or high concentration (300 µM;
B1) of NMDA before (black
traces) and after (gray
traces) PMA. A2, Using the low
concentration of NMDA, the slope of the
Iss/Ip to
[Ca2+]e relationship was significantly
enhanced by PMA (control, black
circles; slope = 0.012
mM1
[Ca2+]e; PMA,
gray squares; slope = 0.045
mM1
[Ca2+]e). B2,
Superimposed traces of the currents before and after PMA
(NMDA, 300 µM) were normalized to their peaks.
C, Potentiation of Ip by PMA
was independent of [Ca2+]e (0.2 mM Ca2+, 26 ± 3%;
n = 6; 1.3 mM Ca2+,
22 ± 4%; n = 6; 5.0 mM
Ca2+, 23 ± 4%; one-way ANOVA,
p = 0.25; n = 6).
D, Depression of Iss was
dependent on [Ca2+]e (0.2 mM Ca2+, control, 0.54 ± 0.06; PMA, 0.43 ± 0.03; 1.3 mM
Ca2+, control, 0.53 ± 0.02; PMA,
0.34 ± 0.03; 5.0 mM
Ca2+, control, 0.49 ± 0.08; PMA,
0.16 ± 0.04; one-way ANOVA, p < 0.001;
n = 6). E, The slope of the
Iss/Ip to
[Ca2+]e relationship was significantly
enhanced by PMA (Control, black
circles; slope = 0.021
mM1
[Ca2+]e; PMA,
gray squares; slope = 0.055
mM1
[Ca2+]e). Con,
Cont, Control.
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Applications of PMA proportionately enhanced
Ip to a similar degree in all three
tested values of
[Ca2+]e (Fig.
4B,C), whereas the depression of
Iss was proportionally greater as
[Ca2+]e was
increased (Fig. 4B,D). The slope of the
Iss/Ip
against [Ca2+]e
relationship was more than doubled after treatment of cells with PMA
(before PMA, k = 0.021
mM1
[Ca2+]e; after
PMA, k = 0.055
mM1
[Ca2+]e;
n = 6; two-way ANOVA, p < 0.05; Fig.
4E). The enhanced
Ca2+-dependent inactivation occurred in
spite of the greater charge transfer and influx of
Ca2+ associated with the control responses
(Fig. 4B1, 5.0 mM
Ca2+) compared with those after PMA
treatment. This suggests that we are likely underestimating the
PMA-induced enhancement of the sensitivity of inactivation because the
size of the calcium signal is less after treatment with PMA than
before. In one group of cells (n = 5) the effects of
PMA on
Iss/Ip
were also examined in the absence of added
Ca2+. Applications of PMA still enhanced
Ip by ~25%, but there was no
depression of Iss (data not shown).
These results also support the hypothesis that the PMA-induced
depression of Iss results from an
enhancement of the sensitivity of Ca2+ inactivation.
Buffering intracellular Ca2+ alters the
PMA-induced depression of Iss
To test the hypothesis that the PMA-induced depression of
Iss results from a modification of
Ca2+ inactivation of NMDA channels, we
made a series of recordings with or without the
Ca2+ buffer EGTA (11 mM) in the patch pipette. In recordings made with
EGTA, applications of PMA enhanced Ip
but had little effect on Iss evoked by
applying relatively intermediate concentrations of agonist and
coagonist (50 µM NMDA and 0.5 µM glycine; Fig. 5A). In contrast, in the
absence of EGTA PMA induced a clear depression of
Iss (Fig. 5B).
Consequently, PMA caused a larger reduction in
Iss/Ip
in recordings without EGTA than in those with EGTA (Fig. 5C), indicating that buffering the influx of
Ca2+ reduced the degree of depression of
Iss. We then examined whether or not a
Ca2+ buffer would block the PMA-induced
depression of Iss recorded in the
presence of a near-saturating concentration of agonist and coagonist
(300 µM NMDA and 3 µM
glycine). To achieve this we used the more rapid
Ca2+ buffer BAPTA (20 mM) in the patch pipette. As shown in Figure 5D the PMA-induced depression of
Iss/Ip
was substantially reduced by BAPTA (two-way ANOVA, p < 0.002; Fig. 5D).
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Figure 5.
Intracellular buffering of
Ca2+ reduced the PMA-induced depression of
Iss. A, With 11 mM EGTA in the patch pipette, NMDA (50 µM;
glycine, 0.5 µM)-evoked currents demonstrated a
potentiation of Ip but no depression of
Iss after applications of PMA.
B, When EGTA was not added to the pipette, both an
enhancement of Ip and a depression of
Iss were observed. C,
PMA-induced changes in
Iss/Ip
with or without EGTA in the pipette are summarized in this graph (with
EGTA, 0.47 ± 0.04; n = 6; without EGTA,
0.28 ± 0.04; n = 5; p < 0.01). D, A plot of
Iss/Ip
recorded from two groups of cells with (gray
squares; n = 6) or without
(black circles; n = 5) inclusion of 20 mM BAPTA in the patch pipette is shown.
Currents were evoked by nearly saturating concentrations of agonist and
coagonist (300 µM NMDA; 3 µM glycine) in
the presence of 1.3 mM
[Ca2+]e. 4-PMA was applied 8-15
min after establishment of the whole-cell patch configuration. The
PMA-induced depression of
Iss/Ip was
reduced (two-way ANOVA, p < 0.002) with the
inclusion of intracellular BAPTA. E, NMDA-evoked
currents in the same neuron at holding potentials ranging from 60 to
40 mV are shown. Top, In the presence of 1.3 mM [Ca2+]e responses
before (Control) and during application of
4-PMA are illustrated. Bottom, Normalized values
of Iss/Ip
before (black circles) and after
(gray squares) PMA are plotted
against the holding potential (Em). The
PMA-induced reduction in
Iss/Ip was
reduced at depolarized potentials.
|
|
We also examined the relationship between
Iss and
Ip under conditions of altered
Ca2+ driving force to confirm the
requirement of an influx of Ca2+ through
NMDA channels. Before applying PMA (the intracellular solution
contained 11 mM EGTA), the values of
Iss/Ip
for the NMDA-evoked current were approximately the same at all holding
potentials demonstrating that little Ca2+
inactivation was present. After treatment of the cells with PMA, the
proportionate increase in Ip was the
same at all potentials, but there was a disproportionate decrease in
Iss at negative holding potentials
(Fig. 5E), conditions that favor a greater influx of Ca2+ via the channels. These results
further support the interpretation that the PMA-induced reduction of
Iss/Ip
is caused by enhancement of Ca2+ inactivation.
CaM depresses Iss and occludes the effects
of PMA
The binding of Ca2+/CaM to the NR1
subunit of the NMDA receptor is thought to underlie
Ca2+ inactivation of NMDA receptors (Zhang
et al., 1998). Therefore, we added CaM (50-100 nM) to the
patch pipettes and reexamined the effects of PMA in the presence of
0.2, 1.3, or 5.0 mM
[Ca2+]e (Fig.
6A). Addition of CaM
caused a reduction of
Iss/Ip
when compared with appropriate control responses. Furthermore, the slope of the
Iss/Ip-[Ca2+]e
relationship was increased from 0.012
mM1 (without CaM)
to 0.031 mM1
(Fig. 6B), demonstrating an enhanced sensitivity of
the inactivation to Ca2+. The subsequent
addition of PMA further depressed
Iss/Ip
but occluded the ability of PMA to enhance the
Ca2+ sensitivity of inactivation (Fig.
6D,E).
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Figure 6.
Intracellular applications of CaM
suppressed Iss and occluded the PMA-induced
and Ca2+-dependent reduction of
Iss/Ip.
A, Example traces of original
(top) and normalized (to peak; bottom)
NMDA currents in the presence of three different
[Ca2+]e. Currents before
(thick lines) and after
(thin lines) PMA are shown with CaM (50 nM) in the patch pipettes. B, Ratios of
peak-to-steady-state currents recorded with the inclusion of CaM
(gray circles;
n = 6) are compared with similar recordings without
CaM (Control, black
circles; the same data used in Fig.
4D) and are plotted against
[Ca2+]e. CaM reduced
Iss/Ip in
a [Ca2+]e-dependent manner (0.2 mM [Ca2+]e,
Control, 0.55 ± 0.05; CaM, 42 ± 0.06; 1.3 mM
[Ca2+]e,
Control, 0.53 ± 0.02; CaM, 38 ± 0.05; p < 0.05; 5.0 mM
[Ca2+]e,
Control, 0.49 ± 0.08; CaM, 27 ± 0.06; p < 0.05). CaM enhanced the slope of the
Iss/Ip-[Ca2+]e
relationship from 0.012 mM1
[Ca2+]e in control
(thin line) to 0.031
mM1 [Ca2+]e
(thick line). C, PMA
enhanced Ip in the presence
(+CaM, black bars;
n = 6) or absence (CaM,
white bars; the same data used in Fig.
4B) of CaM, and the degree of potentiation of
Ip was similar at each
[Ca2+]e. D, In
contrast, intracellular CaM (black bars;
n = 6) enhanced the PMA-induced depression of
Iss in the presence of low
[Ca2+]e, while causing no
further depression in the presence of high
[Ca2+]e. E, With CaM in
the patch pipettes,
Iss/Ip
recorded before (CaM, gray
circles) and after (CaM + PMA,
black squares) PMA treatment is plotted
against [Ca2+]e. Note that in the
presence of CaM, PMA induced only a parallel shift of the
Iss/Ip-[Ca2+]e
relationship (PMA, 0.031 mM1
[Ca2+]e; +PMA, 0.03 mM1
[Ca2+]e; n = 6), demonstrating that CaM occludes the
Ca2+-dependent inactivation of these currents.
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|
We then examined the potential role of endogenous CaM in the
PMA-induced depression of Iss. A
series of recordings was made using patch pipettes containing the
inhibitory CaM-binding peptide KY9 or its ineffective control peptide
KY8 (Liu et al., 1994). In the absence of PMA no changes in
Iss/Ip
were observed over 30 min of recording regardless of whether KY9 or KY8
was included in the patch pipettes (Fig.
7A). The ratio of
Iss to
Ip did decrease by ~11% (Fig.
7B) consistent with the time-dependent change in desensitization observed in control recordings (see Fig.
1B). In contrast, when PMA was applied the depression
of Iss was blocked in the presence of
KY9 (200-300 µM), and PMA reduced
Iss/Ip
by only 18% (before PMA,
Iss/Ip = 0.5 ± 0.03; after PMA,
Iss/Ip = 0.41 ± 0.03; Fig. 7C,E). In recordings with KY8 (300 µM) this ratio was reduced by ~42% (before
PMA,
Iss/Ip = 0.48 ± 0.05; after PMA,
Iss/Ip = 0.28 ± 0.04; Fig. 7D,F).
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Figure 7.
The inhibitory CaM-binding protein
KY9, but not the control peptide KY8, blocked the PMA-induced
depression of Iss. A, Example
traces of NMDA-evoked currents recorded from the same
cell 5, 15, and 25 min after establishment of the whole-cell patch
configuration with KY9 in the pipette. B, Recordings
from two groups of cells, one with KY9 (black
circles; n = 4) and one with KY8
(gray squares;
n = 3).
Iss/Ip was
plotted against the time course of the recordings. C,
NMDA currents recorded from the same cell shown in A
(KY9). Left, Current recorded just before application of
PMA (32 min after establishment of the whole-cell configuration).
Middle, Current recorded after PMA treatment for 8 min.
Right, The two traces superimposed. Note
the blockade by KY9 of the PMA-induced depression of
Iss. D, NMDA-evoked currents
recorded from another cell with KY8 in the patch pipette.
Left, Current before PMA application and recorded 36 min
after establishment of the whole-cell configuration.
Middle, Current recorded after PMA treatment for 6 min.
Right, The two traces superimposed. Note
the depression of Iss by PMA.
E, Summary of a series of recordings of
Iss/Ip
with KY9 in the pipette before (control) and after PMA (control,
0.48 ± 0.03; PMA, 0.41 ± 0.03; n = 6).
F, In another group of cells,
Iss/Ip
recorded with KY8 (control, 0.45 ± 0.05; PMA, 0.28 ± 0.04;
p < 0.05; n = 5).
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We also included either the phosphatase inhibitor cyclosporin A (Tong
et al., 1995) or the Ca2+/CaM-dependent
kinase II (CamK-II) inhibitor KN-93 in pipettes to determine whether
the activity of these Ca2+-dependent
enzymes was required. The presence of cyclosporin A or KN-93 failed to
prevent the PMA-induced Ca2+-dependent
increase in the sensitivity of inactivation (data not shown).
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DISCUSSION |
Phorbol esters such as PMA induce a complex regulation of NMDA
receptors, acting to enhance Ip
and simultaneously depressing Iss
evoked by applications of agonist. Both actions of PMA are mediated via
activation of endogenous PKC because neither the enhancement nor the
depression was mimicked by an inactive phorbol and both actions were
blocked by chelerythrine. Previous experiments demonstrated that the
potentiation of Ip depends on
activation of Src (Lu et al., 1999) and CAK/Pyk2 (Huang et al.,
1999). In the present study we have confirmed that intracellular Src
enhances Ip but also show that it
fails to depress Iss. Furthermore, the potentiation of Ip was blocked by the
Src peptide Src(40-58), whereas the phorbol ester-induced depression of
Iss was not. Nor was the
phorbol-induced depression of Iss
replicated by intracellular applications of the focal adhesion kinase
CAK/Pyk2. These results clearly demonstrate that the depression of
Iss depends on activation of
endogenous PKC by a signaling cascade that bypasses the requirement for
activation of Src and CAK/Pyk2.
Glycine-independent desensitization
The major form of desensitization or inactivation of NMDA
receptors in isolated CA1 neurons is a glycine- and calcium-independent form of desensitization (Sather et al., 1992; McBain and Mayer, 1994;
Lu et al., 1998). This desensitization is accentuated as agonist or
coagonist concentrations are increased (see Fig. 2), suggesting a
concentration-dependent rate of entry into the desensitized state(s) of
the receptor (Sather et al., 1992). In contrast, a glycine-sensitive component of desensitization predominates in whole-cell recordings from cultured cortical and hippocampal neurons (Mayer et al., 1989). In prolonged whole-cell recordings from cultured hippocampal neurons, the glycine-sensitive
component of desensitization of NMDA-evoked currents is
gradually lost (McBain and Mayer, 1994), revealing the glycine-
and Ca2+-insensitive component of
desensitization (Sather et al., 1992; McBain and Mayer, 1994).
A similar loss of this component is observed after excision of
outside-out or nucleated patches (Sather et al., 1992; Tong and Jahr,
1994).
Generally, ratios of
Iss/Ip
were decreased by PMA under all conditions for which
Ip and
Iss could be distinguished, implying a
potential underlying alteration of glycine-independent desensitization. Furthermore, similar reductions in
Iss/Ip
were observed in cultured neurons after the intracellular perfusion of
a constitutively active PKC fragment (Xiong et al., 1998; Lu et al.,
1999). Therefore, the depression of
Iss by phorbol ester might be
accounted for on the basis of an enhancement of glycine-insensitive
desensitization. However, our experiments strongly argue that this can
be only a part of the explanation. For example, PMA depressed
Iss even when the potentiation of
Ip was blocked by a Src inhibitor, and Src itself enhanced Ip without
depressing Iss. The depressant effects
of PMA were also dependent on extracellular and intracellular concentrations of Ca2+ even though it is
generally assumed that glycine-independent desensitization is
Ca2+ independent (Sather et al., 1992;
McBain and Mayer, 1994).
Unlike the glycine-independent form of desensitization, the loss of the
glycine-dependent component of desensitization depends on intracellular
Ca2+ and the activity of
Ca2+-dependent phosphatase (PP2B or
calcineurin) (Tong and Jahr, 1994). Previously it was shown that the
entry of Ca2+ through NMDA receptors acts
via calcineurin to downregulate single-channel activity in
cell-attached patch recordings from acutely isolated dentate gyrus
neurons (Lieberman and Mody, 1994). An elevation of intracellular
Ca2+ also accelerated the appearance of
glycine-independent desensitization, and inhibitors of calcineurin
retarded glycine-independent desensitization in cultured hippocampal
neurons (Tong and Jahr, 1994). In contrast, neither activated PKC nor
autophosphorylated CamK-II was able to regulate glycine-independent
desensitization (Tong and Jahr, 1994). Calcium-induced activation of
calcineurin together with a postulated dephosphorylation of NMDA
receptors was suggested as the mechanism underlying synaptic
desensitization of NMDA receptors (Tong et al., 1995), and
rephosphorylation of the receptor by PKA was suggested as the mechanism
of its recovery (Raman et al., 1996).
PKC enhances Ca2+-dependent inactivation of
NMDA-evoked currents
Changing
[Ca2+]e from 0.2 to 5 mM only slightly reduced
Iss/Ip
(Fig. 4C), demonstrating that with 11 mM EGTA in the patch pipette hippocampal CA1
neurons exhibit little Ca2+-dependent
inactivation (Lu et al., 1998). Nonetheless, the presence of a
Ca2+-dependent inactivation (an decrease
in
Iss/Ip)
of these currents was revealed after applications of PMA. The
proportionate enhancement of Ip by PMA
was approximately the same in all concentrations of extracellular
Ca2+, showing that the depression of
Iss and
Iss/Ip
after activation of PKC was not likely a consequence of an enhanced
glycine-independent desensitization. This result is also consistent
with our previous demonstration that constitutively active PKC enhances
Ip regardless of the presence or
absence of [Ca2+]e
(Xiong et al., 1998). Elevating extracellular
Ca2+ or membrane hyperpolarization, both
of which favor entry of Ca2+ through NMDA
channels, increased the depression of
Iss, whereas increased intracellular
buffering of Ca2+ reduced it. Therefore,
the most parsimonious explanation for our observations is that PMA
acting via endogenous PKC enhances the sensitivity of
Ca2+-dependent inactivation of NMDA
receptor-mediated currents.
Ca2+ inactivation of NMDA channels results
from an inhibition of channel gating associated with the binding of
Ca2+/CaM to the carboxy tail of the NR1
subunit (Zhang et al., 1998; Krupp et al., 1999). In agreement with
this mechanism we found that intracellular application of CaM to
isolated CA1 neurons reduced
Iss/Ip,
indicative of an enhanced Ca2+-dependent
inactivation of these currents. Also the residual PMA-induced inhibition observed in the presence of CaM proved be independent of
[Ca2+]e,
supporting the interpretation that CaM occludes PMA-induced Ca2+-dependent inactivation. This evidence
also demonstrates that a part of the effect of PMA is mediated via a
Ca2+-independent inhibition of NMDA-evoked currents.
Our demonstration that the inhibitory CaM-binding peptide KY9 blocked
the PMA-induced depression of Iss
provides strong support for the role of endogenous CaM in the
PKC-induced potentiation of Ca2+
inactivation. Previously, inclusion of KY9 in patch pipettes was
reported to depress Ip recorded from
human embryonic kidney 293 cells transfected with cDNAs for NMDA
channel subunits (Zhang et al., 1998). This result was interpreted as a
blockade of Ca2+ inactivation because the
reduction of Ip resulted in an
increase of
Iss/Ip.
Krupp et al. (1999) disputed this interpretation and considered the
depression of Ip to be a nonspecific
action resulting from the putative amphipathic helical structure of CaM
inhibitory peptides. However, using intracellular applications of KY9,
we have demonstrated that this peptide was itself without effect on
either the peak or steady-state currents evoked by applications of NMDA
to CA1 neurons, whereas it was effective at blocking the PMA-induced
enhancement of Ca2+ inactivation.
The NR1a subunit possesses two binding sites for CaM, a high-affinity
site in the C1 cassette and a low-affinity site in the C0 region
(Ehlers et al., 1996b); however, it is the low-affinity site that is
responsible for inhibition of channel gating. Inactivation likely
results in part from displacement of the cytoskeletal-linking protein
-actinin by Ca2+/CaM bound to the same
C0 site (Krupp et al., 1999). The function of CaM binding to the
high-affinity site is unknown. In hippocampal neurons it was shown
recently that PKC phosphorylates residues in the C1 cassette of the NR1
subunit and reduces but does not eliminate CaM binding to the NR1
subunit (Hisatsune et al., 1997). These results appear to be contrary
to our observations that PKC enhances
Ca2+/CaM-dependent inactivation.
To interpret this contradiction, it should be noted that the residues
of PKC-induced phosphorylation are in the C1 and not the C0 cassette
where binding of CaM is associated with inhibition of channel gating.
One possible explanation is that phosphorylation in the C1 cassette
displaces CaM at this site, providing a source of CaM for binding at
the C0 site and leading to an enhancement of channel inactivation. In
this way the NR1 subunit might function as a CaM-binding protein,
mimicking proteins such as neurogranin (RC3), neuromodulin (GAP-43),
and the myristoylated; alanine-rich C kinase substrate
(Chakravarthy et al., 1999). The binding of CaM or
Ca2+/CaM to these proteins is also
inhibited by PKC phosphorylation. Alternatively, PKC-induced
phosphorylation within the C1 cassette of the NR1 may enhance the
sensitivity of the C0 cassette to the binding of
Ca2+/CaM or reduce the binding of
-actinin.
Moreover, effects of PKC on NMDA-evoked currents can be affected by
interactions between the channel and postsynaptic density proteins or
cytoskeletal elements. For example, coexpression of NMDA receptor
subunits with PSD-95 in oocytes greatly reduces the degree of
enhancement by phorbol esters of NMDA-evoked currents (Yamada et al.,
1999). Furthermore, in neurons both CaM and activation of PKC inhibit
the binding of the cytoskeleton-linking protein spectrin to the C
terminal of the NR1 subunit (Wechsler and Teichberg, 1998). The
PKC-dependent phosphorylation of the NR1 subunit also inhibits
clustering subunits overexpressed in a cell line (Ehlers et al., 1995;
Tingley et al., 1997). Therefore, PKC-induced phosphorylation of NR1
subunits may enhance dissociation of NMDA channels from their linking
cytoskeletal proteins, subsequently affecting channel location and
physical proximity to other Ca2+-dependent
signaling enzymes (Sattler et al., 1999). Alternatively, PKC may
phosphorylate CaM, -actinin, or other cytoskeletal proteins than in
turn modulate the sensitivity of
Ca2+-dependent inactivation of NMDA receptors.
The role of Ca2+ inactivation in
determining the time course of synaptic events is not well understood.
However, an influx of Ca2+ induced by
repetitive firing can depress the NMDA receptor-mediated component of
synaptic transmission between individual cultured neurons (Medina et
al., 1999). Desensitization of NMDA receptors may also contribute
directly to the time course of synaptic responses (Huganir and
Greengard, 1990; Jones and Westbrook, 1995; Paradiso and Brehm, 1998).
Phosphorylation of NMDA receptors may regulate both their
desensitization (Tong and Jahr, 1994; Tong et al., 1995) and their
inactivation by an influx of Ca2+,
providing multiple mechanisms to limit the time course of excitatory synaptic currents.
| |
FOOTNOTES |
Received Jan. 6, 2000; revised April 4, 2000; accepted April 7, 2000.
The present study was supported by the Medical Research Council (MRC)
of Canada. W.-Y.L. is a fellow of the Heart and Stroke Foundation of
Canada, M.F.J. is a fellow of the Natural Sciences and Engineering
Research Council of Canada, and D.B. is a fellow of the MRC of Canada.
We thank Dr. K.-W. Yau for providing CaM-binding peptide (KY9) and its
control peptide (KY8), Dr. Terukatsu Sasaki for providing recombinant
CAK/Pyk2, and Dr. M. W. Salter for Src(40-58).
Correspondence should be addressed to Dr. Wei-Yang Lu, Department of
Physiology, Medical Sciences Building, University of Toronto, 1 King's
College Circle, Toronto, Ontario M5S 1A8 Canada. E-mail:
w.lu{at}utoronto.ca.
| |
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TOP
ABSTRACT
INTRODUCTION
