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Blocking L-type Voltage-gated Ca
2ⴙ
Channels with
Dihydropyridines Reduces
␥
-Aminobutyric Acid Type A
Receptor Expression and Synaptic Inhibition
*
Received for publication, July 7, 2009, and in revised form, September 22, 2009 Published, JBC Papers in Press, September 24, 2009, DOI 10.1074/jbc.M109.040071
Richard S. Saliba
‡
, Zhenglin Gu
§
, Zhen Yan
§
, and Stephen J. Moss
‡¶1
From the
‡
Department of Neuroscience, Tufts University, Boston, Massachusetts 02111, the
§
Department of Physiology and
Biophysics, State University of New York at Buffalo, Buffalo, New York 14214, and the
¶
Departments of Neuroscience, Physiology,
and Pharmacology, University College, London WC1E 6BT, United Kingdom
␥
-Aminobutyric acid type A receptors (GABA
A
Rs) are the
major sites of fast inhibitory neurotransmission in the brain,
and the numbers of these receptors at the cell surface can deter-
mine the strength of GABAergic neurotransmission. Chronic
changes in neuronal activity lead to an adaptive modulation in
the efficacy of GABAergic synaptic inhibition, brought about in
part by changes in the number of synaptic GABA
A
Rs, a mecha-
nism known as homeostatic synaptic plasticity. Reduction in the
number of GABA
A
Rs in response to prolonged neuronal activity
blockade is dependent on the ubiquitin-proteasome system.
The underlying biochemical pathways linking chronic activity
blockade to proteasome-dependent degradation of GABA
A
Rs are
unknown. Here, we show that chronic blockade of L-type voltage-
gated calcium channels (VGCCs) with nifedipine decreases the
number of GABA
A
Rs at synaptic sites but not the overall number of
inhibitory synapses. In parallel, blockade of L-type VGCCs
decreases the amplitude but not the frequency of miniature inhib-
itory postsynaptic currents or expression of the glutamic acid
decarboxylase GAD65. We further reveal that the activation of
L-type VGCCs regulates the turnover of newly translated GABA
A
R
subunits in a mechanism dependent upon the activity of the pro-
teasome and thus regulates GABA
A
R insertion into the plasma
membrane. Together, these observations suggest that activation of
L-type VGCCs can regulate the abundance of synaptic GABA
A
Rs
and the efficacy of synaptic inhibition, revealing a potential mech-
anism underlying the homeostatic adaptation of fast GABAergic
inhibition to prolonged changes in activity.
␥
-Aminobutyric acid type A receptors (GABA
A
Rs),
2
the
major sites of action for both benzodiazepines and barbiturates,
are Cl
⫺
-selective ligand-gated ion channels that can be assem-
bled from seven subunit classes (
␣
1–6,

1–3,
␥
1–3,
␦
,
⑀
,
, and
), providing the structural basis for extensive heterogeneity of
GABA
A
R structure (1–3). A combination of molecular, bio-
chemical, and genetic approaches suggests that, in the brain,
the majority of benzodiazepine receptor subtypes are com-
posed of
␣
,

, and
␥
2 subunits (1).
␥
2-containing receptors are
highly enriched at synaptic sites in neurons and are responsible
for mediating phasic inhibition (4–6). In contrast, receptors
composed of
␣
,

, and
␦
subunits are believed to form a special-
ized population of extrasynaptic receptors that mediate tonic
inhibition (7). GABA
A
Rs are assembled within the endoplasmic
reticulum (ER) and then transported to the plasma membrane
for insertion, whereas misfolded or unassembled receptor sub-
units are rapidly targeted for ER-associated degradation (5, 6, 8,
9), a process that can be modulated by neuronal activity (9). The
number of GABA
A
Rs on the neuronal cell surface is a critical
determinant for the efficacy of synaptic inhibition and, at steady
state, is determined by the rates of receptor insertion and
removal from the plasma membrane (10, 11). Cell-surface
GABA
A
Rs are dynamic entities that exhibit rapid rates of consti-
tutive endocytosis, with internalized receptors being subject to
rapid recycling or lysosomal degradation (5, 6, 10). Phosphoryla-
tion of the intracellular domains between transmembrane
domains 3 and 4 of the GABA
A
R

and
␥
subunits by serine/
threonine and tyrosine kinases has been shown to alter receptor
function either by a direct effect on receptor properties, such as the
probability of channel opening or desensitization, or by regulating
trafficking of the receptor to and from the cell surface (11, 12).
The activity of neurons in neural circuits is highly regulated,
and when firing frequency either falls below or rises above nor-
mal physiological levels, compensatory mechanisms come into
play to restore normal activity, a process known as homeostatic
plasticity (13, 14). Homeostatic synaptic scaling is one homeo-
static mechanism that involves uniform adjustments in the
strength of all synapses in response to changes in activity while
maintaining the individual weights between synapses (15, 16).
GABA regulates the excitability of neural circuits, and a num-
ber of studies in cultures of dissociated neurons have provided
evidence that chronic changes in neuronal activity can lead to
homeostatic scaling of GABAergic synaptic strength (9, 17–19),
which appears to be dependent on brain-derived neurotrophic
factor and tumor necrosis factor
␣
(19, 20). There is now increas-
ing evidence that homeostatic regulation of GABAergic synaptic
*This work was supported, in whole or in part, by National Institutes of Health
Grants NS047478, NS048045, NS051195, NS056359, and NS054900 from
NINDS (to S. J. M.) and by a fellowship from the American Society for Epi-
lepsy (to R. S. S.).
1
Consultant for Wyeth Pharmaceuticals. To whom correspondence should
be addressed: Dept. of Neuroscience, Tufts University, 136 Harrison Ave.,
Boston, MA 02111. Tel.: 617-636-3976; Fax: 617-636-2413; E-mail: stephen.
moss@tufts.edu.
2
The abbreviations used are: GABA
A
R,
␥
-aminobutyric acid type A receptor;
ER, endoplasmic reticulum; VGCC, voltage-gated calcium channel;
␣
-Bgt,
␣
-bungarotoxin; DIV, days in vitro; PBS, phosphate-buffered saline; NHS,
N-hydroxysuccinimide; RIPA, radioimmune precipitation assay; pH,
pHluorin; BBS, bungarotoxin-binding site; mIPSC, miniature inhibitory
postsynaptic current; TTX, tetrodotoxin; NMDA, N-methyl-D-aspartic acid;
AMPA,
␣
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 47, pp. 32544 –32550, November 20, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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strength is achieved by modulating the numbers of postsynaptic
GABA
A
Rs (9, 17, 19). Furthermore, presynaptic changes underly-
ing activity-dependent scaling of inhibitory synaptic strength have
been reported in which the levels of the GABA-synthesizing
enzyme GAD65, the GABA transporter, and presynaptic GABA
content are modulated by activity (17, 18, 21). Currently, little is
known about the mechanisms underlying the modulation of
synaptic GABA
A
R abundance in response to chronic changes
in neuronal activity. However, it has been recently established
that prolonged alteration in neuronal activity modulates the
ubiquitination and proteasomal degradation of GABA
A
Rs in
the ER, resulting in changes in the number of receptors at syn-
aptic sites and the efficacy of synaptic inhibition (9).
A number of important questions remain. For instance, how
do chronic changes in activity translate into the homeostatic
regulation of GABA
A
R abundance at synaptic sites? In this
study, we have begun to investigate the role of Ca
2⫹
influx
through L-type voltage-gated calcium channels (VGCCs) in the
modulation of activity-dependent expression of GABA
A
Rs and
the efficacy of synaptic inhibition.
EXPERIMENTAL PROCEDURES
Antibodies—Rabbit anti-

3 IgG polyclonal antibodies have
been described previously (22, 23). Rabbit anti-green fluores-
cent protein IgG, mouse anti-synapsin IgG, and rabbit anti-
GAD65 antibodies were purchased from Synaptic Systems.
Peroxidase-conjugated and fluorescent dye-conjugated IgG
secondary antibodies were from Jackson ImmunoResearch
Laboratories. Fluorescently labeled
␣
-bungarotoxin (
␣
-Bgt)
was purchased from Invitrogen.
Neuronal Cell Culture and Transfections—Cultures of hip-
pocampal neurons were prepared from embryonic day 18 rats
(9, 22, 24). Dissociated embryonic day 18 rat cortical neurons
were transfected with 3
g of plasmid DNA/2 ⫻10
6
neurons
using the rat neuron Nucleofector
TM
kit (Lonza). 60-mm
dishes were seeded with 0.4 ⫻10
6
neurons and used in
experiments after 18–21 days in vitro (DIV).
Biotinylation—Hippocampal neurons were chilled on ice for
5 min and then washed twice with phosphate-buffered saline
(PBS) containing 1 mMCaCl
2
and 0.5 mMMgCl
2
(PBS-CM) at
4 °C. Cells were incubated for 15 min at 4 °C in 1 mg/ml sulfo-
N-hydroxysuccinimide (NHS)-biotin (Pierce) dissolved in PBS-
CM. To quench unreacted biotin, neurons were washed three
times (10 min each wash at 4 °C) with PBS-CM ⫹75 mMglycine
and then washed twice with PBS and lysed in radioimmune
precipitation assay (RIPA) buffer (50 mMTris (pH 8), 150 mM
NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 2 mM
EDTA, and mammalian protease inhibitor mixture (Sigma)).
Protein concentrations were determined using the micro BCA
protein assay kit (Pierce), and equal amounts of solubilized pro-
tein were added to UltraLink-immobilized NeutrAvidin biotin-
binding protein (Pierce) for2hat4°C.Avidin beads were
washed twice for 15 min at 4 °C with high salt (500 mMNaCl)
RIPA buffer, followed by a 15-min wash at 4 °C with RIPA
buffer (150 mMNaCl). Precipitated biotinylated proteins and
total proteins were resolved by SDS-PAGE, and

3 was
detected by immunoblotting with rabbit anti-

3 IgG antibodies
(9, 22, 25), followed by peroxidase-conjugated anti-rabbit IgG
antibodies and detection with ECL. Blots were imaged using the
Fujifilm LAS-3000 imaging system, and bands were quantified
with Fujifilm Multi Gauge software.
Immunocytochemistry—Neurons expressing
pH

3 (where
pH is pHluorin; 18–21 DIV) were fixed in 4% paraformalde-
hyde, stained without membrane permeabilization with rabbit
anti-green fluorescent protein IgG antibodies, and then perme-
abilized with 0.1% Triton X-100 for 4 min. Neurons were
labeled with anti-synapsin IgG antibodies, visualized by confo-
cal microscopy, and analyzed using MetaMorph imaging soft-
ware (Molecular Devices). To quantify the fluorescence inten-
sity of cell-surface
pH

3 synaptic staining, images of neurons
were thresholded to a point at which dendrites were outlined.
Synapsin staining was thresholded to a set value and kept con-
stant for control and test neurons. Next, a 25-
m section along
a given proximal dendrite was selected, and a 1-bit binary image
(exclusive) was made of the synapsin staining in the outlined
dendrite. We then subtracted all
pH

3 staining that did not
co-localize with the binarized synapsin staining. As a result,
only
BBS

3 staining that co-localized with synapsin remained,
and the average fluorescence intensity of these
pH

3 puncta was
determined (9). Data were analyzed from 10–12 neurons for
each condition from at least two to three different cultures. To
quantify the number of
pH

3 synapses, thresholds were set and
kept constant for control and test neurons. Receptor clusters
were defined as being 0.5–2
m in length and ⬃2–3-fold more
intense than background diffuse fluorescence and were co-lo-
calized with synapsin staining (9, 26).
pH

3 synaptic puncta
were counted from 1-bit binary masks. Data were analyzed
from 10–12 neurons for each condition (25
m/dendrite/cell).
Analyses were all performed blind to experimental condition.
GABA
A
R
BBS

3 Insertion Assay—Hippocampal neurons
(18–21 DIV) expressing
BBS

3 were first labeled with 10
g/ml
unlabeled
␣
-Bgt for 15 min at 15 °C to block existing cell-sur-
face receptors. The neurons were then washed three times with
PBS at 15 °C, followed by a 5-min incubation at 37 °C with 1
g/ml Alexa 594-conjugated
␣
-Bgt (9). All incubations were
performed in the presence of 200
Mtubocurarine (Sigma) to
block
␣
-Bgt binding to endogenous acetylcholine receptors (25,
27–29). Cells were fixed in 4% paraformaldehyde. Confocal
images were collected using a ⫻60 objective lens acquired with
Olympus FluoView Version 1.5 software, and the same image
acquisition settings for
BBS

3
with or without nifedipine were
used. These images were analyzed using MetaMorph imaging
software. A three-dimensional reconstruction of an imaged
neuron was made from a series of Zsections, and then the
average fluorescence intensity of Alexa 594-conjugated
␣
-Bgt
staining was measured along 30
m of two proximal dendrites/
neuron after subtraction of background fluorescence.
Metabolic Labeling and Immunoprecipitation—Hippocam-
pal neurons were incubated in methionine-free Dulbecco’s
modified Eagle’s medium for 15 min and then labeled with 500
Ci/ml [
35
S]methionine (PerkinElmer Life Sciences) for 30
min. Neurons were washed and incubated in complete Neuro-
basal medium with an excess of unlabeled methionine (100-
fold) for an additional 0–4 h. Neurons were lysed in 1% SDS
and 25 mMTris (pH 7.4), and lysates were diluted 10-fold with
RIPA buffer lacking SDS (50 mMTris (pH 8), 150 mMNaCl, 1%
Dihydropyridines Reduce GABA
A
R Expression/Synaptic Inhibition
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Nonidet P-40, 0.5% sodium deoxycholate, 4 mMEDTA, and
mammalian protease inhibitor mixture). GABA
A
R

3 subunits
were immunoprecipitated with rabbit anti-

3 IgG antibodies
from equal amounts of solubilized protein as detailed previ-
ously (9, 10, 22, 30). Precipitated material was then subjected to
SDS-PAGE, and

3 band intensities were determined by phos-
phorimage spectrometry (Bio-Rad).
Electrophysiological Recordings—Patch-clamp recordings in
the whole cell mode were used to measure the properties of
miniature inhibitory postsynaptic currents (mIPSCs) in cul-
tured hippocampal neurons. mIPSCs were isolated by the
inclusion of tetrodotoxin (TTX; 0.5
M), D-2-amino-5-phos-
phopentanoic acid (20
M), and 6,7-dinitroquinoxaline-2,3-di-
one (20
M) to block action potentials, N-methyl-D-aspartic
acid (NMDA), and
␣
-amino-3-hydroxy-5-methyl-4-iso-
xazolepropionic acid (AMPA)/kainate receptors, respectively,
as detailed previously (9). The cell membrane potential was
held at ⫺70 mV. A mini analysis program (Synaptosoft, Leonia,
NJ) was used to analyze the spontaneous synaptic events. Sta-
tistical comparisons of the amplitude and frequency of mIPSCs
were made using Student’s ttest. The threshold for detection of
mIPSC amplitude was 15 pA.
RESULTS
Blocking Ca
2⫹
Influx through L-type VGCCs Reduces the
Expression Levels of GABA
A
Rs—The synaptic expression levels
of GABA
A
Rs are dynamically regulated in response to chronic
changes in neuronal activity (9, 17, 19). Furthermore, bidirec-
tional changes in neuronal activity modulate the ubiquitin-de-
pendent proteasomal degradation of GABA
A
Rs (9). Given that
L-type VGCCs in neurons couple membrane depolarization to
numerous processes, including gene expression (31) and
changes in synaptic efficacy (32, 33), we speculated that Ca
2⫹
influx through L-type VGCCs may play a role in regulating
activity-dependent expression of GABA
A
Rs. To determine the
effects of blocking Ca
2⫹
influx through L-type VGCCs on
GABA
A
R expression, we used a class of compounds known as
dihydropyridines, which block L-type VGCCs (34). Cultured
hippocampal neurons (18–21 DIV) were incubated for 24 h
with or without 10
Mnifedipine. We chose a time point of 24 h
given that homeostatic scaling of GABAergic synaptic strength
occurs over hours to days (9, 17, 19). Cell-surface proteins were
labeled with Sulfo-NHS-biotin and isolated using immobilized
avidin, and precipitated cell-surface proteins were then
resolved by SDS-PAGE. GABA
A
R

3 subunits were detected by
immunoblotting with rabbit anti-

3 IgG antibodies. Nifedipine
reduced the cell-surface expression and the total pool of
GABA
A
R

3 subunits by 46 ⫾7.3 and 45 ⫾3.9%, respectively,
compared with control levels (Fig. 1A).
FIGURE 1. Blockade of L-type VGCCs reduces expression of GABA
A
Rs.
A, hippocampal neurons (18 –21 DIV) were treated with 10
Mnifedipine (Nif)
for 24 h and then biotinylated with Sulfo-NHS-biotin and lysed in RIPA buffer.
Cell-surface proteins were isolated with immobilized avidin. Immunoblots
show the total and cell-surface levels of GABA
A
R

3 subunits as indicated.
Graphs represent quantification of band intensities of cell-surface and total

3 subunits normalized to controls (Ctrl). Data represent the mean ⫾S.E.
percentage of control values. *, significantly different from the control (p⬍
0.01; ttest; n⫽4). B, nimodipine reduced the expression of the cell-surface
and total levels of GABA
A
R

3 subunits. Hippocampal neurons were incu-
bated with 15
Mnimodipine for 24 h and then biotinylated with Sulfo-NHS-
biotin and lysed in RIPA buffer. Immunoblots show the cell-surface and total
levels of GABA
A
R

3 subunits as indicated. Graphs represent quantification of
band intensities, and data represent the mean ⫾S.E. percentage of control
values. *, significantly different from the control (p⬍0.05; ttest; n⫽3).
FIGURE 2. Blockade of L-type VGCCs reduces the synaptic expression of

3-containing GABA
A
Rs. A, images of hippocampal neurons (18 –21 DIV)
expressing GABA
A
R
pH

3 treated with or without nifedipine (Nif;10
M) for
24 h as indicated. Neurons were fixed, and non-permeabilized cells were
stained with rabbit anti-green fluorescent protein IgG antibodies and Rhoda-
mine Red-X-conjugated anti-rabbit IgG antibodies to label cell-surface
pH

3
subunits (shown in red). pHluorin fluorescence is not shown. Cells were then
permeabilized and incubated with mouse anti-synapsin IgG antibodies and
Cy5-conjugated anti-mouse IgG antibodies to label synaptic sites (shown in
green). The boxed areas in the left panels are magnified in the right panels.
Scale bars ⫽10
m. B, quantification of the fluorescence intensity of
pH

3at
synaptic sites. Data represent the mean ⫾S.E. percentage of control (Ctrl)
values. *, significantly different from the control (p⬍0.001; ttest; n⫽10 –12
neurons in two independent experiments). C, quantification of the number of
synaptic sites containing GABA
A
R
pH

3 subunits (no significant difference;
n⫽10 –12 neurons in two independent experiments).
Dihydropyridines Reduce GABA
A
R Expression/Synaptic Inhibition
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We also determined the effects of another member of the
dihydropyridine class of compounds on GABA
A
R expression
levels, nimodipine, which is cell-impermeable, unlike nifedip-
ine. These experiments revealed that a 24-h treatment with
nimodipine (15
M) reduced the cell-surface and total levels of
GABA
A
R

3 subunits by 42 ⫾10.8 and 41 ⫾6.2%, respectively,
compared with control levels (Fig. 1B).
To determine whether GABA
A
Rs localized at synaptic sites
were also regulated by Ca
2⫹
influx through L-type VGCCs, we
used a construct containing superecliptic pHluorin encoded
within the N terminus of the GABA
A
R

3 subunit (
pH

3) in our
experiments (9, 26, 29). Embryonic day 18 hippocampal neu-
rons were nucleofected with
pH

3 and cultured for 18 –21 DIV.
Neurons were then incubated with nifedipine (10
M) for 24 h and
fixed in 4% paraformaldehyde. To label only the cell-surface pop-
ulation of
pH

3, rabbit anti-green fluorescent protein IgG antibod-
ies were used on non-permeabilized neurons, and synaptic sites
were labeled with mouse IgG antibodies to the presynaptic marker
synapsin-1 following permeabilization (Fig. 2A). We chose to stain
synapsin because the levels of this synaptic marker are unaltered
by chronic changes in neuronal activity (17). Images were collected
using confocal microscopy, and based on their morphology, only
pyramidal cells were chosen. These studies revealed that nifedip-
ine reduced the levels of
pH

3-containing GABA
A
Rs at synaptic
sites by 47.2 ⫾6.15% compared with control levels (Fig. 2B). It
should be noted that the nifedipine-induced reduction in the
expression levels of total and synaptic GABA
A
R

3 subunits is
approximately equal to the reduction observed following chronic
blockade of neuronal activity with TTX (9).
The number of GABAergic inhibitory synapses in cultured
neurons is modulated by activity blockade (9, 17). Therefore, we
determined whether blocking Ca
2⫹
influx had any effect on the
number of synaptic sites containing GABA
A
Rs. We used the
synaptic marker synapsin-1 to label presynaptic sites in neu-
rons expressing
pH

3 following treatment with nifedipine for
24 h. We observed no change in the number of synaptic sites
containing
pH

3 (Fig. 2C). Collectively, these data suggest that
Ca
2⫹
influx through L-type VGCCs modulates the total pool
and the synaptic expression levels of GABA
A
Rs but not the
number of GABAergic synapses.
Blocking Ca
2⫹
Influx through L-type VGCCs Reduces the Effi-
cacy of Synaptic Inhibition—Given the robust effects of block-
ing L-type VGCCs on the synaptic accumulation of GABA
A
R

3 subunits, the effect of nifedipine on the efficacy of synaptic
inhibition was determined. Analysis of mIPSCs in cultured hip-
pocampal neurons (18–21 DIV) was performed following nife-
FIGURE 3. Blockade of L-type VGCCs reduces GABAergic synaptic transmission. Aand B, representative mIPSC traces (A) and cumulative distribution of
mIPSC amplitude (B) from control and nifedipine-treated (10
M, 24 h) hippocampal neurons (18 –21 DIV). The threshold for mIPSC detection was 15 pA.
C, scatter plot of mIPSC amplitudes from control and nifedipine-treated neurons. The mean amplitude in each neuron was the average of all the mIPSC events
above 15 pA. D, bar graph of the average (mean ⫾S.E.) mIPSC amplitude and frequency in control (n⫽16) and nifedipine-treated (n⫽16) neurons. *,
significantly different from the control (p⬍0.001; ttest).
Dihydropyridines Reduce GABA
A
R Expression/Synaptic Inhibition
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dipine treatment (10
Mfor 24 h) using patch-clamp recording
in the whole cell mode as detailed previously (9). mIPSCs were
recorded from cultured hippocampal pyramidal neurons in the
presence of TTX (500 nM) and in the presence of D-2-amino-5-
phosphonopentanoate and 6,7-dinitroquinoxaline-2,3-dione
to inhibit neuronal depolarization and ionotropic glutamate
receptor activation, respectively. mIPSC amplitudes and fre-
quencies were compared between control neurons and those
treated with nifedipine (Fig. 3, A–D). The data revealed that
mIPSC amplitude was reduced from 57.6 ⫾3.6 pA (n⫽16) in
control neurons to 38.3 ⫾1.9 pA (n⫽16) in nifedipine-treated
neurons, which represents a 33.5% decrease (Fig. 3, Cand D).
Also, nifedipine shifted the entire distribution of amplitudes to
the left, toward smaller values (Fig. 3B), suggesting that there is
a uniform reduction in synaptic inhibition. Nifedipine treat-
ment did not significantly alter mIPSC frequency (control,
1.68 ⫾0.17 Hz (n⫽16); nifedipine-treated, 1.35 ⫾0.23 Hz (n⫽
16)). Overall, the above data suggest that the efficacy of synaptic
inhibition can be modulated by prolonged blockade of Ca
2⫹
influx through L-type VGCCs.
Previous reports have shown that chronic changes in neuro-
nal activity regulate the expression levels of GAD65 (17, 18), the
main enzyme involved in GABA synthesis, leading to reduced
levels of GABA and decreased synaptic inhibition (18). We
therefore tested whether Ca
2⫹
influx through L-type VGCCs
had any effect on GAD65 expression levels. We treated hip-
pocampal neurons with 10
Mnifedipine for 24 h and deter-
mined the expression levels of GAD65 by Western blotting. We
observed no change in the expression levels of GAD65 (Fig. 4A)
but found reduced steady-state levels of GAD65 when neurons
were treated with 1
MTTX for 24 h (Fig. 4B), in agreement
with previous studies (17, 18). These data suggest that Ca
2⫹
influx through L-type VGCCs does not regulate GAD65
expression levels and consequently suggest that GABA synthe-
sis is unaffected under these conditions.
Ca
2⫹
Influx through L-type VGCCs Regulates the Proteasome-
dependent Turnover and Membrane Insertion of GABA
A
Rs—
Given that the steady-state levels of GABA
A
Rs were reduced by
nifedipine, we tested whether Ca
2⫹
influx through L-type
VGCCs directly influenced the turnover of newly synthesized
GABA
A
R

3 subunits within the ER/secretory pathway. To
assess this, we used metabolic labeling with [
35
S]methionine in
pulse-chase experiments. Hippocampal neurons were treated
with nifedipine for 24 h and then labeled with [
35
S]methionine
for 30 min and chased for 4 h (Fig. 5A). Therefore, at the time
point we chose (4 h post-labeling),

3-containing GABA
A
Rs
would not have yet reached the cell surface because they take up
to6htoreach this compartment from the ER (8). Following
lysis of neurons, GABA
A
R

3 subunits were immunoprecipi-
tated with anti-

3 IgG antibodies and resolved by SDS-PAGE.
At 4 h, a 26.7 ⫾1.94% decrease in

3 subunits was observed in
FIGURE 4. GAD65 expression is unaltered by L-type VGCC blockade.
A, hippocampal neurons (18 –21 DIV) were treated with 10
Mnifedipine (Nif)
for 24 h and then lysed in RIPA buffer. Western blotting was performed with
rabbit anti-GAD65 IgG antibodies. The graph represents quantification of
band intensities, and data represent the mean ⫾S.E. of control (Ctrl) values.
No significant difference from the control was observed (ttest; n⫽4). B, neu-
ronal activity blockade with TTX reduced GAD65 expression. Hippocampal
neurons were treated with 1
MTTX for 24 h and then lysed in RIPA buffer.
Western blotting was performed with rabbit anti-GAD65 IgG antibodies. The
graph represents quantification of band intensities, and data represent
the mean ⫾S.E. percentage of control values. *, significantly different from
the control (p⬍0.05; ttest; n⫽3).
FIGURE 5. Blockade of L-type VGCCs increases the turnover of GABA
A
Rs.
A, hippocampal neurons (18 –21 DIV) were treated with 10
Mnifedipine for
24 h, followed by a pulse chase with [
35
S]methionine. Neurons were lysed in
1% SDS and diluted in RIPA buffer, and GABA
A
R

3 subunits were immuno-
precipitated with anti-

3 IgG antibodies or nonspecific IgG antibodies (as
indicated) and subjected to SDS-PAGE. Band intensities were quantified by
phosphorimage spectrometry, and data represent the mean ⫾S.E. percent-
age of levels at time 0. *, significantly different from0h(p⬍0.05; ttest; n⫽3).
B, inhibition of proteasome activity blocked the effects of nifedipine on
GABA
A
R

3 expression. Hippocampal neurons (18 –21 DIV) were treated with
or without 10
Mnifedipine for 24 h, and 10
MMG132 was added for the last
8 h of the nifedipine incubation as indicated. Neurons were lysed, and West-
ern blots were probed with anti-

3 IgG antibodies. Data represent the
mean ⫾S.E. percentage of control (Ctrl) values. *, significantly different (p⬍
0.05); **, significantly different (p⬍0.01; one-way analysis of variance and
Bonferroni post-test; n⫽3).
Dihydropyridines Reduce GABA
A
R Expression/Synaptic Inhibition
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neurons previously treated with nifedipine, whereas in control
neurons at 4 h, there was no significant degradation (Fig. 5A).
These data implicate Ca
2⫹
influx through L-type VGCCs in
modulating ER-associated degradation of de novo synthesized
GABA
A
R

3 subunits.
We next tested whether proteasome activity was involved in
the increased turnover of GABA
A
Rs following blockade of
L-type VGCCs in cultured hippocampal neurons. We treated
neurons with nifedipine (10
M) for 24 h and added the protea-
some inhibitor MG132 (10
M) during the last8hofthenife-
dipine treatment. These experiments revealed that the effects
of nifedipine on the steady-state levels of GABA
A
R

3 subunits
were blocked by MG132 (Fig. 5B). Nifedipine decreased the
expression levels of

3 subunits by 38 ⫾4.6% compared with
control levels, whereas the addition of MG132 brought

3 sub-
unit levels up to 85 ⫾5.77% of control levels (Fig. 5B). Together,
these data suggest that Ca
2⫹
influx through L-type VGCCs can
directly regulate the turnover of newly synthesized GABA
A
Rs
and that proteasome activity is required for this process.
As activity-dependent L-type Ca
2⫹
influx modulates the
turnover of GABA
A
Rs, we speculated that this may influence
the rate of insertion of receptors into the plasma membrane. To
test this, we used a construct that encodes a bungarotoxin-
binding site and pHluorin tag within the N terminus of the
GABA
A
R

3 subunit (
BBS

3) in an insertion assay as described
previously (9, 29). Hippocampal neurons (18 –21 DIV) express-
ing
BBS

3 were incubated with 10
Mnifedipine for 24 h. Neu-
rons were then incubated with excess unlabeled
␣
-Bgt at 15 °C
to block existing receptors and then incubated at 37 °C with
Alexa 594-conjugated
␣
-Bgt for a number of time points (0–10
min) to label newly inserted
BBS

3 at the plasma membrane
(Fig. 6A). Excess unlabeled
␣
-Bgt blocked accumulation of
Alexa 594-conjugated
␣
-Bgt staining on the plasma membrane,
and
␣
-tubocurarine (200
M) was added to block
␣
-Bgt binding
to endogenous acetylcholine receptors. At different time
points, neurons were fixed and imaged by confocal microscopy.
The insertion of
BBS

3 increased linearly over a 10-min time
period, which was reduced by nifedipine treatment (Fig. 6B).
We therefore used a time point of 5 min to assess the influence
of Ca
2⫹
influx on the insertion of
BBS

3 and normalized data to
those seen in untreated neurons, which were assigned a value of
100% (Fig. 6C). These experiments revealed that blocking Ca
2⫹
influx with nifedipine significantly reduced the insertion of
BBS

3-containing GABA
A
Rs into the membrane by 52.7 ⫾5%
compared with control levels (Fig. 6C).
DISCUSSION
In this study, we have shown that prolonged blockade of
Ca
2⫹
influx through L-type VGCCs leads to reduced total and
synaptic expression of GABA
A
Rs and diminished GABAergic
synaptic transmission without affecting the levels of GAD65.
Reduced GABA
A
R expression following L-type VGCC block-
ade is a result of increased turnover of de novo synthesized
GABA
A
Rs, which is dependent on the activity of the protea-
some. In addition, chronic blockade of Ca
2⫹
influx reduces the
insertion of GABA
A
Rs into the neuronal membrane. Overall,
prolonged reduction in L-type Ca
2⫹
influx decreases the num-
bers of synaptic GABA
A
Rs and reduces synaptic inhibition,
highlighting a possible role for L-type VGCCs in the activity-
dependent scaling of GABAergic synaptic strength.
The number of GABA
A
Rs is a critical factor in determining
the strength of GABAergic synaptic inhibition (35). It has been
previously reported that prolonged changes in neuronal activity
lead to an adaptive modulation of the numbers of GABA
A
Rs at
synaptic sites and the efficacy of synaptic inhibition (9, 17, 19,
36). Therefore, a critical expression locus of activity-dependent
scaling of GABAergic synaptic strength is the postsynaptic
change in GABA
A
R numbers. Furthermore, the direct ubiquiti-
nation of GABA
A
Rs and the ubiquitin-proteasome system play
a critical role in the activity-dependent modulation of the abun-
dance of these receptors at synaptic sites (9). In this study, we
have shown that blocking Ca
2⫹
influx through L-type VGCCs
in cultured hippocampal neurons mimics the effects of TTX on
GABA
A
R turnover, accumulation at synaptic sites, and efficacy
of synaptic inhibition (9). These observations suggest that
activity-dependent scaling of GABAergic synaptic strength is
mediated in part by Ca
2⫹
influx through L-type VGCCs. Inter-
estingly, a recent report has shown that the increase in AMPA
receptor number brought about by activity blockade with TTX
is mediated by somatic Ca
2⫹
influx and that TTX prevents
these action potential-triggered Ca
2⫹
transients in the cell
soma (37). In addition, the effects of TTX on AMPA receptor
accumulation could be partially mimicked by blocking L-type
VGCCs, but the authors speculate that other VGCCs (T- and
R-type) contribute to this phenomenon (37). Furthermore,
Ca
2⫹
influx through L-type VGCCs is critical for activity-de-
pendent changes in the composition of AMPA receptors (33).
Thus, these studies highlight the importance of L-type Ca
2⫹
FIGURE 6. Insertion of GABA
A
Rs is modulated by Ca
2ⴙ
influx through
L-type VGCCs. A, images of pHluorin fluorescence and Alexa 594-conjugated
␣
-Bgt staining at 5 min of insertion in the presence or absence of nifedipine (Nif;
10
M) as indicated. Hippocampal neurons expressing
BBS

3 (18 –21 DIV) were
treated with or without nifedipine (10
M) for 24 h. Neurons were then incubated
with 10
g/ml unlabeled
␣
-Bgt to block existing cell-surface
BBS

3, washed, incu-
bated for 5 min with 1
g/ml Alexa 594-conjugated
␣
-Bgt to label newly inserted
BBS

3, and fixed. The boxed areas in the upper right-hand corners show pHluorin
fluorescence. The rectangles in the left panels (Alexa 594-conjugated
␣
-Bgt stain-
ing) are enlarged in the right panels.B, increase in
BBS

3 insertion over time with
or without nifedipine (10
M). The assay was performed as described for A.
C, graph showing quantification of Alexa 594-conjugated
␣
-Bgt fluorescence
intensity at 5 min of insertion for control (Ctrl) and nifedipine (10
M)-treated
neurons. Data represent the mean ⫾S.E. percentage of control
BBS

3 values. *,
significantly different from the control (p⬍0.01; ttest; n⫽six to eight neurons
from two independent cultures).
Dihydropyridines Reduce GABA
A
R Expression/Synaptic Inhibition
NOVEMBER 20, 2009•VOLUME 284• NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 32549
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influx in the homeostatic scaling of glutamatergic synaptic
strength.
In addition to the changes in abundance of GABA
A
Rs, a
number of presynaptic factors also determine GABAergic syn-
aptic strength. We did not observe a change in GAD65 expres-
sion or the frequency of mIPSCs when L-type Ca
2⫹
influx was
blocked. This is in stark contrast to the presynaptic changes
that have been reported when neuronal activity was blocked
with TTX. Activity blockade in cultured neurons with TTX
induces down-regulation of GAD65 and vesicular inhibitory
amino acid transporter expression (17–19, 21), reduces the
number of GABAergic synapses (9, 17), and results in a smaller
mIPSC frequency (9, 17). Thus, Ca
2⫹
influx through L-type
VGCCs may be responsible for only mediating activity-depen-
dent postsynaptic changes in GABAergic synaptic strength.
However, it appears that other mechanisms regulate presynap-
tic factors governing the homeostatic scaling of GABAergic
synaptic strength. Perhaps activity-dependent changes in the
expression of GAD65 are mediated by Ca
2⫹
influx through
NMDA receptors and not L-type VGCCs, as one report has
shown that NMDA receptor activation dominates Ca
2⫹
influx
in interneurons (38) and that chronic blockade of NMDA
receptors reduces GAD67 expression (39).
This study has begun to delineate the biochemical pathways
underlying the scaling of the strength of GABAergic synaptic
neurotransmission. However, there are a number of unanswered
questions. For instance, we have revealed that Ca
2⫹
influx can
regulate the turnover of GABA
A
Rs, but the exact processes leading
to the modulation of receptor turnover and insertion still remain
to be determined but could possibly involve Ca
2⫹
-dependent
phosphorylation of GABA
A
R subunits and subsequent modula-
tion of receptor ER export/degradation.
In conclusion, this study has demonstrated the importance of
L-type Ca
2⫹
influx in regulating the abundance of GABA
A
Rs at
synaptic sites and the efficacy of synaptic inhibition. Further-
more, this work emphasizes the importance of Ca
2⫹
signaling
in mediating activity-dependent scaling of GABAergic synaptic
strength.
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Dihydropyridines Reduce GABA
A
R Expression/Synaptic Inhibition
32550 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 47• NOVEMBER 20, 2009
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Stephen J. Moss
Richard S. Saliba, Zhenglin Gu, Zhen Yan and
Expression and Synaptic Inhibition
-Aminobutyric Acid Type A Receptor γChannels with Dihydropyridines Reduces
2+
Blocking L-type Voltage-gated Ca
and Biogenesis:
Membrane Transport, Structure, Function,
doi: 10.1074/jbc.M109.040071 originally published online September 24, 2009
2009, 284:32544-32550.J. Biol. Chem.
10.1074/jbc.M109.040071Access the most updated version of this article at doi:
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