Department of Neurobiology, University of Alabama School of
Medicine, Birmingham, Alabama
Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate
induced a continuous decrease in the
-aminobutyric acid
(GABA)-activated current amplitude from recombinant GABA receptors
(formed by
1 or 

subunits) expressed in
Xenopus oocytes. This decline was due to internalization
of receptors from the plasma membrane as confirmed by a decrease in
surface fluorescence with green fluorescence protein-tagged receptors as well as a concomitant decrease in surface [3H]GABA
binding. PMA specifically caused internalization of GABA receptors, but
not neuronal acetylcholine receptors (
7 or
4
2), indicating the internalization was
not a general, nonspecific phenomenon. Mutation of
1 PKC
phosphorylation sites, identified by in vitro phosphorylation, did not
prevent GABA receptor internalization, nor did coexpression of the
1
M3-M4 intracellular loop along with
1 GABA receptors. It is likely
that PKC-mediated phosphorylation of other proteins, rather than
1
itself, was required for the internalization. Both
1 and 

receptors did not degrade after phorbol 12-myristate 13-acetate-induced
internalization, but returned to the membrane surface within 24 h.
These data suggest internalized receptors can exist in an intracellular
compartment that can be delivered back to the plasma membrane. Thus, by
regulating GABA receptor surface expression, PKC may play a key role in
the regulation of GABA-mediated inhibition.
 |
Introduction |
-Aminobutyric
acid (GABA)-activated chloride channels represent a pharmacologically
diverse class of receptors that mediate inhibitory synaptic activity in
the central nervous system (CNS). At least two classes of fast
inhibitory GABA receptors exist in the CNS: GABAA
receptors composed of
,
,
,
,
,
, and
subunits (Schofield et al., 1987
; Lolait et al., 1989
; Pritchett et al., 1989
;
Shivers et al., 1989
; Ymer et al., 1989
; Hedblom and Kirkness, 1997
;
Bonnert et al., 1999
) and GABAC receptors
presumably composed of
subunits (Cutting et al., 1991
). Both
classes have a Cl
-selective pore, but in
contrast to GABAA, GABAC
receptors are insensitive to bicuculline, barbiturates and
benzodiazepines (Sivilotti and Nistri, 1989
; Polenzani et al., 1991
;
Johnston, 1996
).
The distribution of GABAA and
GABAC receptors in the CNS is well documented
(Richards et al., 1987
; Houser et al., 1988
; Benke et al., 1991
;
Zimprich et al., 1991
; Wisden et al., 1992
; Enz et al., 1996
; Koulen et
al., 1998
), although the mechanism responsible for delivery of
receptors to and from the membrane surface is poorly understood.
Although GABAA and GABAC
receptors can be localized in the same neuron, they exist at separate
synaptic sites (Koulen et al., 1998
). In addition to surface
receptors, a significant cytoplasmic pool exists in cells (Mammen et
al., 1997
; Wan et al., 1997
). Whether these receptors can be
dynamically shuttled back and forth between an intracellular pool and
the cell surface is not well established.
GABAA receptors can be potentiated or inhibited
by direct phosphorylation via protein kinase C (PKC), PKA, or tyrosine
protein kinases (Moss et al., 1992
; Moss et al., 1995
; Lin et al.,
1996
; McDonald et al., 1998
). The results from these studies, however, have been controversial. Recombinant
1
1
2 receptors expressed in HEK293 cells and GABAA (mRNA injection) or
recombinant
1
1
2S receptors expressed in Xenopus
oocytes demonstrated a reduction in the amplitude of the GABA-activated
current in response to PKC activators (Sigel and Baur, 1988
;
Kellenberger et al., 1992
; Krishek et al., 1994
; Chapell et al., 1998
).
In contrast, introduction of catalytically active PKC into L929
fibroblasts expressing recombinant
1
1
2L receptors caused an
enhancement of the GABA-mediated current (Lin et al., 1996
).
Previously, for GABAC receptors, we provided evidence that internalization is a potential mechanism for
phosphorylation-dependent inactivation of
1 receptors transfected in
HEK cells (Filippova et al., 1999
). Both recombinant
GABAA and GABAC receptors
are inactivated in a PKC-dependent manner by phorbol 12-myristate 13-acetate (PMA) in Xenopus oocytes (Sigel and Baur, 1988
;
Chapell et al., 1998
; Kusama et al., 1998
).
To gain insight into the mechanism of PKC-dependent modulation of
recombinant GABAA and GABAC
receptors, we used: 1) the two-electrode voltage clamp to measure
GABA-activated currents, 2) fluorescence microscopy to visualize green
fluorescence protein (GFP)-tagged GABA receptor subunits on the
membrane surface, and 3) [3H]GABA binding in
intact oocytes to estimate changes in the number of receptors on the
cell surface. We found that activation of PKC induced the
internalization of GABA receptors and these internalized receptors
could, in time, return back to the cell surface. Thus, by regulating
GABA receptor surface expression, PKC may play a key role in the
regulation of GABA-mediated inhibition.
 |
Materials and Methods |
Clones, Constructs, and In Vitro Transcription.
The human
1 and rat
1,
2,
2 subunits were obtained via the polymerase
chain reaction as previously described (Amin et al., 1994
; Amin and
Weiss, 1994
), and subcloned into the p-ALTER-1 vector (Promega,
Madison, WI). Henceforth,
1,
2, and
2 will be referred to as
,
, and
, respectively. Altered Sites (Promega) was used for
the site directed mutagenesis that was verified by cDNA sequencing.
The
2 subunit was subcloned into the pEGFP-N1
vector (Clonetech Laboratories, Palo Alto, CA) between the
EcoRI and BamHI sites, leaving GFP attached to
the carboxy terminus. For in vitro transcription, the
-GFP construct
was subcloned into the pcDNA3.1 vector (Promega). The 6HIS construct
consisted of the wild-type
1 subunit with 10 glycine residues
followed by six histidine residues at the C terminus. For expression of
the M3-M4 intracellular (IC) loop of the
1 receptor, a cDNA fragment
encoding residues 347 to 436 was subcloned into the pGEM vector
(Promega). For the in vitro phosphorylation assays, the same segment of
the M3-M4 IC loop was cloned into pGEX-2T (Pharmacia, Piscataway, NJ),
producing a fusion protein with glutathione
S-transferase (GST) at the amino terminus of the IC
loop. The clones for the rat
7,
4, and
2 subunits of
the neuronal acetylcholine (nACh) receptor were kindly provided by Dr.
M Quick (University of Alabama School of Medicine, Birmingham,
AL) in the pCMV vector.
cDNAs of each clone were linearized and run-off capped cRNA was
transcribed from the linearized cDNAs with standard in vitro transcription procedures. Integrity and yield of the cRNA were verified
on a 1% agarose gel.
Xenopus Oocyte Expression.
X.
laevis (Xenopus I, Ann Arbor, MI) were anesthetized by MS-222, and
oocytes were surgically removed and placed in a solution that consisted
of 85.5 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 1 mM
CaCl2, 1 mM MgCl2, 1 mM
Na2HPO4, 50 U/ml
penicillin, and 50 µg/ml streptomycin, pH 7.5. Oocytes were dispersed
in this solution without Ca2+, but in the
presence of 0.3% Collagenase A (Boehringer-Mannheim, Indianapolis,
IN). After isolation, stage 6 oocytes were thoroughly rinsed and
maintained at 18°C in the above-mentioned solution plus 1 mM
Ca2+. Micropipettes for injecting cRNA were
pulled on a Sutter P87 horizontal puller. cRNA was diluted 2- to
10-fold with pyrocarbonate-treated water. A total of 0.5 to 5 ng of
cRNA was injected into each oocyte.
Voltage Clamp of Oocytes.
Two-electrode voltage-clamp
procedures were used for current recording 3 or 4 days after cRNA
injection. Oocytes were placed on a 300-µm nylon mesh suspended in a
small volume chamber (<100 µl). The oocyte was perfused continuously
with a solution containing 92.5 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 1 mM
CaCl2, 1 mM MgCl2, and 0.5 mM Na2PO4, pH 7.5. The
solution was switched to the test solution, which is identical with the
perfusion solution plus drug (e.g., GABA, carbachol). In experiments
with nACh receptors, atropine (0.5 µM) was added to block muscarinic
ACh receptors. All experiments were performed at room temperature.
Recording microelectrodes were pulled on a P87 Sutter horizontal puller
and filled with 3 M KCl. The electrode resistances ranged from 1 to 3 M
. Standard two-electrode voltage-clamp techniques (GeneClamp 500; Axon Instruments, Foster City, CA) were used
to record currents at a holding potential of
70 mV. The current signal was filtered at 10 Hz and recorded on paper with a Gould EasyGraf chart recorder (Gould Instrument Systems Inc., Valley View,
OH). At the same time, on-line digitization of the signal at 20 Hz with
12-bit resolution was carried out by using the MacADIOS data
acquisition board (GW Instruments, Somerville, MA), Igor software
(Wavemetrics, Lake Oswego, OR) in conjunction with a set of macros to
drive the GW board (provided by Bob Wyttenbach, Cornell University,
Ithaca, NY) in a Macintosh (Apple Computer, Cupertino, CA).
Dose-response and dose-inhibition relationships were fit with the
following Hill equation with a nonlinear least-squares method:
|
(1)
|
|
(2)
|
where I is the peak current response at a given
concentration of agonist (A), Imax is the
maximum current response, EC50 is the
concentration of the agonist with a half-maximal activation, IC50 is the concentration of the agonist with a
half-maximal inhibition, and n is the Hill coefficient.
For the PMA experiments, oocytes were incubated in 100 nM PMA for 10 min. Calphostin C (1 µM) was added in the perfusion solution or injected into the oocytes. The final dimethyl sulfoxide dilution for
the oocyte injection was 1000-fold. The injection of oocytes with
dimethyl sulfoxide diluted 1000-fold did not modify the GABA-activated currents. The degree of the PMA effect on GABA-activated currents varied between batches of oocytes. For example, the percentage of
inactivation in the presence of 100 nM PMA varied between 54 and 98%.
Comparison of the Zn2+ sensitivity of the
wild-type and 6HIS
1 receptors was accomplished by coapplying
different concentrations of Zn2+ along with 3 µM GABA. Oocytes expressing the 6HIS construct were exposed to the
exoprotease, carboxypeptidase A (Sigma Chemical Co., St. Louis, MO) for
~120 min then thoroughly rinsed before subsequent two-electrode
voltage-clamp analysis. All results are presented as mean ± S.E.
Data were compared statistically by the Student's t test.
Statistical significance was determined at the 5% level.
Fluorescence Analysis.
The intensity of the fluorescence
from 
-GFP
receptors was analyzed with a Diaphot 300 fluorescence microscope (Nikon, Tokyo, Japan). The
GABA-activated current from oocytes injected with 
-GFP
cRNA was tested using two-electrode voltage clamp before examining the fluorescence.
Western Blot Analysis.
Noninjected oocytes and oocytes
injected with the cRNA coding for the
1 IC loop (40 per each
condition) were homogenized, centrifuged, and layered on a
discontinuous 20% sucrose gradient in the presence of a protease
inhibitor cocktail (Sigma Chemical Co.), 1 mM dithiothreotol, and 5 mM
MgCl2 in a Tris-buffered saline. After
centrifugation, the top layer (cytosol) and pellet (membrane) fractions
were collected. The proteins from the membrane fraction were
solubilized by 60 mM urea. The membrane and cytosolic fractions were
then boiled in SDS-polyacrylamide gel electrophoresis loading buffer
and separated on a 10% polyacrylamide gel and transferred to a
nitroceullulose membrane. The immunoblotting procedure included a 2-h
incubation with a mouse polyclonal antibody (1:500) raised against the
M3-M4 IC loop of the
1 receptor followed by a 1-h incubation in an
alkaline phosphatase-conjugated anti-IgG antibody diluted 1:1000. A
14-kDa band from the cytosol fraction was detected as the IC loop protein.
[3H] Binding Assay.
To estimate
[3H]GABA binding, we used the single-oocyte
binding method as previously described (Chang and Weiss, 1999
).
Briefly, single oocytes injected with
1 cRNA were held by suction at
the end of a pipette tip. The oocyte was first incubated in
[3H]GABA for 60 to 90 s (specific
activity = 94 Ci/mmol; Amersham life Sciences, Arlington Heights,
IL), then rinsed for 6 s in a 150 ml of ice-cold stirring bath to
remove free [3H]GABA from the oocyte surface.
Next, the oocyte was held in a 250-µl incubation solution for 90 s to allow the bound [3H]GABA to dissociate
from the receptor. The released counts per minute were then determined
in a liquid scintillation counter.
In Vitro Phosphorylation.
The GST-IC loop fusion
protein was purified from the bacterial cell line Epicurian Coli
BL21-Gold(DE3)pLysS (Stratagene, La Jolla, CA) with
glutathione-Sepharose (Amersham, Uppsala, Sweden) under nondenaturing
conditions. The in vitro phosphorylation assay was carried out with the
fusion protein still bound to the Sepharose. The catalytic domain of
protein kinase C (Calbiochem, San Diego, CA) was added to the beads in
the presence of [
-32P]ATP for 25 min. The Sepharose
was then rinsed, boiled, and ran on a denaturing 15% polyacrylamide
gel and subjected to autoradiography.
 |
Results |
PMA-Induced Inactivation of Homomeric
1 GABAC and


-GABAA Receptors.
The GABA-activated current
from homomeric
1 receptors expressed in Xenopus oocytes
was stable during long-term recording (up to 2 h). The activator
of PKC, PMA (100 nM; 10-min application) induced a continuous decrease
in the amplitude of the GABA-activated current (normalized value was
0.32 ± 0.02; n = 4 after 55 min of recording)
(Fig. 1, A and B). The inactive analog of
PMA,
PMA (1 µM; 10-min application), had no significant effect on
the GABA-evoked current (1.03 ± 0.06; n = 4)
after 30 min of recording (Fig. 1, A and B).

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Fig. 1.
PMA-induced inactivation of recombinant 1
receptors. A, PMA (100 nM; 10 min) decreased the GABA-evoked current to
0.36 of its original value after 35 min of recording. The inactive
analog of PMA, PMA (1 µM; 10 min), was ineffective during 55 min
of recording. B, average of the normalized GABA-activated current
amplitude after PMA ( ) and PMA ( ) treatment during prolonged
recording. Note the amplitude of the GABA-activated current after
PMA treatment did not significantly change (1.03 ± 0.06;
n = 4, P < .05). After PMA
treatment, however, the amplitude declined to 0.32 ± 0.02 (n = 4) of the initial value.
|
|
A similar experimental paradigm was used to study PKC activation on


-GABAA receptors. PMA (100 nM; 10-min
treatment) induced a continuous inactivation of recombinant
GABAA receptors expressed in Xenopus
oocytes (Fig. 2A). The time course of
inactivation was similar to the PMA-induced inactivation of
1
receptors. After 30 min from the end of PMA treatment, the amplitude of
the GABA-activated current was 0.22 ± 0.06 (n = 5) from the initial value (Fig. 2C).
PMA (1 µM; 10-min
application) had no significant effect on the current amplitude (Fig.
2, B and C).

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Fig. 2.
PMA-induced inactivation of recombinant  
receptors. A, PMA (100 nM; 10 min) decreased the GABA-evoked current to
0.1 of the original value during 45 min of recording. B, inactive
analog of PMA, PMA (1 µM; 10 min), was ineffective during 45 min
of recording. C, average of the normalized GABA-activated current
amplitude after PMA and PMA treatment. Note the amplitude of the
GABA-activated current declined to 0.22 ± 0.06 (n = 5) of the initial value during 45 min of
recording after PMA treatment. With PMA treatment, the amplitude was
0.95 ± 0 0.03 (n = 5) after 45 min of
recording.
|
|
PKC Is Involved in PMA-Induced Inactivation of
1 and 

Receptors.
To verify the role of PKC in the PMA-induced
inactivation of GABA receptors, the degree of inactivation was
determined in the presence of the PKC inhibitor calphostin C (1 µM),
injected into the oocyte 1 h before PMA (100 nM) treatment (Fig.
3, A and B). Calphostin C, on its own,
did not significantly modify the amplitude of the GABA-activated
current (0.96 ± 0.06; n = 5; data not shown),
although it decreased the degree of PMA-induced inactivation of
1
and 

receptors (Fig. 3, B and C, respectively). It is possible
that calphostin C slowed the rate of the PMA effect. Nevertheless,
these data confirm that PKC is involved in the time-dependent inactivation.

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Fig. 3.
PKC is involved in the modulation of GABA receptors.
A, current traces evoked by GABA (20 µM) from   receptors
after 1 nM (top) and 5 nM (middle) PMA treatment. The lower traces are
in the presence of 5 nM PMA and 1 µM calphostin C. B, plot of the
amplitude of the GABA-activated current from   -GABA receptors
as a function of time in the presence of 20 nM PMA either with ( ) or
without ( ) 1 µM calphostin C. After 55 min of recording, the
normalized amplitudes were 0.12 ± 0.05 (n = 4) and 0.37 ± 0.11 (n = 4) in the absence and
presence of calphostin C, respectively. C, plot of the amplitude of the
GABA-activated current from 1 GABA receptors as a function of time
in the presence of 50 nM PMA either with ( ) or without ( ) 1 µM
calphostin C. After 40 min of recording, the normalized amplitudes were
0.10 ± 0.05 (n = 7) and 0.44 ± 0.10 (n = 5) in the absence and presence of calphostin
C, respectively.
|
|
PMA-Induced Inactivation Was Not Caused by Direct Phosphorylation
of
1 Receptor.
We next set out to determine whether the
PMA-induced inactivation was the result of a direct phosphorylation of
the GABA receptor. The
1 subunit has three consensus PKC sites in
the IC loop between the third and fourth transmembrane domains (S410,
S419, and S426) (Cutting et al., 1991
) and it has been demonstrated
that mutation of these three sites does not prevent the PMA-induced
inactivation (Kusama et al., 1998
). However, it is not known which, if
any of these three sites can be phosphorylated. In addition, other nonstandard PKC consensus sites may exist (Kennelly and Krebs, 1991
).
To identify potential PKC sites, we carried out an in vitro phosphorylation assay with a GST
1 IC loop fusion protein. A summary
autoradiogram is presented in Fig. 4A.
Mutation of the serines at positions 419 and 426 to alanines eliminated
a majority of the phosphorylation (compare lanes 1 and 2). Most of the
phosphorylation occurred at residue 419 (data not shown). Mutation of
the serine doublet at positions 422 and 423 to alanines eliminated the
remainder of the phosphorylation (lane 3). At present, we do not know
which of these two serines (422 or 423) is the actual site of
phosphorylation (perhaps it is both), but nevertheless, mutation of
these four serines produced an IC loop that was not phosphorylated in
vitro.

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Fig. 4.
Mutation of the PKC phosphorylation sites on the 1
receptor did not prevent the inactivation of IGABA. A,
lower gel is an autoradiogram from an in vitro phosphorylation assay of
the GST- 1 IC loop fusion protein. Note that the S419A and S426A
mutations eliminated a majority of the phosphorylation, whereas the
S422SA and S423A mutations eliminated the remainder of the
phosphorylation. The gel on top is the corresponding coomassie stain of
the autoradiogram, indicating similar amounts of protein were loaded in
each lane. Alignment of the autoradiogram with the stained gel, as well
as a comparison of the predicted and observed molecular weight of the
GST-IC loop fusion protein, indicated that the upper band (indicated by
the arrow) corresponded to the phosphorylated band in the
autoradiogram. Additional nonphosphorylated bands could be identified
as smaller fragments of the fusion protein without the PKC
phosphorylation sites. B, average of the normalized GABA-activated
current amplitude from S419A/S422A/S423A/S426A receptors after PMA (100 nM; 10 min) treatment. Note the decline in the current amplitude was
similar to that of the wild-type 1 receptor, indicating that these
mutations did not alter the PMA-induced inactivation of
IGABA.
|
|
We next tested if these four mutations altered the PMA-induced
inactivation of the
1 receptor. PMA (100 nM; 10 min) induced inactivation of the GABA-activated current of the
1 mutant
(0.29 ± 0.05; n = 5) after 50 min of recording
following PMA treatment (Fig. 4B) similar to that observed for
wild-type
1 receptors (0.27 ± 0.11; n = 3).
For these studies, we have assumed the four transmembrane domain model
of the GABA receptor (Schofield et al., 1987
) and that only domains
accessible from inside the cell have the potential to be phosphorylated
by intracellular kinases. Besides the M3-M4 linker, the M1-M2 linker is
assumed to face the intracellular environment. In this 11-residue
domain, there is one serine (S281) residue with nearby basic amino
acids (R303 and R304). Although this is not a PKC consensus sequence,
we mutated this serine to alanine and examined the PMA-induced
inactivation. PMA decreased the maximum current amplitude of the S281A
1 mutant to a similar fraction (0.13 ± 0.1; n = 3) as that of the wild-type
1 receptor (0.10 ± 0.1;
n = 3). These results, along with the data in Fig. 4,
suggest the PMA-induced inactivation was not the result of direct
phosphorylation of the
1 receptor.
PMA Does Not Alter Sensitivity for GABA.
One possible
explanation for the PMA-induced inactivation could be a change in the
sensitivity for GABA. To investigate this possibility, we recorded the
GABA sensitivity of
1 and 

receptors before and after
inactivation (Fig. 5). In control
conditions,
1 and 

receptors have a GABA
EC50 of 51 ± 5 and 0.87 ± 0.05 µM,
respectively (Fig. 5A). Based on these mean EC50
values, we selected two GABA concentrations (dashed lines in Fig. 5A)
and compared the ratio of the amplitudes at these concentrations before and after inactivation. The ratio between the current amplitude activated at 1 and 5 µM GABA for
1 receptors was similar before and after PMA treatment (Fig. 5B and Fig. 5C, left), although the
current amplitude decreased to 0.23 ± 0.06 (n = 5) of its pretreatment value (Fig. 5C, right). The normalized traces in Fig. 5B are the scaled and superimposed responses to 1 µM GABA before
and after PMA treatment. Note the responses are identical, indicating
that there was no change in the activation and deactivation kinetics.
The ratio between current amplitude activated at 20 and 50 µM GABA
for 

receptors was similar before and after PMA treatment
(Fig. 5D, left), although the current amplitude decreased to 0.04 ± 0.02 (n = 4) of its pretreatment value (Fig. 5D,
right). Thus, PMA does not alter the GABA sensitivity of
1 and


receptors.

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Fig. 5.
PMA did not alter agonist sensitivity. A,
dose-response relationship for 1 (left) and   (right)
receptors. The continuous line is the best fit of the Hill equation
(eq. 1) to the data. The vertical dashed lines are the responses at 1 and 5 µM GABA for 1 and 20 and 50 µM GABA for   used to
assess changes in sensitivity. B, current traces from  
receptors at 1 and 5 µM GABA before (left) and after (middle) PMA
exposure (100 nM; 10 min). On the right are the 1 µM GABA traces
before and after PMA trratment superimposed. C, graph on the left plots
the ratio of the 1 and 5 µM response before and after PMA treatment;
the right graph plots the normalized amplitude of the current in
response to 5 µM GABA before and after PMA treatment. Note the ratio
did not significantly change after PMA-induced inactivation with a
normalized value of 0.9 ± 0.11 (n = 5) after
PMA exposure. The current amplitude, however, decreased to 0.23 ± 0 0.06 (n = 5) of the initial value. D, graph on
the left plots the ratio of the 20 and 50 µM response before and
after PMA treatment. The right graph plots the normalized amplitude of
the current in response to 50 µM GABA before and after PMA treatment.
Note the ratio did not significantly change after PMA-induced
inactivation with a normalized value of 0.93 ± 0.15 (n = 4) after PMA exposure. The current amplitude,
however, decreased to 0.04 ± 0.02 (n = 4) of
its initial value.
|
|
Internalization Is a Possible Mechanism of PMA-Induced Inactivation
of
1 and 

Receptors.
A second possible mechanism of
the PMA-induced inactivation of the GABA-activated current is removal
of GABA receptors from the membrane surface. To estimate changes in the
number of receptors on the membrane surface, we examined
[3H]GABA binding to surface
1 receptors and
fluorescence of GFP-tagged 

-GABAA
receptors during PMA-induced inactivation.
Measurement of the surface binding of [3H]GABA
and functional inactivation in individual (the same) oocytes expressing
1 receptors demonstrated that PMA decreased the current and binding
with a similar time course and magnitude. Normalized to the initial
value, the current amplitude and [3H]GABA
binding were 0.18 ± 0.03 (n = 5) and 0.16 ± 0.03 (n = 5) after 30 min from the end of PMA
treatment, respectively (Fig. 6). The
current and surface binding decayed along a similar time course, with
time constants of 71 and 50 min, respectively (single exponential
component fitted to the mean of five oocytes). Previous studies have
demonstrated that the degree of surface binding in oocytes
expressing
1 receptors not treated with PMA was stable over extended
time periods (Chang and Weiss, 1999
). The concomitant reduction in
surface binding and current amplitude suggests an internalization of
1 receptors as a potential mechanism of PMA-induced inactivation.

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Fig. 6.
PMA reduced surface binding of [3H]GABA
to 1 receptors. Oocytes were exposed to PMA (100 nM; 10 min) and the current amplitude and surface binding of
[3H]GABA (Chang and Weiss, 1999 ) were examined
concomitantly at 10-min intervals. The average of the normalized
amplitude ( ) and [3H]GABA binding ( ) are plotted.
Note the similar time course of the decline.
|
|
To visualize changes in surface GABAA receptors,
GFP was attached to the C terminus of the
subunit. Figure
7A shows examples of GABA-activated
currents formed by 
-GFP
receptors. Except for a reduced
expression level, the properties of the 
-GFP
receptors were
indistinguishable from the wild-type 

receptors. The top
micrograph in Fig. 7B shows a patch of fluorescence on the surface of
an oocyte expressing 
-GFP
receptors. After PMA treatment (100 nM; 10 min) the intensity of the fluorescence continuously decreased
and disappeared from the surface. Similar results were observed in five
oocytes. The data in Figs. 6 and 7 suggest that PMA induced the
internalization of
1 and 

receptors expressed in
Xenopus oocytes.

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Fig. 7.
PMA-induced internalization of
 -GFP -receptors. A, GFP was fused to the C terminus of the subunit and the , -GFP, and subunits were coexpressed in
Xenopus oocytes. Representative currents in response to
20 and 500 µM GABA from the  -GFP construct. Although the
expression level was reduced somewhat in comparison to wild-type
  receptors, the activation features of the GFP-tagged
receptors were similar to that of the wild type. B, surface
fluorescence from an oocyte expressing  GFP receptors. Note
the continuous decline of fluorescence after PMA treatment.
|
|
Specificity of PMA-Induced Internalization of GABA Receptors.
To examine the specificity of PMA-induced internalization, we
coexpressed nACh receptors (
7 or
4
2) along with
1
receptors. Carbachol (1 mM) or GABA (20 µM) were applied at 15-min
intervals to oocytes expressing both nACh and GABA receptors. PMA (100 nM; 10-min treatment) produced inactivation of
1 receptors (Fig. 8A) but did not significantly change the
current amplitude from
7 or
4
2 receptors (Fig. 8,
B and C). After 30 min of recording, the amplitude normalized to the
initial value was 0.36 ± 0.05 (n = 4) for
1
receptors, 1.24 ± 0.18 (n = 4) for
7 receptors, and 1.01 ± 0.1 (n = 5) for
4
2 receptors,
respectively (Fig. 8D). Thus, PMA specifically internalizes GABA
receptors but does not modulate the amplitude of neuronal nACh
receptors. These data suggest that the internalization of GABA
receptors was not simply due to a PKC-dependent nonspecific plasma
membrane retrieval.

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Fig. 8.
PMA induced inactivation of 1 receptors but did
not decrease the current amplitude of neuronal nACh receptors. A-C,
traces are currents activated by 20 µM GABA for 1 receptors and 1 mM carbamycholine for 7 and
4 2 nACh receptors, respectively. D,
normalized averages of the current amplitudes from 1,
7, and 4 2 receptors after
PMA treatment (50 nM; 10 min).
|
|
Coexpression or Coinjection of Large IC Loop.
The IC loop of
1 and
2 subunits has been shown to interact with the cytoskeleton
(Hanley et al., 1999
; Wang et al., 1999
) and this interaction may be
important in the PKC-dependent receptor internalization. To test this
possibility, we coexpressed
1 receptors with the M3-M4 IC loop in
Xenopus oocytes. If the IC loop of the
1 receptor
interacts with a component of the cytoskeleton and this interaction was
necessary for the PMA-induced inactivation, then overexpression of the
IC loop should compete with the PMA-induced inactivation. Figure
9A shows a Western blot from oocytes
confirming the expression of the IC loop in the cytosol. With PMA
treatment (0.1 µM; 10 min), the amplitude of the GABA-activated
current normalized to the initial value was 0.22 ± 0.03 (n = 5) and 0.24 ± 0.08 (n = 4)
after 50 min of recording with and without the coexpression of the IC
loop, respectively (Fig. 9B, right). Similar results were obtained with
direct injection of recombinant IC loop protein. These data suggest
that the PMA-induced inactivation of
1 receptors may not require a
specific interaction with the M3-M4 IC loop. We cannot rule out the
possibility, however, that for reasons of aberrant IC loop conformation
or improper subcellular location, the IC loop protein was unable to
compete with the intact IC loop.

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Fig. 9.
Coexpression of the IC loop with 1 receptors did
not prevent the PMA-mediated inactivation. A, Western blot from oocytes
expressing the 1 IC loop. An ~15-kDa band was detected in the
cytosol that was not present in the membrane fraction or the cytosolic
fraction of noncRNA-injected oocytes. B, current amplitude before and
after PMA treatment in oocytes expressing the 1 receptor and oocytes
expressing the 1 receptor along with the IC loop. The degree of
PMA-induced inactivation was similar, indicating that the IC loop did
not compete with the receptor internalization.
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|
Receptors Could Return to Cell Surface after PMA-Dependent
Internalization.
We next examined if the receptors that were
internalized by PKC activation could return to the cell surface. To
answer this question, we assessed the recovery of functional
GABA-activated currents after PMA-induced internalization. The
amplitude of the GABA-evoked current from
1 receptors normalized to
the initial value recovered from 0.08 ± 0.03 to 0.8 ± 0.1 (n = 9) after 24 h (PMA = 100 nM; 10 min)
(Fig. 10, A and B). The GABA-activated current did not significantly change during 24 h in oocytes
without PMA treatment [amplitude normalized to the initial value was
0.98 ± 0.1 (n = 5); Fig. 10B, right].
Twenty-four hours after the PMA-induced internalization, the
GABA-activated current from 

receptors recovered from
0.06 ± 0.05 (n = 4) to 0.60 ± 0.12 (n = 4) of the initial value. These data suggest that
receptors internalized by PMA activation can exist as a pool that can
be returned to the cell surface.

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Fig. 10.
Recovery from PMA-induced internalization of
GABA
receptors.A,GABA-activated
current from 1 receptors before and after PMA treatment (100 nM; 10 min). Note that the current almost completely recovered within 20 h. B, plot of the normalized current amplitude before and after PMA
treatment. represent the GABA-activated current amplitude in
a group of oocytes that were not exposed to PMA. Note that in the
absence of PMA, the current amplitude remained stable over this time
course. C, GABA-activated current from   receptors before and
after PMA treatment (100 nM; 10 min). As for the 1 receptors, the
current almost completely recovered within 20 h. D, plot of the
normalized current amplitude before and after PMA treatment in
  receptors. represent the GABA-activated current amplitude
in a group of oocytes that were not exposed to PMA.
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|
Further evidence that it was the internalized receptors that returned
to the cell surface is provided by the data in Fig. 11. The 6HIS receptor includes 10 glycines followed by six histidines at the C terminus of the wild-type
1 receptor. This subunit has a GABA sensitivity identical with that
of the wild type (data not shown), but displays a higher
Zn2+ sensitivity, probably due to an additional
Zn2+ binding site(s) formed by the histidine
residues (Fig. 11A). At 2.5 µM Zn2+, the
fractional block of the 6HIS receptor was 0.25 ± 0.02 (n = 4) (Fig. 11B, left traces; Fig. 11C, open column)
compared with the wild-type fractional block of 0.04 ± 0.02 (n = 4) (Fig. 11C, striped column). After exposure of
the 6HIS receptor to the exoprotease carboxypeptidase A, the maximum
amplitude of the current was unchanged but the
Zn2+ sensitivity reverted to that of the
wild-type receptor, 0.04 ± 0.002 (n = 4; Fig.
11B, middle traces; Fig. 11C, stippled column), indicating histidines
were cleaved from the C terminus. After carboxypeptidase exposure, the
oocytes were exposed to 100 nM PMA and the maximum current amplitude
decreased to 0.22 ± 0.04 (n = 4) of the prePMA
value (Fig. 11D, stippled column). Twenty-four hours later, the current
amplitude recovered to 0.70 ± 0.1 (n = 4) of the
original value (Fig. 11D, filled column). If it is new receptors that
have come to the cell surface (either newly synthesized or maintained
in an intracellular compartment), they should be intact 6HIS receptors
and therefore display the higher Zn2+
sensitivity. In contrast, if it is the internalized receptors that
returned to the cell surface, the Zn2+
sensitivity should be similar to that of the wild-type receptor. Figure
11B (right traces) and Fig. 11C (filled column) demonstrate that the
Zn2+ sensitivity was similar to that of the wild
type. Assuming carboxypeptidase A does not cross the cell membrane, the
most straightforward interpretation of these results is that receptors
that were previously on the cell surface and cleaved by extracellular
carboxypeptidase A were reinserted into the plasma membrane.

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Fig. 11.
Internalized receptors can return to the cell
surface. A, traces show the block of a 3 µM GABA-activated current by
different concentrations of Zn2+ for the wild-type (top)
and 6HIS (bottom) constructs. The graph to the right plots the
fractional block as a function of the Zn2+ concentration.
The continuous lines are fits of eq. 2 to these data, which yielded
IC50 values/Hill coefficients of 44 ± 3/1.8 ± 0.2 (n = 4) and 10.2 ± 0.1/1.0 ± 0.05 (n = 8) for the wild-type and 6HIS receptors,
respectively. B, block of the 6HIS receptor by 2.5 µM GABA before
(left) and after (middle) a 120-min incubation with the exoprotease
carboxypeptidase A that cleaved the histidines and reverted the
Zn2+ sensitivity to that of the wild-type receptor. The
rightmost traces show the Zn2+ sensitivity after the same
oocyte was exposed to 100 nM PMA causing internalization and was then
allowed to recover for 24 h. Note that the Zn2+
sensitivity was similar to that of the wild type (cleaved 6HIS),
indicating the internalized receptors were returned to the cell
surface. C, averages from four oocytes showing the fractional
activation of a 3 µM GABA-activated current by 2.5 µM
Zn2+ before exposure to carboxypeptidase A ( ), after
exposure ( ), and after exposure to 100 nM PMA with a subsequent 24-h
recovery period ( ). Percentage of inactivation by Zn2+
is shown for the wild-type 1 receptor for comparison ( ). D, plot
of the fraction of current remaining after 100 nM PMA ( ) and after
the 24-h recovery period ( ). The averages are for the same data set
as in Fig. 11C.
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Discussion |
Previous investigations on the PKC-dependent regulation of
GABAA receptors have been controversial because
both an inhibition (Sigel and Baur, 1988
; Kellenberger et al., 1992
;
Chapell et al., 1998
) and potentiation (Lin et al., 1996
) have been
observed. For GABAC receptors, a negative
modulation by PKC has been reported for both native
GABAC (Feigenspan and Bormann, 1994
) and
recombinant
1 (Kusama et al., 1998
; Filippova et al., 1999
) receptors.
Our results document a role for PKC in the modulation of recombinant
GABA receptors expressed in Xenopus oocytes. PKC induces receptor internalization as revealed by a decrease in surface binding
of [3H]GABA, and the disappearance of
GFP-tagged GABA receptors from the cell surface. This confirms previous
findings that activation of PKC can reduce the amplitude of
GABA-activated currents in oocytes (Sigel and Baur, 1988
) and is in
agreement with the conclusion that receptor internalization is the
mechanism of this inactivation for GABAA
receptors expressed in oocytes (Chapell et al., 1998
) and
1
receptors expressed in HEK293 cells (Filippova et al., 1999
). That a
similar effect of PKC activation was observed for native
GABAC receptors (Feigenspan and Bormann, 1994
) as
well as recombinant
1 receptors transiently expressed in HEK293
cells (Filippova et al., 1999
) indicates that this effect is not unique to the oocyte expression system.
Internalization of membrane receptors by PKC activation is not without
precedent because PMA has been shown to internalize the transferrin
receptor, epidermal growth factor receptor, sodium, K-ATPase, a
retinal taurine transporter, and the Na+/glucose
transporter (for review, see Backer and King, 1991
). In many cases, and
similar to our findings, this internalization does not depend on
receptor phosphorylation (Backer and King, 1991
). Internalization of
receptors in Xenopus oocytes is not, however, a nonspecific
phenomenon because PKC-internalized GABA receptors but not nACh
receptors expressed in the same oocyte. Although this phenomenon may
reflect a specificity for interaction with the internalization
machinery or with PKC (receptor for activated C kinase proteins;
for review, see Mochly-Rosen, 1995
), we cannot rule out the possibility
that the nACh and GABA receptors are clustered in different regions of
the oocyte and that endocytosis is region- (e.g., pole-) specific.
Although we have not mapped the location of the GABA and nACh
receptors, the 
-GFP
receptors are clearly clustered on the
cell surface (Fig. 7).
The details of PKC-dependent modulation of GABAC
receptors are still unclear. Assuming the residues identified in the in
vitro phosphorylation studies are the same as in the full-length
1 receptor, direct phosphorylation of the
1 receptor itself was not
required for the PKC-dependent internalization. Our present working
hypothesis is that other protein(s) along the pathway for
internalization require phosphorylation by PKC. Phorbol esters have
been shown to alter the organization of microtubules, microfilaments, and other components of the cytoskeletal network (Phaire-Washington et
al., 1980
; Backer and King, 1991
). Presumably, an
interaction between GABA receptors and the cytoskeleton could be
crucial for receptor shuttling to and from the cell surface. Recently,
two proteins have been identified, mitogen-activated protein-1B and GABARAP, that are candidates for linking GABAC
and GABAA receptors to the cytoskeleton,
respectively (Hanley et al., 1999
; Wang et al., 1999
). The cytoskeleton
has already been implicated in membrane receptor modulation (Connolly,
1984
; Rosenmund and Westbrook, 1993
), including recombinant
1
GABAC receptors expressed in HEK293 cells (Filippova et
al., 1999
).
Concerning the particular domains of the GABA receptor that could
interact with the machinery responsible for internalization and
retrieval, the obvious candidate was the putative large IC loop
between M3 and M4. This region has already been implicated in the
interaction with mitogen-activated protein-1B (Hanley et al., 1999
).
Coexpression or coinjection of the
1 IC loop did not interfere with
the PMA-induced internalization although several technical problems
could account for these findings. First, the amount of IC loop could
have been insufficient to block the requisite protein-protein
interactions. Second, the coexpressed IC loop may exist in a different
subcellular compartment than the
1 receptors (e.g., cytosol versus
membrane). Third, the conformation of the IC loop protein could be
different from that of the IC loop in the
1 receptor. Alternatively,
other putative intracellular regions such as the loop between the first
and second membrane-spanning domains could serve as a tether for
internalization. And last, the present picture of the membrane topology
of the GABA receptor subunit is only a model based on a hydrophobicity
analysis. It is possible that other regions of the subunit are
available for intracellular protein-protein interactions.
The reported half-life of receptors on the membrane surface of native
neurons varies from 12 to 30 h at different stages of development
(Steinbach, 1981
; Killisch et al., 1991
; Mammen et al., 1997
). During
this time, receptors can change their surface distribution (Mammen et
al., 1997
; O'Brien et al., 1997
). In addition to the surface
receptors, a second intracellular pool exists (Mammen et al., 1997
).
Whether receptors can be dynamically shuttled between the membrane
surface and an intracellular pool during their lifetime is still
controversial. Concerning GABA receptors, activation of tyrosine
kinases has been shown to recruit GABAA receptors to the postsynaptic region (Wan et al., 1997
). In this study, we
demonstrated a PKC-dependent internalization of GABA receptors from
the cell surface and, as evidenced by functional recovery, these
internalized receptors can be reinserted in the plasma membrane. A
similar recovery of internalized receptors has been observed for
1
receptors expressed in HEK293 cells (Filippova et al., 1999
). Thus, by
controlling surface expression, PKC may play a key role in regulating
GABA-mediated inhibition in the CNS.