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The Journal of Neuroscience, July 1, 1998, 18(13):4993-5007
GABAA Receptor Pharmacology and Subtype mRNA
Expression in Human Neuronal NT2-N Cells
Torben R.
Neelands1,
L.
John
Greenfield Jr2,
Jie
Zhang1, 2,
R. Scott
Turner1, 2, 4, and
Robert L.
Macdonald1, 2, 3
1 Neuroscience Program and Departments of
2 Neurology and 3 Physiology, University of
Michigan, and 4 Veterans Affairs Medical Center Geriatric
Research, Education, and Clinical Center, Ann Arbor, Michigan,
48104-1687
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ABSTRACT |
Human NT2 teratocarcinoma cells differentiate into neuron-like
NT2-N cells when treated with retinoic acid. GABA evoked
concentration-dependent whole-cell currents in NT2-N cells with an
EC50 of 21.8 µM and a Hill slope of 1.2. GABAA receptor (GABAR) currents reversed at
ECl and did not display voltage-dependent
rectification. GABAR single channels opened in bursts to a 23 pS main
conductance level and a 19 pS subconductance level, with infrequent
openings to a 27 pS conductance level. Kinetic properties of the main
conductance level were similar to other native and recombinant GABAR
channels. Diazepam and zolpidem enhanced GABAR currents with
moderate affinity, whereas
methyl-6,7-dimethoxy-4-ethyl- -carboline-3-carboxylate inhibited
GABAR currents. Loreclezole enhanced GABAR currents with high affinity,
but furosemide antagonized GABAR currents with low affinity. The
neurosteroids alphaxalone and pregnenolone sulfate appropriately
modulated GABAR currents. Zinc blocked GABAR currents with
low affinity, but lanthanum did not significantly alter NT2-N GABAR
currents. Reverse transcription PCR (RT-PCR) performed on RNA from
NT2-N cells clearly detected transcripts encoding human  2, 3,
5, 3,  3, and  subtypes. The combined pharmacological and
RT-PCR results are most consistent with a single or predominant GABAR
isoform composed of an 2 and/or 3 subtype combined with the 3
and 3 subtypes. The data do not rule out receptors containing
combinations of 2 and/or 3 subtypes with the 5 subtype or
receptors with both 1 and 3 subtypes. The presence or absence or
the subunit in functionally expressed receptors could not be
determined.
Key words:
GABA; electrophysiology; patch clamp; Ntera2; barbiturate; benzodiazepine; neurosteroid; single channel
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INTRODUCTION |
GABA is the major inhibitory
neurotransmitter in the vertebrate brain. Fast IPSPs are mediated
by GABAA receptors (GABARs), which contain
binding sites for many modulators including benzodiazepines, barbiturates, general anesthetics, penicillin, picrotoxin, bicuculline, and zinc. GABARs consist of five subunits that form a chloride ion
channel. Four different subunit families (, , , and  ) have
been studied extensively (Macdonald and Olsen, 1994 ), and two new
subunit families, (Hedblom et al., 1997) and  (Davies et al.,
1997), have been identified recently. In addition, in mammals,
including humans, six (1-6), three (1-3), and three (1-3) subtypes have been described. Pharmacological studies of recombinant receptors have shown that individual subunits and their subtypes confer different sensitivities to GABAR modulators such as benzodiazepines (Pritchett et al., 1989; Wieland et al., 1992)
and zinc (Draguhn et al., 1990). The subunit subtypes are differentially expressed throughout development and in different CNS
regions (Wisden et al., 1992), reducing the total number of possible
isoforms that can be formed in different brain regions and in
individual cells.
Studies of GABARs in human CNS neurons have been difficult
because of heterogeneity of cell types and unknown GABAR subtype expression. For example, in isolated human central neurons, the heterogeneity of cell types has led to a wide range of responses to
GABAR modulators, presumably because of differing subunit composition (Gibbs et al., 1996). It has not been possible to isolate a homogeneous population of postmitotic neurons from human brain and maintain them in
culture for detailed pharmacological and biochemical studies. Electrophysiological studies of neuronal GABARs, therefore, have relied
on nonhuman neurons in primary cell cultures or acutely isolated neuron
preparations. However, the transcriptional, translational, and
post-translational processing, assembly, transport, and subcellular localization performed by a heterogeneous population of rodent neurons
may not adequately reflect the behavior of human neurons.
The NT2-N human neuronal cell line offers a useful bridge between
studies of defined recombinant receptors expressed in non-neuronal cell
lines and studies of native nonhuman receptors. Prolonged treatment of
the human NT2 teratocarcinoma cell line with retinoic acid causes these
pluripotential cells to become terminally differentiated into neurons
termed NT2-N cells (Pleasure et al., 1992). After differentiation,
NT2-N cells extend both dendritic and axonal processes and express
neuron-specific cell surface, cytoskeletal, and secretory markers and
functional neurotransmitter receptors (Pleasure et al., 1992; Younkin
et al., 1993; Munir et al., 1995; Beczkowska et al., 1997). Levels of
protein kinases A and C have also been shown to increase in NT2 cells
during differentiation (Abraham et al., 1991), which may be important
in regulating the function of these receptors and other cellular
processes. In addition, recent reports have shown the existence of
synaptic connections between cells in culture (Hartley et al., 1997).
In this study we demonstrated that NT2-N cells expressed functional
human GABARs with relatively homogeneous properties, suggesting that
NT2-N cells may provide a useful model system for investigation of
human GABARs assembled and expressed in a homogeneous population of human neurons.
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MATERIALS AND METHODS |
Cell culture. NT2 cells were grown and maintained in
DMEM high glucose (HG) with 10% fetal bovine serum, and penicillin and streptomycin were added as previously described (Andrews, 1984). Cells
were plated at 2 × 106 cells/75
cm2 flask and differentiated by treatment with 1 µM retinoic acid (RA) for 4 weeks. After RA treatment,
cells were washed with versene and then treated with trypsin to
dislodge the cells. Cells were resuspended after being triturated and
replated at a 1:10 dilution with DMEM HG and maintained in 5%
CO2. The following day the media was removed and saved as
conditioned media to feed replates II and III. Cells were again treated
with trypsin and then spun for 5 min at 1000 rpm. The pellet was
resuspended in 1 ml of media containing mitotic inhibitors (in
µM: 10 5-fluoro-2'deoxyuridine plus 10 uridine and 1 cytosine arabinoside) and replated (replate II). The same treatment was
performed again after 1 week in culture (replate III) to obtain
~100% pure neurons. These cells were replated onto 35 mm Corning
(Corning, NY) dishes for electrophysiological recording. For these
experiments we limited selection of cells to those that were isolated
and had neuronal morphology. NT2-N cells tend to form clusters as they
differentiate in culture, and cell-cell interactions could possibly
effect the differentiation process and resulting cell morphology and
might affect neuronal properties. In addition, cell cultures were at
best only 99% pure neurons. The contaminating non-neuronal cells were
morphologically distinct and could be eliminated by visual selection
for electrophysiological experiments.
Solutions and drug application. Cells were removed from the
5% CO2 incubator, and the feeding medium was replaced with
recording medium (in mM: 142 NaCl, 1 CaCl2, 6 MgCl2, 8.09 KCl, 10 glucose, and 10 HEPES, 315-325 mOsm, pH 7.4). Patch-clamp electrodes
of 5-10 M were filled with pipette solution (in mM:
153.33 KCl, 1 MgCl2, 10 HEPES, 5 EGTA, and 2 ATP,
300-310 mOsm, pH 7.3). This combination of solutions results in nearly
equivalent intracellular and extracellular chloride ion concentrations,
hence an ECl of ~0 mV, and produced an
inward current when cells were voltage clamped at negative
potentials.
Compounds were applied to cells using a modified U-tube
"multipuffer" application system (Greenfield et al., 1996). The
U-tube system enabled us to position a micropipette with a 40-50 µm
tip next to the cell for the duration of the recording and apply
multiple concentrations of individual drugs to each cell. Stock
solutions of GABA, diazepam, lanthanum, pentobarbital, phenobarbital,
picrotoxin, 3-5-pregnen-3-ol-20-one sulfate sodium salt
(pregnenolone sulfate), zinc, and zolpidem were made by dissolving each
in sterile water. Stock solutions of
(3)-hydroxy-(5)-pregnan-11,20-dione (alphaxalone), bicuculline,
methyl-6,7-dimethoxy-4-ethyl--carboline-3-carboxylate (DMCM),
furosemide, and loreclezole were dissolved in dimethylsulfoxide (DMSO)
and diluted with sterile water (final DMSO concentration was <0.1%
v/v). Individual drugs were diluted in recording medium to their final
concentration. Alphaxalone and pregnenolone sulfate were purchased from
Research Biochemicals (Natick, MA). Loreclezole was obtained from
Janssen Biochimica (Berse, Belgium). Bicuculline was
purchased from Calbiochem (La Jolla, CA). All other compounds were from
Sigma (St. Louis, MO).
Electrophysiology. Whole-cell voltage-clamp and
single-channel recordings using the patch-clamp technique were obtained
as described previously (Hamill et al., 1981). Patch-clamp electrodes were pulled from Labcraft microhematocrit capillary tubes (Curtin Matheson Scientific, Inc., Houston, TX) or WPI (Sarasota, FL) borosilicate electrode glass (1B150F-3) using a Flaming-Brown P-87
micropipette puller (Sutter Instrument Co., San Rafael, CA). Borosilicate pipettes were fire-polished on a Narashige MF-3 microforge and subsequently coated with polystyrene Q-dope (GC Electronics, Rockford, IL) to reduce electrode tip capacitance.
Whole-cell recordings were performed using an Axopatch 1-B amplifier
(Axon Instruments, Foster City, CA). Signals were digitized on-line at
200 Hz using a Labmaster TL-1 analog-to-digital converter (ADC),
recorded, and subsequently analyzed off-line using Axotape 2.0 software
(Axon Instruments). Single-channel recordings were obtained from
"outside-out" patches formed using standard techniques (Hamill et
al., 1981) with an Axopatch 200A amplifier. Single-channel currents
were low-pass-filtered at 2 kHz using an eight pole Bessel filter and
subsequently digitized on line at 20 kHz using Axotape 2.0 software.
For some later patch recordings, digitization was performed using a
Digidata 1200A ADC and Axoscope software (Axon Instruments).
Data analysis. The magnitude of the enhancement or
inhibition of GABAR current by a drug was measured by dividing the peak amplitude of GABAR currents elicited in the presence of a given concentration of the drug (with GABA) by the peak amplitude of control
current elicited by GABA alone and multiplying the fraction by 100 to
express it as percent of control. Thus the control response was 100%.
Peak GABAR currents at various drug concentrations were fitted to a
sigmoidal function using a four-parameter logistic equation (sigmoidal
concentration-response) with a variable slope. The equation used to
fit the concentration-response relationship was:
where I was the GABAR current at a given GABA
concentration, and Imax was the maximal GABAR
current. Maximal current and concentration-response curves were
obtained after pooling data from all cells tested for GABA and for all
drugs. Concentration-response curves were also obtained from
individual cells. The curve-fitting algorithm minimized the sum of the
squares of the actual distance of points from the curve. Convergence
was reached when two consecutive iterations changed the sum of the
squares by <0.01%. The curve fit was performed on an IBM PC
compatible personal computer using Prism 1.0 or 2.0 (Graph Pad, San
Diego, CA). Data are presented as mean ± SEM.
To quantify whole-cell current rectification, peak amplitudes of
responses to GABA (at EC50 concentrations) were measured at
holding potentials of  50 and +50 mV. These responses exhibited no
visible desensitization. An amplitude ratio (+50:50 mV) was calculated, and rectification was determined with respect to a linear
ratio of 1.0 using the predicted ECl of 0 mV. An
amplitude ratio >1.0 indicated outward rectification. To quantify
voltage-dependent differences in desensitization, the amount of
desensitization produced by a high concentration of GABA (100 µM) was first determined at holding potentials of 50
and +50 mV. Desensitization was expressed as (1 amplitude at
end of GABA application)/(peak GABA amplitude). A desensitization ratio
(+50:50 mV) was then calculated using the method used to characterize
rectification. A desensitization ratio <1.0 was indicative of less
desensitization at depolarized membrane potentials.
Single-channel current analysis. Analysis of single-channel
currents off-line consisted of amplitude and interval detection with
Fetchan 6.0 (Axon Instruments) using a 50% threshold detection system,
followed by pStat 6.0 (Axon Instruments) for amplitude histograms, and
subsequent kinetic investigation using Interval 5 (Dr. Barry S. Palotta, University of North Carolina, Chapel Hill, NC) and software
developed in our laboratory (Macdonald et al., 1989). To reduce errors
resulting from multichannel patches, recordings were included in the
kinetic analysis only if overlaps of simultaneous openings were very
brief and occurred in <1% of openings. Open and shut duration
histograms were constructed as described by Sigworth and Sine (1987)
and fit by the maximum likelihood method. The number of exponential
functions required to fit the binned data was increased until
additional components did not significantly improve the fit, as
determined by the log-likelihood ratio test (Horn 1987; McManus et al.,
1988). Intervals with durations <1.5 times the system dead time were
displayed in the interval histograms but not included in the fit.
Bursts were defined as clusters of openings separated by closures
longer than the two shortest closed components, which were thus
considered intraburst closures. A critical gap for each patch was
calculated from the closed interval distribution to equalize the
proportion of misclassified events (Colquhoun and Sakmann, 1985).
Prolonged (30 sec to several minutes) drug applications were used to
ensure stationarity of channel activity.
Reverse transcription PCR. Total RNA was isolated from NT2-N
neurons using the Ultraspec method by Biotecx (Houston, TX). One
microgram of total RNA was treated with DNase in a total volume of 10 µl composed of 1 µl of 10× DNase I buffer, 1 µl of DNase I (1 U/µl), and 7 µl of DEPC water. This mixture was incubated for 15 min at 25°C. Addition of 1 µl of 25 mM EDTA was
followed by a 10 min incubation at 70°C to heat inactivate the DNase
I. The reaction mixture was then chilled immediately on ice. One microliter of a random primer (0.6 µg/µl) was then added to the mixture that was then incubated at 25°C for 15 min. Reverse
transcription used the product of the above reaction mixed with the
following: 1 µl of 5× first-strand buffer, 2 µl of 0.1 M DTT, 1 µl of 10 mM dNTP mix, and 1 µl of
RNase inhibitor (10 U/µl). The mixture was prewarmed for 2 min at
42°C before addition of 1 µl of Superscript II (10 U/µl) and then
was incubated for 45 min at 42°C. Using the same procedure as
described above, with the exception that 1 µl of DEPC water was
substituted for Superscript II, acted as a negative control. The
reverse transcription product was heat-inactivated for 15 min at 70°C
before PCR. The PCR reaction was performed for each subunit in 100 µl
of the following mixture: 1 µl of a 20 µM primer
mixture (3' primer and 5' primer), 2 µl of the reverse transcription
product, 10 µl of 10× buffer, 16 µl of 25 mM
MgCl2, 1 µl of 25 mM dNTP mixture, 0.5 µl of Amplitaq (Perkin-Elmer, Norwalk, CT; 5 U/µl), and 72.5 µl
of DEPC-treated water. The templates for the PCR reaction using cDNA
for each subunit subtype were used as positive controls. PCR was
performed as follows: a 2 min period at 94°C to denature the mixture,
35 cycles at 94°C for 1 min, 55°C for 2 min, 72°C for 1 min, and
a 7 min extension period at 72°C. PCR products (5 µl for positive
controls, 15 µl for both test and negative controls) were mixed with
dye and run on a 1.5% agarose gel at 120 V for 40 min and then stained
with ethidium bromide and photographed. Reaction reagents were
purchased from Amplitaq; random primer, Superscript II, RNase
inhibitor, and DNase I were purchased from Life Technologies
(Gaithersburg, MD); and dNTP mix and DNA molecular weight marker VI
were purchased from Boehringer Mannheim (Indianapolis, IN).
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RESULTS |
NT2-N cell GABAR current
NT2-N cells were voltage-clamped in the whole-cell configuration.
Neurons can first be isolated 5 weeks after retinoic acid treatment has
begun (Pleasure et al., 1992). At this time multipolar cells were the
predominant cell type found in the cultures, and they were readily
identifiable. Immediately on rupturing the membrane for intracellular
access, the resting membrane potential was measured for each NT2-N cell
by shifting to current-clamp mode with current set at zero. Resting
membrane potentials ranged from 49 to 33 mV with an average of
40.9 ± 0.4 mV (n = 118). Cells were then voltage-clamped at 75 mV, and 300 µM GABA was
applied for 6-10 sec to determine whether functional GABARs were
expressed on the membrane surface. Whole-cell currents resembled those
seen in recordings of native GABARs in mouse cortical neurons (Kume et al., 1996) and recombinant receptors (Angelotti et al., 1993) with a
rapid concentration-dependent rising phase, followed by a decrease in
current (despite continued application of GABA), consistent with
desensitization. The apparent desensitization occurred at GABA
concentrations >10 µM and was typically monoexponential in time course, with faster rates at increased GABA concentrations. Typical current traces from an NT2-N cell are shown in Figure 1A. Peak amplitudes of
NT2-N cell responses (n = 12) to a range of GABA
concentrations (300 nM to 300 µM) were pooled
and fitted to a sigmoidal logistic function (EC50,
21.8 µM; Hill slope, 1.2) (Fig. 1B).
Individual EC50 values ranged from 7 to 80 µM
with an average of 31.3 ± 8.3 µM. NT2-N cells had
an average maximal current of 472.4 ± 70.5 pA at 300 µM GABA (n = 12).
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Figure 1.
GABA concentration-response characteristics for
NT2-N cell GABARs. A, Representative traces of
GABAR responses to increasing concentrations of GABA
(Vh = 75 mV). Horizontal bar, GABA
application (3, 30, or 300 µM). B,
Concentration-response curve of average peak GABA-evoked currents
(n = 12) fitted with a four-parameter logistic
equation. Data are mean ± SEM.
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Voltage dependence of GABAR currents
The degree and direction of GABAR current rectification depended
on the GABAR isoform (Burgard et al., 1996). Whole-cell
current-voltage (I-V) relations were generated in
NT2-N cells by measuring peak currents evoked by 30 µM
GABA at holding potentials from 100 to +75 mV at 25 mV increments. In
all cells studied (n = 6), there was no evidence of
inward or outward current rectification. A plot of the average peak
current at each holding potential for the four cells in which the
complete I-V relations were generated resulted in a linear
I-V plot (p < 0.001) (Fig.
2). An amplitude ratio of 0.90 ± 0.90 was calculated for six cells by dividing the peak current evoked
at +50 mV by the peak current evoked at 50 mV. This again indicates
that there was no rectification in either direction, because the
amplitude ratio was not significantly different from unity (see
Materials and Methods).
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Figure 2.
Voltage-dependent properties of NT2-N cell
GABARs. A, Current-voltage relationship for five NT2-N
cells. Peak currents evoked by 30 µM GABA, represented as
mean ± SEM, are plotted against holding potential showing no
rectification. B, NT2-N cells show no rectification or
voltage-dependent desensitization. Amplitude ratio
(filled bar) and desensitization ratio
(hatched bar) were not significantly different from 1. Bars represent mean ± SEM. Larger amplitude ratios indicate
greater outward rectification, and larger desensitization ratios
indicate more desensitization at positive membrane potentials.
C, Membrane currents recorded in NT2-N cells in response
to applications of 100 µM GABA (horizontal
bar). Responses were obtained at Vh levels of both
+50 and 50 mV. The response recorded at 50 mV has been inverted and
superimposed on the +50 mV trace for comparison. The degree of
desensitization was calculated by dividing the current remaining at the
end of GABA application (arrow) by the peak response
(see Materials and Methods).
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Voltage-dependent desensitization to high concentrations of GABA was
also dependent on the GABAR isoform expressed (Burgard et al., 1996).
In the continued presence (15 sec) of 100 µM
and higher GABA concentrations, there was a prominent exponential decrease in amplitude from peak until the agonist was removed. The rate
of apparent desensitization increased with higher GABA concentration.
With our application system, rapid desensitization (time constants <50
msec) could not be recorded, but slower desensitization (time constants
>100 msec) could be characterized. To determine whether this apparent
desensitization was voltage-dependent, cells were held at +50 and 50
mV for sequential 15 sec applications of 100 µM GABA. The
current amplitude at the end of the application was divided by the peak
(initial) current for each holding potential. The desensitization ratio
for the four cells tested was 1.01 ± 0.12 (see Materials and
Methods), indicating no voltage dependence of desensitization during
applications of 100 µM GABA at these holding potentials.
Single-channel currents
Single-channel currents were recorded in the outside-out
configuration from a total of 116 GABA applications onto 19 excised patches in six separate experiments. However, most of these patches were not used for kinetic analysis because of the large number of
overlapping or multiple openings, suggesting a high density of GABAR
channels in the NT2-N cell membrane. Five patches were obtained in four
of these experiments in which single openings predominated during
applications of 1, 3, and 10 µM GABA. Data from
these patches were used for most of the kinetic and amplitude analysis
(see Materials and Methods).
GABAR single-channel currents opened singly and in bursts of multiple
openings (Fig. 3A). At least
two conductance levels were observed at most holding potentials: a main
conductance level of 23 pS in which the majority of openings were
classified and a subconductance level of ~19 pS (Fig. 3B).
Rare openings to a conductance level of ~27 pS were observed more
commonly at negative than positive holding potentials. A
current-voltage relationship was obtained by holding the membrane
potential at voltages from 80 to +80 mV in 20 mV steps during
sequential applications of 3 µM GABA. Openings were
detected using a 50% threshold based on the amplitude of the main (23 pS) conductance level and thus included both the subconductance (19 pS)
and larger conductance level (27 pS) openings. Event amplitude
histograms were best fit using three Gaussian functions; the
statistical means of these functions for a single patch are displayed
in the current-voltage relationship shown in Figure 3B.
Linear regressions of mean amplitudes at each holding potential showed
reversal of single-channel currents at ~0 mV for all three
conductance levels, consistent with the reversal potential for chloride
with these solutions. None of the three conductance levels demonstrated
either inward or outward rectification at the holding potentials
examined. An example of the amplitude histogram for openings in a
different patch held at +50 mV (Fig. 3C) and 50 mV (Fig.
3D) demonstrates symmetrical conductance levels (i.e., no
inward or outward current rectification), although the relative
proportion of main to subconductance level openings was somewhat larger
at more negative potentials. The relative proportion of 27 pS
openings was small (5-10%) at both positive and negative holding
potentials in most patches.
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Figure 3.
Single-channel currents obtained in the
outside-out configuration from NT2-N cells. A,
Representative raw data traces of channel openings in NT2-N cells
illustrating the typical bursting pattern of openings to two different
conductance states (openings to the subconductance state are indicated
by asterisks). The same sample data are
displayed on three different time scales (see calibration bars).
B, Current-voltage relationship for single-channel
currents evoked by 3 µM GABA. Amplitudes at each holding
potential were obtained from amplitude histograms that were best fit
with the means of three Gaussian distributions. C, D,
Amplitude histograms of single-channel openings. The amplitudes of
individual openings were measured and placed into 0.05 pA bins. The
resulting frequency histograms were best fit with three Gaussian
distributions of 27, 23, and 19 pS.
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The frequency of single-channel openings increased with increasing GABA
concentration, from 10.4 openings/sec during application of 1 µM GABA to 28.8 openings/sec with 10 µM
GABA (Table 1). Mean open time also
increased slightly from 1.4 to 1.6 msec, and the percentage of time
open increased from 1.8 to 5.2%, consistent with both longer burst
openings and increased opening frequency at increased GABA
concentration. The mean closed time diminished as GABA concentration
increased. Open-duration histograms from selected patches with minimal
overlapping or multiple openings were fitted best with three
exponential functions, suggesting three open time constants with
durations of 0.2, 1.5, and 4.5 msec (Fig.
4A, Table
2). These time constants did not change significantly with GABA concentration, but the percentage of openings classified in each exponential distribution varied with GABA
concentration. With 1 µM GABA, most of the openings were
classified in the shortest (O1) distribution (57%) with smaller
proportions of openings in the two longer opening classes (23% O2
openings and 12% O3 openings). As GABA concentration increased, the
percentage of O1 openings decreased, and the percentage of longer
openings increased (Fig. 4A), as predicted by models
in which the binding of more than one GABA molecule drives the channel
into longer open states (Macdonald et al., 1989). However, with
10 µM GABA, a concentration at which almost all openings
would be expected to be in the O2 and O3 classes, there were still a
large number of O1 openings. This may have resulted from "trapping"
of liganded channels in a desensitized distal closed state, from which
recovery was slow enough that brief applications of GABA (lasting
30-60 sec) were biased toward sampling channels in the O1 and O2
states.
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Figure 4.
Open and closed histograms of single-channel
currents evoked by 1, 3, and 10 µM GABA.
A, Open-duration histograms for a single patch exposed
to 1, 3, and 10 µM GABA. Open times were best fit by
three exponential distributions with time constants and proportions as
reported in Table 2. B, Closed-duration histograms for a
single patch exposed to 1, 3, and 10 µM GABA. Closed
times were best fit by five exponential distributions with time
constants and proportions as reported in Table 2.
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Closed-duration histograms were fitted best with five exponential
functions (Fig. 4B). As GABA concentration increased,
the durations of the three longer closed states (C3, C4, and C5)
decreased, whereas the durations of the two short states (C1 and C2)
did not change (Fig. 4B). This was consistent with
previous observations in our laboratory (Twyman et al., 1990; Angelotti
and Macdonald, 1993) and the interpretation that the C1 and C2 states
represent intraburst closures. Histograms of burst durations pooled for each GABA concentration (based on a critical gap determined for each patch) were fitted best with three exponential distributions whose time constants did not change significantly between 1 and 3 µM GABA (1.27, 3.07, and 24.4 msec for 1 µM
GABA; 1.96, 2.32, and 24.7 msec for 3 µM GABA) (data not
shown), but the proportions shifted from shorter to longer burst
durations (64.5, 22.5, and 12.8% for shortest to longest durations
for 1 µM GABA vs 31.0, 28.2, and 40.6% for 3 µM GABA). There was an increase in mean burst duration
from 3.9 at 1 µM GABA to 10.8 msec at 3 µM
GABA, with a concomitant increase in mean openings per burst from 3.6 to 4.9 (Table 1). At 10 µM GABA, however, mean burst
duration and the number of openings per burst decreased to 6.6 and 3.1 msec, respectively, again probably because of entry into desensitized states from which recovery may be slow.
Pharmacology of GABAR currents
Individual NT2-N neurons were tested for their responsiveness to a
variety of GABAR modulators. In each of the cells tested with all of
the following drugs (n = 3), the positive allosteric modulators diazepam (300 nM), loreclezole (3 µM), and pentobarbital (100 µM) enhanced
currents evoked by 10 µM GABA (Fig.
5A), whereas the negative
allosteric modulators zinc (100 µM), bicuculline (100 µM), and picrotoxin (30 µM) inhibited
currents evoked by 30 µM GABA (Fig. 5B). GABAR
currents in NT2-N cells were further studied to characterize the
pharmacology of NT2-N GABARs in greater detail with a variety of
compounds that modulate GABAR function (Table
3).
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Figure 5.
Pharmacological responses of an individual neuron
to a variety of allosteric modulators that act at distinct GABAR sites.
A, Coapplication of GABA plus positive allosteric
modulators. Membrane currents were recorded in response to 10 µM GABA, followed by 10 µM GABA plus 300 nM diazepam (DZ), 3 µM loreclezole
(LOR), and 100 µM pentobarbital
(PB), and then 10 µM GABA alone.
B, Coapplication of GABA with negative allosteric
modulators recorded in the same neuron as shown in A.
Current traces were recorded in response to 30 µM GABA,
followed by GABA plus 100 µM zinc
(Zn2+), GABA plus 100 µM
bicuculline (BIC), GABA alone, and finally 30 µM GABA plus 30 µM picrotoxin
(Picro). Horizontal bars, Drug applications
made at 2 min intervals.
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Barbiturates
Both pentobarbital (Fig.
6A) and phenobarbital
(Fig. 6B) enhanced whole-cell GABAR currents evoked
by 10 µM GABA. Pentobarbital maximally enhanced
GABA-evoked currents by 434 ± 60% at 200 µM (n = 6) with an apparent EC50 of 41.3 µM and Hill slope of 1.47 (Fig. 6C).
Phenobarbital maximally enhanced GABAR current to 331 ± 37% of
control at 3 mM but was ~10-fold less potent with an apparent EC50 of 412 µM and a Hill slope of
2.21 (n = 5) (Fig. 6C).
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Figure 6.
Enhancement of GABA-evoked currents by
barbiturates with different affinities in NT2-N cells.
A, Representative current traces showing current evoked
by 10 µM GABA and enhancement by 30 and 300 µM pentobarbital (PB) (showing maximal
enhancement). B, Representative current traces showing
current evoked by 10 µM GABA and enhancement by 300 µM and 3 mM phenobarbital
(PhB). Horizontal bars, Drug applications
made at 2 min intervals. C, Concentration-response
curves for enhancement of 10 µM GABAR peak currents by PB
(n = 6) and PhB (n = 5).
Ordinate, Percent of peak response to 10 µM GABA is displayed. Data are mean ± SEM.
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Benzodiazepines
Diazepam (300 nM) enhanced whole-cell GABAR currents
evoked by 10 µM GABA (Fig.
7A). Diazepam maximally
enhanced GABAR current to 340 ± 42% (n = 5) of
control at 100 nM diazepam with an apparent EC50 of 74.2 nM and a Hill slope of 1.08 (Fig. 7D). Concentration-response curves were obtained
in five cells by coapplication of diazepam with 10 µM
GABA.
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Figure 7.
Benzodiazepine, imidazopyridine, and
-carboline modulation of NT2-N cell GABAR currents.
A-C, Representative current traces from three
different NT2-N cells showing maximal effect of three different
modulators at the benzodiazepine site. The first pair of traces
(A) shows control (10 µM
GABA) and maximal enhancement by diazepam (DZ) (3 µM); the second pair (B)
demonstrates 10 µM GABA control current and maximal
enhancement by zolpidem (ZOL) (10 µM); and the
third pair (C) shows the control response to 30 µM GABA followed by maximal inhibition by DMCM (3 µM). Horizontal bars, Drug applications
made at 2 min intervals. D, Concentration-response
relationships for enhancement of peak 10 µM GABAR
currents by diazepam (n = 5) and zolpidem
(n = 9) and inhibition of 30 µM GABAR
currents by DMCM (n = 9). Ordinate,
Percent of peak response to 10 µM GABA (diazepam or
zolpidem) or 30 µM (DMCM). Data are mean ± SEM.
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Zolpidem
The imidazopyridine zolpidem is another GABAR benzodiazepine site
agonist sensitive to the subunit subtype. Zolpidem (10 µM) enhanced GABAR currents evoked by 10 µM
GABA in all but one of the NT2-N cells studied (eight of nine) (Fig.
7B). Zolpidem enhanced GABAR currents with moderate affinity
when coapplied with 10 µM GABA (EC50,
527 nM; Hill slope, 0.99) (Fig. 7D).
DMCM
DMCM is a -carboline that acts as an inverse agonist at the
benzodiazepine site. DMCM (3 µM) reduced whole-cell GABAR
currents evoked by 30 µM GABA (Fig. 7C). DMCM
produced a maximal reduction of 44.6 ± 5.2% of GABAR current
(n = 9) with an IC50 of 44.8 nM and Hill slope of 1.12 (Fig. 7D). Eight of the nine cells
tested showed inhibition of the current across the range of DMCM
concentrations tested. However, in one cell GABAR currents were
enhanced at concentrations up to 30 nM and inhibited at
higher concentrations.
Loreclezole
The novel anticonvulsant drug loreclezole
interacts with a site on the 2 and 3 subtypes to enhance GABAR
currents (Wafford et al., 1994). Loreclezole enhanced GABAR currents
evoked by 10 µM GABA in every cell tested
(n = 5) (Fig.
8A). Peak enhancement occurred at 10 µM loreclezole (240 ± 46%), but the
degree of enhancement decreased at 30 µM loreclezole
(148 ± 23%) for all five cells tested (Fig. 8), as previously
reported for recombinant 532 GABARs (Donnelly and Macdonald,
1996). To determine the EC50 for loreclezole, only the
responses between 100 nM and 10 µM were used
to generate the concentration-response relationship. Under these
conditions, the loreclezole EC50 was 1.22 µM
with a Hill slope of 3.26 (n = 5) (Fig.
8B).
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Figure 8.
NT2-N cell GABAR current enhancement by
loreclezole. A, Current traces from a single cell
showing concentration-dependent enhancement of 10 µM
GABAR currents by loreclezole (100 nM to 30 µM). Concentrations of loreclezole applied with 10 µM GABA are shown above the traces. Horizontal
bars, Drug applications made at 2 min intervals.
B, Loreclezole concentration-response relationship for
GABAR current enhancement (n = 5). Data are
mean ± SEM. The EC50 was derived from a
four-parameter logistic equation fit to points up to 10 µM loreclezole. Inhibitory effects of higher
concentrations of loreclezole were not investigated but may have
reduced the maximal enhancement seen at 10 µM loreclezole
and thus affected the calculated EC50.
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Neurosteroids
Neurosteroids have been shown to differentially
modulate GABAR currents. Alphaxalone
(5-pregnan-3-ol-11,20-dione) and pregnenolone sulfate
(5-pregnan-3-ol-20-one sulfate sodium salt) are neurosteroids that
enhance and inhibit GABAR currents, respectively. Alphaxalone (10 µM) enhanced currents elicited by GABA (10 µM) by 431 ± 31% (n = 4).
Increasing concentrations of alphaxalone (30 nM to 30 µM) resulted in greater enhancement of the peak current
(Fig. 9A). A
concentration-response curve of the enhancement of peak GABAR current
relative to control was fit with a four-parameter logistic equation
yielding an apparent EC50 of 414 nM and a Hill slope of 0.94 (Fig. 9B). Pregnenolone sulfate (10 pM to 10 µM) decreased GABAR currents (Fig.
9C) in a concentration-dependent manner starting at 1 nM with a maximal inhibition of 67.5 ± 1.9% at 10 µM (Fig. 9D). An accurate IC50 for
pregnenolone sulfate could not be calculated, because 10 µM was near the limit of solubility. However, fitting the
data with a logistic equation where Imax was set
at 0% yielded an equation with an apparent IC50 of 4.9 µM and a Hill slope of 0.50.
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Figure 9.
Modulation of NT2-N cell GABAR currents by
neurosteroids. A, Representative current traces
demonstrating enhancement of 10 µM GABAR currents by
alphaxalone (ALX, 1 and 10 µM).
B, ALX concentration-response relationship for GABAR
current enhancement (n = 4). Data are mean ± SEM. C, Representative current traces of pregnenolone
sulfate (PS) inhibition of 30 µM
GABA-evoked currents at control levels (GABA alone), moderate
inhibitory concentration (+100 nM PS), and maximal
concentration (+10 µM PS). D, Pregnenolone
sulfate concentration-response relationship for GABAR current
inhibition (n = 4). Data are mean ± SEM. An
apparent IC50 was calculated by a logistic equation where
Imax was set at 0%.
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Bicuculline
Bicuculline is a competitive GABAR antagonist and has
no effect on GABAB receptors, GABAC receptors,
or the phylogenetically and structurally similar glycine receptor
(Macdonald and Olsen, 1994). Bicuculline inhibits current at any
functional GABAR isoform, irrespective of subunit composition. Currents
evoked by 30 µM GABA in NT2-N cells were inhibited by
bicuculline (10 nM to 30 µM)
(EC50, 1.1 µM; Hill slope, 0.72)
(Fig. 10A,C).
Currents elicited by 30 µM GABA were completely blocked
by coapplication of 100 µM bicuculline.
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Figure 10.
Inhibition of NT2-N cell GABAR currents by
competitive (BIC) and noncompetitive
(FUR) antagonists. A, Representative
current traces of BIC inhibition of 30 µM GABA-evoked
currents at control levels (GABA alone), IC50 concentration
(+1 µM BIC), and a near maximal concentration (+30
µM BIC). B, Representative current traces
of furosemide inhibition of 30 µM GABA-evoked currents at
control levels (GABA alone), +100, 300 µM FUR and maximal
concentration (+3 mM FUR). C,
Concentration-response curves for inhibition of peak current
amplitudes by BIC (n = 5) and FUR
(n = 5). Either BIC or FUR was coapplied with 30 µM GABA. Data are mean ± SEM.
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Furosemide
Furosemide, an anthranilic acid derivative, inhibits
recombinant GABAR currents with IC50s in the micromolar
range, but only when either an 4 or 6 subunit is present (Wafford
et al., 1996). Furosemide (10 µM to 3 mM)
inhibited NT2-N GABAR currents evoked by 30 µM GABA only
at concentrations >100 µM (Fig. 10B).
The greatest inhibition observed was 78.2 ± 1.2% at 3 mM furosemide; concentrations >3 mM could not
be achieved because of limits of solubility; hence, true maximal
inhibition could not be assessed. However, fitting the data with a
logistic equation where Imax was set at 0%
produced an apparent IC50 of 1.4 mM and a Hill
slope of 1.3 (Fig. 10C).
Picrotoxin
Picrotoxin, a noncompetitive GABAR antagonist, also inhibited
NT2-N GABAR currents. Picrotoxin (30 µM) blocked 71.5%
of the peak current evoked by 30 µM GABA
(n = 8) (data not shown). A complete
concentration-response curve was not generated because of the
long-lasting effects of moderate concentrations of picrotoxin (Table
3).
Polyvalent cations
The divalent cation zinc (Zn2+) is a
noncompetitive antagonist of GABAR currents. Zn2+
(300 nM) reduced whole-cell GABAR currents evoked
by 30 µM GABA (Fig.
11A). Inhibitory
concentration-response curves were generated by increasing the
concentration of Zn2+ (100 nM to 1 mM) coapplied with a concentration of GABA near the
EC50 for NT2-N cells (30 µM) (Fig.
11C). The IC50 for Zn2+ was
14.7 µM with a Hill slope of 0.43 (n = 5). GABAR currents were almost completely blocked by 1 mM
Zn2+ (94.8 ± 1.7%).
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Figure 11.
Effects of polyvalent cations on GABAR currents.
A, Representative current traces of
Zn2+ inhibition of 30 µM GABA-evoked
currents at control levels (GABA alone), IC50 concentration
(+10 µM Zn2+), and maximal
concentration (+1 µM Zn2+).
B, Representative current traces of
La3+ inhibition of 30 µM GABA-evoked
currents at control levels (GABA alone), +100 µM
La3+ and maximal concentration (+1 mM
La3+). Horizontal bars, Drug
applications made at 2 min intervals. C,
Concentration-response curves for the effects of the polyvalent
cations Zn2+ (n = 5) and
La3+ (n = 11) on peak current
amplitudes. Either Zn2+ or La3+
was coapplied with 30 µM GABA. Data are mean ± SEM.
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The trivalent cation lanthanum (La3+), when
coapplied with a GABA concentration near the EC50,
inhibits 6-containing GABARs and enhances 1-containing
GABARs (Saxena et al., 1997). La3+ (100 nM to 1 mM) was coapplied with either 10 µM (data not shown) or 30 µM GABA to NT2-N
cells (Fig. 11B). La3+ had little
effect on peak GABAR currents elicited by either GABA concentration.
There were no statistically significant changes at lower
La3+ concentrations. A small (22.5 ± 4.8%)
but significant inhibition of GABAR currents evoked by 30 µM GABA was seen at 1 mM
La3+ (n = 11) (Fig.
11C).
Distribution of maximal effects on individual neurons
The pharmacological data for the allosteric modulators that were
tested are summarized in Table 3. The maximal effect was statistically
compared with control GABA currents for each cell using paired
t tests. Statistical significance was reached for each
compound (*p < 0.05; **p < 0.01; and
***p < 0.001). Normalized maximal effects of all
compounds tested on individual cells were evenly distributed around the
mean and not separated into distinct groups.
PCR analysis of GABAR subunit mRNAs
Total RNA was isolated from cultures of NT2-N cells at the same 5 week time point that electrophysiological studies were performed. Reverse transcription PCR (RT-PCR) was used to determine the presence or absence of GABAR subunits using primers specific for human 1-6,
1-3, 1-3, , and subunit subtypes (Table
4). In repeated experiments, major bands
were found for 2, 3, 5, 3, 3, and subtypes (Fig.
12), whereas bands for 1, 2,
1, and 2 subtypes were not found. Faint bands for the remaining
subtypes (4, 6, and 1) were inconsistently found. Primers
designed to amplify the subunit did not amplify products of the
predicted molecular size. Multiple primers were designed for the 3
subtype. The molecular masses of the PCR products were slightly larger
than the cDNA-positive control when the primers amplified the entire
N-terminal domain to the first intracellular loop [amino acids (aa)
18-267]. However, when the primers were designed to amplify only part
of the N-terminal domain (aa 110) to midway through the large
intracellular loop between M3 and M4 (aa 413), the PCR products were
the same molecular mass as the cDNA control (data not shown). These
same primers were unable to amplify either the 1 or 2 subtype,
suggesting that the 3 subtype expressed in NT2-N cells may represent
a longer splice variant than previously described.
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Figure 12.
RT-PCR reaction amplification of GABAR subunits
in NT2-N cells. Lanes are designated as follows:
M, marker; C, human cDNA for each subunit
subtype used as a positive control; +, NT2-N RNA with Superscript II;
, NT2-N RNA without Superscript II used as a negative control.
A, Agarose gel of GABAR subunits showing the
presence of 2, 3, and 5 but not 1. (1,
lanes 2-4; 2, lanes 5-7;
3, lanes 8-10; 5,
lanes 11-13; lanes 1 and 14 are
standards). B, Non- subunits showing major bands for
3, 3, and but only a faint band for 1.
(1, lanes 2-4; 3,
lanes 5-7; 3, lanes 8-10;
lane 1 standard). C, Major band for the subunit (, lanes 2-4; lane 1 standard). In
some lanes, bands of lower molecular mass than that predicted for the
product of interest, which are nonspecific PCR amplification products,
were stained (Bloch, 1991).
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DISCUSSION |
NT2-N cells express primarily 2, 3, 5, 3, 3, and GABARs subtype mRNAs
High mRNA levels for the 2, 3, 5, 3, 3, and subtypes were found using RT-PCR performed on total RNA isolated from
NT2-N cells. Minimal mRNA levels for 4, 1, and 1 subtypes were
found in some, but not all, of the experiments, and no transcripts
encoding human 1, 6, 2, or 2 subtypes were detected. The
pharmacological data suggested that a limited number of subunit
subtypes were endogenously expressed in functional GABARs in NT2-N
cells, which was confirmed by the use of RT-PCR. These subtype mRNAs
were consistent with most of the pharmacological properties of GABAR
currents recorded from NT2-N cells. Because RNA was isolated from an
entire 10 cm dish of cells with no preference for cell type, we cannot rule out the possibility that there were subpopulations of NT2-N cells
that expressed different isoforms based on either the stage of
differentiation or differences in cell fate. It is unlikely that
retinoic acid treatment alone would cause multiple fates of clonal NT2
stem cells. However, it is possible that multiple cell fates could
occur during the differentiation process caused by secondary factors
such as cell-cell or cell-substrate interactions that may alter the
major GABAR isoforms being expressed. The possible existence of
subpopulations of cells may also explain the low levels of 4, 6,
and 1 mRNA and the inconsistency of their presence across RNA
preparations. Although it is unlikely that NT2-N cells differentiate
into a specific neuronal population found in the developing vertebrate
brain, it is interesting to note that the subunits found expressed at
high levels in NT2-N cells at this time point are located on
chromosomes 15 (5, 3, and 3) and X (3). With the exception
of the 2 subtype mRNA, those barely detected were located on
chromosome 4 (2, 4, 1, and 1), and those not expressed at
all were located on chromosome 5 (1, 6, 2, and 2) (Rabow et
al., 1995). The subunits expressed in NT2-N cells have been shown to be
primarily expressed early in the development of the rat brain (Laurie
et al., 1992), whereas those located on chromosome 5 are the
predominate subunits found in adult rat brains (Wisden et al., 1992).
NT2-N cells resemble immature CNS neurons, as determined by markers
such as fetal  , the incomplete phosphorylation state of high
molecular weight neurofilament protein, and no MAP2b expression
(Pleasure et al., 1992). Further studies are under way to determine
whether all NT2-N cells contain all subtype mRNAs or whether there are
multiple populations of cells with identifiable morphologies that
contain different combinations of messages and whether the NT2 cell
line undergoes a developmental switch in subunit expression similar to
that found in rat brain.
NT2-N cells express GABARs likely composed primarily of 2, 3,
5, 3, and 3 subtypes
Differentiated NT2-N neurons expressed GABARs with a
distinct set of pharmacological and biophysical properties and
expressed a limited number of GABAR subtype mRNAs. High levels of 2,
3, 5, 3, 3, and subunit subtype mRNAs were consistently
amplified using RT-PCR performed on total RNA isolated from NT2-N
cells. These data suggest that NT2-N cells express a limited number of GABAR isoforms with relatively uniform biophysical and pharmacological properties. NT2-N cells constitute the only known human-derived clonal
cell line that express functional GABARs; therefore, they provide a
unique preparation for investigation of human GABAR expression,
assembly, structure, and regulation.
Studies using nonhuman recombinant GABAR subunits have shown that the
affinity and efficacy for many GABAR modulators, as well as certain
biophysical properties, depend on the expressed isoform. Although no
definitive conclusions about the subunit subtype composition of GABARs
can be made by determination of pharmacology and kinetic properties,
several pharmacological and biophysical properties of NT2-N GABARs
limit the possible expressed subtypes. Possible subtypes can be further
limited by comparison of the pharmacological, biophysical, and RT-PCR
data.
All NT2-N cells responded to GABA with a mean GABA EC50 of
21.8 µM, and individual EC50 values and
maximal peak amplitudes were evenly distributed around the mean.
Recombinant GABARs expressing rat or bovine , , and subunits
had EC50 values ranging from 1-50 µM,
whereas GABARs expressing , , or subunits
alone had lower GABA EC50 values, ranging from 0.5 to 5 µM (Angelotti et al., 1993; Saxena and Macdonald, 1994)
(T. R. Neelands and R. L. Macdonald, unpublished results).
Thus, NT2-N cell GABARs had a GABA EC50 that was consistent
with an isoform containing , , and subunits.
NT2-N GABARs likely contain a 3 subtype
Enhancement of GABAR currents by benzodiazepine site ligands
requires the presence of a subunit in the receptor (Knoflach et
al., 1991; Luddens et al., 1994; Hadingham et al., 1995; Benke et al.,
1996). Because NT2-N cells expressed high levels of mRNA for 3, but
not 1 or 2 subtypes, and GABAR currents were enhanced by
diazepam, zolpidem, and reduced by DMCM, it is likely that NT2-N GABARs
contain a 3 subtype.
NT2-N GABARs likely contain 2 and/or 3 subtypes
Recombinant GABARs with high affinity for benzodiazepines contain
the 1 subtype, and low-affinity receptors contain either 4 or
6 subtypes when coexpressed with and subtypes. Receptors with moderate affinity for diazepam can be further divided based on
moderate affinity (2- or 3-containing receptors) or insensitivity (5-containing receptors) to zolpidem. In addition, DMCM, an inverse agonist at the benzodiazepine site, inhibited GABAR |
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Abstract
Introduction
