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The Journal of Neuroscience, August 15, 1999, 19(16):7057-7065
GABAA Receptors Expressed in Undifferentiated Human
Teratocarcinoma NT2 Cells Differ from Those Expressed by Differentiated
NT2-N Cells
Torben R.
Neelands1,
Jie
Zhang1, and
Robert L.
Macdonald2, 3
1 Graduate Program in the Neurosciences and Departments
of 2 Neurology and 3 Physiology, University of
Michigan Health Sciences Center, Ann Arbor, Michigan 48104-1687
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ABSTRACT |
During CNS development, changes occur in expression of
GABAA receptor subunit subtypes and GABAA
receptor pharmacological and biophysical properties. We used reverse
transcription PCR and whole-cell-recording techniques to
determine whether GABAA receptor expression and function
also changed during retinoic acid-induced differentiation of human
Ntera 2 (NT2) teratocarcinoma cells into neuron-like cells
(NT2-N cells). In undifferentiated NT2 cells only  5,  3,  3, and
 subtype mRNAs were detected. NT2 GABAA receptor
currents had a maximal amplitude of 52 pA and an EC50 of
4.0 µM, were relatively insensitive to enhancement by
zolpidem and diazepam, and were enhanced by loreclezole and inhibited
by lanthanum, zinc, and furosemide. In contrast, in NT2-N cells after
13 weeks of retinoic acid treatment, all GABAA receptor
subtype mRNAs were detected. Maximal peak whole-cell currents were
~50-fold larger than NT2 cell currents, and the GABA EC50
was higher (39.7 µM). In 13 week NT2-N cells, diazepam, zolpidem, loreclezole, and lanthanum had only small effects on GABAA receptor currents, and the zinc IC50 for
current inhibition was significantly higher than that for NT2 cells. In
a previous study, we showed that NT2-N cells after 5 weeks of retinoic
acid treatment had moderate peak currents, GABA EC50, and
zinc IC50 but that currents were robustly enhanced by
diazepam, zolpidem, and loreclezole. During differentiation of NT2
cells to NT2-N cells, GABAA receptors underwent changes in
subunit expression and pharmacology that were similar to many of the
developmental changes in GABAA receptors that occur in CNS neurons.
Key words:
GABA; GABAA receptor; electrophysiology; patch clamp; RT-PCR; Ntera2; loreclezole; benzodiazepine; zinc; differentiation; NT2 cells; NT2-N cells
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INTRODUCTION |
The Ntera 2/cl.D1 subclone (NT2) of
neuronal precursor cells was isolated from the TERA2 cell line for its
ability to differentiate into postmitotic neuron-like cells (NT2-N
cells) when treated with retinoic acid (Pleasure et al., 1992 ).
Therefore, NT2 cells provide a potentially interesting cell line for
studies of the regulation of neuronal development (Andrews, 1998). We
reported previously that after 5 weeks of retinoic acid treatment,
differentiated NT2-N cells expressed a limited number of
GABAA receptor (GABAR) subtype mRNAs and that the
pharmacological and biophysical properties of GABAR currents were
consistent with those of GABARs assembled from these subunit mRNAs
(Neelands et al., 1998). In the CNS, expression of GABAR subunits is
developmentally and regionally regulated and may be associated with
changes in GABAR function and pharmacology (Laurie et al., 1992;
Poulter et al., 1992; Fritschy et al., 1994; Oh et al., 1995). Little
is known, however, about the developmental regulation of expression and
assembly of CNS GABARs.
GABARs are composed of five subunits that together form a chloride ion
channel that mediates fast IPSPs in mature CNS. Six different
subunit families (, , ,  , , and  ) and multiple subtypes (1-6, 1-4, and 1-4) have been identified
(Macdonald and Olsen, 1994; Davies et al., 1997; Hedblom and Kirkness,
1997). Each subtype has a unique regional expression in the brain, and individual neurons often express multiple subtypes. The assembly (Angelotti et al., 1993; Saxena and Macdonald, 1994; Burgard et al.,
1996; Neelands et al., 1999) and stoichiometry (Chang et al., 1996;
Tretter et al., 1997) of GABARs are regulated so that all the potential
combinations of subunits do not form functional channels. Studies of
recombinant GABARs have shown that the subunit composition of the
receptor isoform can influence their pharmacological and biophysical
properties (Pritchett et al., 1989a; Angelotti and Macdonald, 1993;
Macdonald and Olsen, 1994). The regional and developmental regulation
of GABAR subunit subtypes regulates the GABAR isoforms expressed and,
therefore, the function of the receptors.
The majority of GABAR subunit subtype genes are found in four clusters
on human chromosomes 4 (2, 4, 1, and 1), 5 (1, 6,
2, and 2), 15 (5, 3, and 3) (McLean et al., 1995; Rabow et al., 1995), and X (3, , and 4) (Wilke et al., 1997). The subunit is also located on chromosome 5 but is not tightly linked with the GABAR gene cluster (E. Kirkness, personal
communication), and the subunit has been mapped to human
chromosome 1 (Sommer et al., 1990). In general, GABAR subunits whose
genes are located on chromosomes 4 and 15 are expressed early in the
developing rat brain, and those on chromosome 5 are expressed in adults
(Laurie et al., 1992). In this study we determined whether retinoic
acid-induced differentiation of NT2 cells into neuron-like NT2-N cells
produced changes in GABAR subunit mRNA expression and in GABAR
biophysical and pharmacological properties that were similar to those
occurring during neuronal development.
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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 added as described previously (Andrews,
1984). NT2 cells were plated directly onto 35 mm dishes for
electrophysiological recordings or were plated at 2 × 106 cells/75
cm2 flask and differentiated by treatment
with 1 µM retinoic acid for 4 weeks. After retinoic acid
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 medium was removed and
saved as conditioned medium 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 medium containing mitotic
inhibitors (10 µM FUDR + 10 µM
uridine and 1 µM cytosine arabinoside) and replated
(replate II). The same treatment was performed again after 1 week in
culture (replate III) to obtain nearly 100% pure neuron-like cells.
These cells were plated onto 10 cm dishes and kept in DMEM HG and
maintained in 5% CO2 for an additional 8 weeks.
During the 13th week after the start of retinoic acid treatment, the 10 cm dishes of NT2-N cells were either treated with Ultraspec to
isolate total RNA or cut into sections for electrophysiological recording.
Solutions and drug application. Cells were removed from the
5% CO2 incubator, and the feeding medium was
replaced with recording medium containing (in mM): 142 NaCl, 1 CaCl2, 6 MgCl2,
8.09 KCl, 10 glucose, and 10 HEPES, 315-325 mOsm, with a pH of 7.4. Patch-clamp electrodes of 5-10 M were filled with pipette solution
containing (in mM): 153.33 KCl, 1 MgCl2, 10 HEPES, 5 EGTA, and 2 ATP, 300-310 mOsm, with a pH of 7.3. This combination of solutions results in nearly
equivalent intracellular and extracellular chloride ion concentrations,
hence an ECl near 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 and Macdonald, 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, zinc, and zolpidem were made by dissolving
each in sterile water. Stock solutions of loreclezole were dissolved in
dimethylsulfoxide (DMSO) and diluted with sterile water (final DMSO
concentration was less than 1:1000). Individual drugs were diluted in
recording medium to their final concentration. Loreclezole was obtained
from Janssen Biochimica (Berse, Belgium). 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, Houston TX) using a P-87 Flaming-Brown
micropipette puller (Sutter Instrument Company, San Rafael, CA).
Whole-cell recordings were performed using an Axopatch 200A amplifier
(Axon Instruments, Foster City, CA). Signals were digitized on-line at
200 Hz using a TL-1 Labmaster analog-to-digital converter, 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 with an
Axopatch 200A amplifier.
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 2.0 (Graph Pad, San Diego,
CA). Data were presented as mean ± SEM. To quantify whole-cell
current rectification, peak amplitudes of responses to GABA were
measured at holding potentials of  75 and +75 mV. An amplitude ratio
(+75 mV/75 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.
Reverse transcription PCR. Total RNA was isolated
from NT2 cells and NT2-N cells using the Ultraspec method by Biotecx
(Houston, TX). One milligram of total RNA was treated with DNase in a
total volume of 10 ml composed of 1 ml of 10× DNase I buffer, 1 ml of DNase I (1 U/ml), and 7 ml of DEPC water. This mixture was incubated for 15 min at 25°C. Addition of 1 ml 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 milliliter of a random primer (0.6 mg/ml) 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 ml
of 5× first-strand buffer, 2 ml of 0.1 M DTT, 1 ml of 10 mM dNTP mix, and 1 ml of RNase inhibitor (10 U/ml). The
mixture was prewarmed for 2 min at 42°C before addition of 1 ml of
Superscript II (10 U/ml) and then was incubated for 45 min at 42°C.
Using the same procedure as described above, with the exception that 1 ml 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. PCR was performed for each
subunit in 100 ml of the following mixture: 1 ml of a 20 mM
primer mixture (3' primer and 5' primer), 2 ml of the reverse transcription product, 10 ml of 10× buffer, 16 ml of 25 mM
MgCl2, 1 ml of 25 mM dNTP mixture, 0.5 ml of Amplitaq (5 U/ml), and 72.5 ml of DEPC-treated water. Positive controls were
performed using cDNA for each subunit subtype as the PCR template. 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, and 72°C
for 1 min; and a 7 min extension period at 72°C. PCR products (5 ml
for positive controls; 15 ml 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 as follows: Amplitaq from Perkin-Elmer (Norwalk, CT);
random primer, Superscript II, RNase inhibitor, and DNase I from Life
Technologies (Gaithersburg, MD); and the dNTP mix and DNA molecular
weight marker VI from Boehringer Mannheim (Indianapolis, IN).
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RESULTS |
Whole-cell GABAR currents in NT2 cells and 13 week NT2-N cells
NT2 cells and 13 week NT2-N cells were voltage clamped at 75 mV,
and increasing concentrations of GABA were applied for 6-10 sec (Fig.
1A). Whole-cell GABAR
currents recorded from both NT2 cells and 13 week NT2-N cells had
concentration-dependent activation rates (Fig. 1A).
At the higher GABA concentrations, the currents decreased in the
continued presence of GABA, consistent with desensitization. The rate
of apparent desensitization increased in a GABA concentration-dependent manner. The GABAR currents were similar to those seen in recordings from 5 week NT2-N cells, human neurons, and rat dentate granule cells
(Sah, 1995; Kapur and Macdonald, 1996; Neelands et al., 1998). Peak
current amplitudes evoked by a range of GABA concentrations (100 nM to 1 mM) were pooled and
fitted to a sigmoidal logistic function (Fig. 1B).
The maximal current was substantially larger for 13 week NT2-N cells
(2894 ± 504 pA; n = 4) than for NT2 cells (52 ± 11 pA; n = 5). Peak currents evoked at each
GABA concentration were normalized to the maximal current obtained from
each cell. The averaged data were plotted and fitted to a sigmoidal
logistic function (Fig. 1C). Plotting the data as a percent
maximum illustrated the 10-fold shift in the GABA
EC50 between the NT2 cells
(EC50 = 4.0 µM;
nH = 1.1) and the 13 week NT2-N
cells (EC50 = 39.7 µM; nH = 1.3). Included in the two plots
are the data we reported previously for the 5 week NT2-N cells
(Imax = 472 ± 71 pA;
EC50 = 21.8 µM;
nH = 1.2) that fall between the other
two stages of differentiation (Neelands et al., 1998).
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Figure 1.
GABA concentration-response characteristics for
GABARs expressed in NT2 cells and 5 and 13 week neurons.
A, Representative traces of GABAR
currents from NT2 cells (left) and 13 week NT2-N cells
(right) in response to application of increasing
concentrations of GABA are shown. Horizontal bars, GABA
application. B, Peak currents as a function of GABA
concentration were fit with a four-parameter logistic equation. Data
are presented as mean ± SE (Vh = 75 mV). Peak
currents increased during differentiation with retinoic acid.
C, Normalized concentration-response curves show an
increase in the GABA EC50 during differentiation by
retinoic acid. Data are mean ± SE.
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Whole-cell GABAR current-voltage (I-V) relations
were obtained for NT2 cells and 13 week NT2-N cells by measuring peak
currents evoked by GABA at holding potentials from 75 to +75 mV.
Plots of the average peak currents at each holding potential resulted in slight outward rectification for 13 week NT2-N cells and large outward rectification for NT2 cells (Fig.
2). An amplitude ratio was calculated for
each stage of differentiation by dividing the peak current evoked at
+75 mV by the peak current evoked at 75 mV. The amplitude ratios were
3.75 ± 0.25 for NT2 cells (n = 3) and 1.37 ± 0.26 for 13 week NT2-N cells (n = 5). Amplitude
ratios >1 indicated that there was outward rectification for cells
from both stages of differentiation (Fig. 2, inset) (see
Materials and Methods). In contrast, 5 week NT2-N cells showed no
evidence of inward or outward rectification of peak current (Fig. 2,
dashed line) as is illustrated by an amplitude ratio of
1.06 ± 0.12 (inset).
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Figure 2.
Whole-cell current-voltage relationships for NT2
cells and 5 and 13 week NT2-N cells. Peak GABAR, represented as
mean ± SEM, was plotted against the holding potential
(HP). Inset, Amplitude ratios were
calculated by dividing the peak current at +75 mV by the peak current
at 75 mV (see Materials and Methods). Larger amplitude ratios
indicate greater outward rectification. Data represent mean ± SEM.
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Pharmacology of GABAR currents in NT2 cells and 13 week
NT2-N cells
Loreclezole
The novel anticonvulsant drug loreclezole has been shown to
interact with a site on the 2 and 3 subtypes to enhance GABAR currents (Wafford et al., 1994). Loreclezole enhanced whole-cell GABAR
currents recorded from NT2 cells and 13 week NT2-N cells in a
concentration-dependent manner (Fig.
3A). Average normalized data
(excluding the data point for 30 µM
loreclezole) were fit with logistic equations with
EC50 values of 1.0 µM for
NT2 cells (n = 5) and 1.1 µM
for 13 week NT2-N cells (n = 3) (Fig. 3B). Loreclezole (3 µM) maximally enhanced GABAR
currents by 78.2 ± 24.8% in NT2 cells and by only 18 ± 7%
in 13 week NT2-N cells. We reported previously that loreclezole
enhanced GABAR currents from 5 week NT2-N cells in a
concentration-dependent manner (EC50 of 1.2 µM) and 3 µM
loreclezole increased currents by 133 ± 34% (Neelands et al.,
1998).
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Figure 3.
Enhancement of GABAR currents by loreclezole.
A, Representative current traces from NT2
cells (left) and 13 week NT2-N cells
(right) showing enhancement of GABAR currents (1 and 10 µM GABA, respectively) by coapplication of 3 µM loreclezole (LOR). Horizontal
bars, Drug applications. B, Loreclezole
concentration-response relationship for GABAR current enhancement of
NT2 cells (n = 5), 5 week NT2-N cells
(n = 5), and 13 week NT2-N cells
(n = 4). Data are mean ± SEM.
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Diazepam
The benzodiazepine diazepam only enhanced currents from
recombinant GABARs containing a subunit (Pritchett et al., 1989a), and the relative diazepam EC50 varied with the
subunit subtype expressed (Pritchett et al., 1989b). Diazepam
enhanced whole-cell GABAR currents in 13 week NT2-N cells in a
concentration-dependent manner (Fig.
4A).
Concentration-response curves were obtained in three cells by
coapplication of diazepam with 10 µM GABA and fitted with an EC50 of 27.9 nM. Diazepam maximally enhanced GABAR currents by
20 ± 12% of control at 1 µM diazepam
(Fig. 4A). Similarly, 1 µM
diazepam minimally enhanced currents evoked by 1 µM GABA in NT2 cells by 25 ± 8% (Fig.
4B). Full concentration-response curves for diazepam
were not generated for NT2 cells because of the small maximal effect in
combination with the small peak currents (~20 pA). Enhancement of
GABAR currents by 1 µM diazepam was
significantly less in both the NT2 cells and 13 week NT2-N cells
compared with the enhancement reported for GABAR currents in 5 week
NT2-N cells (232 ± 34%).
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Figure 4.
Enhancement of GABAR currents by diazepam.
A, Concentration-response curve for the enhancement by
diazepam of currents evoked by 10 µM GABA from 13 week
NT2-N cells (n = 3). Data are mean ± SEM.
B, Comparison of the ability of a saturating
concentration of diazepam (1 µM) to enhance GABAR
currents from NT2 cells (n = 3), 5 week NT2-N cells
(n = 5), and 13 week NT2-N cells
(n = 3). Data are mean enhancement ± SEM.
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Zolpidem
The imidazopyridine zolpidem is another GABAR benzodiazepine site
agonist that has a higher affinity for GABARs containing an 1
subtype (BZ 1 receptors). GABARs containing 5 subtypes can be
distinguished from other benzodiazepine-sensitive isoforms based on
their insensitivity to zolpidem (BZ 2c receptors) (Pritchett and
Seeburg, 1990). Zolpidem enhanced GABAR currents evoked by 10 µM in Figure 5A GABA in 13 week NT2-N
cells in a concentration-dependent manner (Fig.
5A). Zolpidem (3 µM) maximally enhanced GABAR currents by
23 ± 6%. Average normalized data were fit with a logistic
equation with an EC50 of 455 nM (n = 3) (Fig. 5A).
Coapplication of 1 µM zolpidem with 1 µM GABA to NT2 cells did not produce a
significant effect (2 ± 11%) (Fig. 5B). As for
diazepam, full concentration-response curves were not generated
because the high zolpidem concentration (1 µM)
did not alter GABAR currents. The ability of 1 µM zolpidem to enhance GABAR currents was
significantly less in both the NT2 cells and 13 week NT2-N cells
compared with that reported previously for 5 week NT2-N cells (98 ± 14%) (Neelands et al., 1998).
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Figure 5.
Enhancement of GABAR currents by zolpidem.
A, Concentration-response curve for the enhancement by
zolpidem of currents evoked by 10 µM GABA from 13 week
NT2-N cells (n = 3). Data are mean ± SEM.
B, Comparison of the ability of a high concentration of
zolpidem (1 µM) to enhance GABAR currents from NT2 cells
(n = 3), 5 week NT2-N cells (n = 5), and 13 week NT2-N cells (n = 3). Data are
mean enhancement ± SEM.
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Lanthanum
The trivalent cation lanthanum (La3+)
either potentiated (1 subtype) or inhibited (5 and 6 subtypes)
GABAR currents depending on the subtype composition of the receptor
(Saxena et al., 1997; Neelands and Macdonald, 1998). Lanthanum
increased peak whole-cell GABAR currents from 13 week NT2-N cells in a
concentration-dependent manner but inhibited currents evoked from NT2
cells (Fig. 6A). Concentration-response curves were generated for both NT2 cells and 13 week NT2-N cells by increasing the concentration of lanthanum (100 nM TO 1 mM) coapplied with
an EC equivalent concentration of GABA (Fig. 6B).
Average normalized data were plotted and fit with a four-parameter
logistic equation (see Materials and Methods). Lanthanum inhibited NT2
cell GABAR currents (IC50 = 137 µM; nH = 0.7; n = 2-3) and enhanced 13 week neuron
NT2-N cell GABAR currents (EC50 = 96.5 µM; n = 3). GABAR currents from
the NT2 cells were inhibited 79 ± 4% by 1 mM lanthanum in contrast to currents evoked from
the 13 week NT2-N cells that were enhanced by 9 ± 4% by 1 mM lanthanum. Lanthanum had been shown previously to have no significant effect on GABAR currents from 5 week NT2-N cells
(Fig. 6B, open diamonds, dashed line).
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Figure 6.
Effects of lanthanum on GABAR currents.
A, Current traces from NT2 cells
(left) and 13 week NT2-N cells (right)
showing concentration-dependent effects of GABAR currents (3 or 30 µM GABA, respectively) by coapplication of 1 mM lanthanum (La3+). Horizontal
bars, Drug applications. B,
Concentration-response curves for the effects of lanthanum on NT2
cells (n = 2-3), 5 week NT2-N cells
(n = 11), and 13 week NT2-N cells
(n = 3) on peak GABAR current amplitudes. Data are
mean ± SEM.
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Zinc
The divalent cation zinc has been shown to be an antagonist of
GABAR currents. Zinc reduced whole-cell GABAR currents from both NT2
cells and 13 week NT2-N cells in a concentration-dependent manner (Fig.
7A). Inhibitory
concentration-response curves were obtained by increasing the
concentration of zinc (100 nM to 1 mM) coapplied with a high concentration of GABA
for NT2 cells (10 µM) and 13 week NT2-N cells
(30 µM) (Fig. 7B). Average
normalized data were plotted and fitted by a four-parameter logistic
equation. The IC50 for zinc inhibition of GABAR
currents was less for NT2 cells (IC50 = 5.4 µM; nH = 0.8; n = 2-4) than for 13 week NT2-N cells
(IC50 = 64.7 µM;
nH = 0.4; n = 3).
NT2 cell GABAR currents were maximally inhibited by 100 µM zinc (96% of control); in contrast, currents evoked from the 13 week NT2-N cells were blocked only 85% by
1 mM zinc. Zinc has been shown previously to
inhibit GABAR currents from 5 week NT2-N cells with a moderate
IC50 of 14.7 µM
(nH = 0.9) and a maximal inhibition
by 1 mM zinc of 95% (Fig. 7B,
open diamonds, dashed line).
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Figure 7.
Inhibition of GABAR currents by zinc.
A, Current traces from NT2 cells
(left) and 13 week NT2-N cells (right)
showing concentration-dependent inhibition of GABAR currents (10 or 30 µM GABA, respectively) by coapplication of either 3 or 30 µM zinc. The current trace from the NT2
cell was abnormally large but illustrated the inhibition by zinc better
than the typical 20-40 pA currents. Horizontal bars,
Drug applications. B, Concentration-response curves for
the effects of zinc on NT2 cells (n = 2-4), 5 week
NT2-N cells (n = 5), and 13 week NT2-N cells
(n = 3) on peak GABAR current amplitudes. Data are
mean ± SEM.
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Furosemide
The anthranilic acid derivative furosemide inhibited recombinant
GABAR currents with apparent IC50 values in the
micromolar range when an 4 or 6 subtype was expressed and in the
millimolar range when other subunits are present (Wafford et al.,
1996). Furosemide inhibited GABAR currents evoked from both NT2 cells and 13 week NT2-N cells (Fig.
8A). Data were
normalized to control currents, and the average data were fit with a
logistic equation with apparent IC50 values of
873 µM (nH = 0.9; n = 3) for NT2 cells and 1.5 mM (nH = 0.6;
n = 4) for 13 week NT2-N cells (Fig. 8B). In individual fits of the four 13 week NT2-N
cells in which a full furosemide concentration response was obtained,
there were two distinct populations of IC50
values (~600 µM and 1.5 mM). Maximal inhibition of control currents from
NT2 cells was 53.8 ± 5.7% at 1 mM
furosemide and for 13 week NT2-N cells was 64.4 ± 4.0% at 3 mM furosemide (the solubility limit of furosemide in 0.1% DMSO). Furosemide had been shown previously to inhibit GABAR
currents from 5 week NT2-N cells with a high IC50
(IC50 = 1.4 mM;
nH = 1.3) and a maximal inhibition
at 3 mM furosemide of 78.2 + 1.2%
(Fig. 8B, open diamonds, dashed line).
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Figure 8.
Inhibition of GABAR currents by furosemide.
A, Current traces from NT2 cells
(left) and 13 week NT2-N cells (right)
showing concentration-dependent inhibition of GABAR currents (10 or 30 µM GABA, respectively) by coapplication of 1 mM furosemide (FUR). Horizontal
bars, Drug applications. B,
Concentration-response curves for the effects of furosemide on NT2
cells (n = 3), 5 week NT2-N cells
(n = 3), and 13 week NT2-N cells
(n = 3) on peak GABAR current amplitudes. Data are
mean ± SEM.
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The pharmacological properties of GABARs at these three stages of
differentiation are summarized in Table
1.
PCR analysis of GABAR subunit mRNAs in NT2 cells and 13 week
NT2-N cells
Total RNA was isolated from the same cultures of NT2 cells and 13 week NT2-N cells that were used for the electrophysiological studies.
Reverse transcription (RT)-PCR was used to determine the presence or
absence of GABAR subunit mRNAs using primers specific for human
1-6, 1-3, 1-3, and subunit subtypes (Table
2) (Neelands et al., 1998). In repeated
experiments with RNA isolated from NT2 cells, major bands were found
for the 5, 3, and subtype, and a faint band was found for the
3 subtype (Fig. 9). No bands were
found for 1, 2, 3, 4, 6, 1, 2, 1, and 2
subtype mRNAs (Table 2). The mRNA expression of GABAR subunits was
qualitatively similar for both 10 and 13 week NT2-N cells; major bands
were found for 2, 3, 5, 2, 3, 1, 3, and subtype mRNAs, whereas faint bands were found for 1, 4, 6,
1, and 2 subtype mRNAs (Table 2). The emergence of the previously
unexpressed 4, 1, and 1 GABAR subtype mRNAs after at least 10 weeks of retinoic acid treatment was illustrated in Figure
10 (RT-PCR of 10 week RNA shown) along
with 3 and 3 subtype mRNAs that remained throughout differentiation in these cultures. The change in expression of GABAR
subunit mRNAs as a function of retinoic acid treatment was shown in
Table 2 (including 5 week NT2-N cells that we reported previously).
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Figure 9.
RT-PCR reaction amplification of GABAR subunits in
NT2 neuronal precursor 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; and , NT2-N RNA without Superscript II used as a
negative control. A, Agarose gel of GABAR subunits
showing the presence of 5 but not 2 or 3 [2, lanes
2-4 (numbered from the left); 3,
lanes 5-7; 5, lanes 8-10; molecular
weight standards, lane 1]. B, and
subunits showing major bands for 3, only a faint band for 3,
and no band for 1 (1, lanes 2-4; 3,
lanes 5-7; 3, lanes 8-10; molecular
weight standards, lane 1). C, Agarose gel
showing a major band for the subunit (, lanes
2-4; molecular weight standards, lane 1). In
some lanes bands of lower molecular mass than that
predicted for the product of interest were stained that are nonspecific
PCR amplification products (Bloch, 1991).
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Figure 10.
RT-PCR reaction amplification of GABAR subunits
in 10-13 week NT2-N cells. Total RNA was isolated from NT2-N cells
treated with retinoic acid for 10-13 weeks. RT-PCR amplified all the
GABAR subunit mRNAs tested at these developmental stages. No changes in
the subunit subtype expression pattern were detected over this time
period (results from 10 week NT2-N cells are shown).
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; and , NT2-N RNA
without Superscript II used as a negative control. Top,
Agarose gel of GABAR and subunits showing only faint bands for
4, 1, and 3 [4, lanes 2-4 (numbered from
the left); 1, lanes 7-9; 3,
lanes 10-12; molecular weight standards, lanes
1, 5, 6, and 13].
Bottom, subunits showing bands for 1 and 3
(1, lanes 2-4; 3, lanes 5-7;
molecular weight standards, lanes 1 and
8).
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DISCUSSION |
GABAR subunit subtype expression changed during differentiation of
NT2 cells
Untreated NT2 cells strongly expressed 5, 3, and subtype
mRNAs and weakly expressed 3 subtype mRNA. The 5, 3, and 3 subtype genes are clustered on human chromosome 15, whereas the subunit gene is on chromosome 5, unassociated with any gene cluster. We
reported previously that 5 week NT2-N cells expressed high levels of
2, 3, 5, 3, 3, and subunit mRNAs (Neelands et al.,
1998). In addition to the subtype mRNAs whose genes are located on
chromosome 15 (5, 3, and 3 subtypes), some mRNAs were present
in the 5 week NT2-N cells from genes located on human chromosomes 4 (2 subtype) and X (3 subtype). Conspicuously absent, however,
were subtype mRNAs whose genes are in the chromosome 5 gene cluster
(1, 6, 2, or 2 subtypes). RT-PCR of total RNA isolated from
13 week NT2-N cells amplified mRNA for all , , , and subunit subtypes.
Cultures of NT2-N cells after 5 weeks of retinoic acid treatment were
reported to be 99% pure (Pleasure et al., 1992) with minimal
contamination by NT2 cells. The time point at which an individual NT2
cell begins differentiation during the 4 weeks of retinoic acid
treatment, before replating, is unknown. Therefore, it is possible that
persistence of some NT2 cells and the potential difference in "age"
of NT2-N cells at the 5 and 13 week time points may contribute to the
high level of expression of the subtypes found in "immature" cells
(5, 3, and ) in RNA preparations from these cultures. The
results of RT-PCR experiments alone could not determine whether all the
GABAR subunits were expressed in individual cells, whether there was a
subset of younger NT2-N cells contaminating the RNA, or whether during
differentiation separate populations of NT2-N cells with different
GABAR subunit expression patterns emerged.
Changes in pharmacological properties associated with
differentiation of NT2 cells
The calculated half-maximal responses (EC50
or IC50) of the GABARs for allosteric modulators
were evenly distributed around the mean for individual NT2 cells and 5 week NT2-N cells. In contrast, the effects of many of the allosteric
modulators tested were more varied among individual 13 week NT2-N
cells. This was clearly illustrated by furosemide that inhibited GABAR
currents with two IC50 values of ~600
µM and 2 mM when tested on four NT2-N cells from the same culture dish. In addition, there were large variations in
the effects of diazepam on GABAR currents across the whole range of
concentrations. Most of the pharmacological changes reported in 13 week
NT2-N cells were likely caused by expression of all the GABAR subunit
subtypes present in all RT-PCR reactions. The large number of GABAR
subunits expressed at this stage of differentiation may also be
responsible for the increased variability in responses of the 13 week
cells to allosteric modulators. Expression of the 1 subtype should
have increased the affinity of modulation by benzodiazepine site
ligands. The slight decrease in the diazepam and zolpidem
EC50 values for 13 week NT2-N cells compared with that for 5 week NT2-N cells may have been attributable therefore to
incorporation of an 1 subtype into some of the 13 week NT2-N cell
GABARs. In addition, formation of benzodiazepine-insensitive GABAR
isoforms containing 4 and/or 6 subtypes in 13 week NT2-N cells
may be responsible for the decreased diazepam and zolpidem enhancement
of GABAR currents. The expression of 4 and 6 subtypes may also
have been responsible for the variability in furosemide inhibition of
GABAR currents seen among individual 13 week NT2-N cells. Recombinant
GABAR isoforms had a lower IC50 for furosemide when either an 4 or 6 subtype was expressed (160 and 6 µM, respectively) compared with when other subtypes
were expressed (~1.5 mM). The much smaller effect of
loreclezole on currents recorded from 13 week NT2-N cells could have
been caused by increased expression of the 1 subtype. When 1 and
3 subtypes were coexpressed in recombinant receptors, loreclezole
sensitivity was similar to that in loreclezole-insensitive 1
subtype-containing isoforms (Fisher and Macdonald, 1997). The increase
in expression of 1 and 2 subtypes may have accounted for the
increase in the IC50 for zinc inhibition of GABAR
currents in 13 week NT2-N cells and the rightward shift in the GABA
EC50. The calculated GABA
EC50 for recombinant GABARs, however, has been
shown to depend on the and subunit subtypes and whether or not
a , , , or subunit was incorporated. Therefore, it was not
possible to determine which subunit(s) produced the rightward shift in
the GABA EC50 of 13 week NT2-N cells compared
with NT2 cells; however, the change occurred as the number of GABAR
subunit subtype mRNAs increased and as the cellular morphology became
more neuronal.
What GABAR isoform(s) was assembled in NT2 cells?
The subtype composition of NT2 cell GABARs could not be determined
unambiguously by characterizing their pharmacological properties or by
determining mRNA expression alone. However, the homogeneity of the
pharmacological properties suggested a single isoform, and the mRNA
expression pattern placed considerable constraints on the GABAR
isoforms that could be expressed. Recombinant receptors expressed in
mammalian cell lines typically require at least an and a subunit to form functional GABARs (Angelotti et al., 1993; Neelands et
al., 1998). We have demonstrated that the subunit can incorporate
into recombinant GABARs composed of the 53 and 533
subunit combinations (Neelands and Macdonald, 1998). With the
restrictions imposed by the limited number of subtype mRNAs expressed
by NT2 cells, only four potential GABAR isoforms could likely be
formed: 53, 533, 53, and 533 GABARs. The limited number of possible isoforms allowed determination of the isoform(s) that was likely to be responsible for the
electrophysiological properties of NT2 cell GABARs by comparing these
results with those from recombinant GABARs. Identification of specific
isoforms in most native neurons would be too complex because of the
large number of possible GABAR isoforms.
Incorporation of the 5 subtype into NT2 cell GABARs was supported by
inhibition of currents by lanthanum and insensitivity to zolpidem
(Pritchett and Seeburg, 1990; Neelands and Macdonald, 1998).
Functional expression of the 3 subtype along with the 5 subunit
in NT2 cell GABARs was likely because GABAR currents were enhanced by
loreclezole (Wafford et al., 1994) and 3 homomers were not activated
by GABA (Wooltorton et al., 1997). Incorporation of both 5
and 3 subtypes was likely because all GABARs formed from 5, 3,
2L, and subtype combinations contained both 5 and 3
subtypes (Burgard et al., 1996; Neelands and Macdonald, 1998). The
relative insensitivity of GABAR currents to enhancement by both
diazepam and zolpidem suggested that although low levels of 3
subtype mRNA were detected, the 3 subtype did not assemble into the
major GABAR isoform expressed by NT2 cells. Recombinant GABAR currents
containing the subunit were insensitive to benzodiazepine site
ligands and had a low zinc IC50 but did not
display as much outward rectification as NT2 cell currents (Neelands
and Macdonald, 1998). Therefore, the large degree of outward
rectification of whole-cell currents, the relative insensitivity to
benzodiazepine site ligands, and the low zinc
IC50 suggest that the 53 isoform was the
predominant GABAR isoform expressed by NT2 cells. In addition, preliminary results showed the single-channel conductance of NT2 cell
GABARs (~15 pS; data not shown) was more similar to that of the
recombinant 53 isoform (15.2 pS) than to that of the 53
(23.8 pS) or 533 (26.9 pS) isoforms (Neelands and Macdonald, 1998). These differences suggested that although the subunit was
highly expressed, it was not incorporated into NT2 cell GABARs. These
data do not rule out the possibility that other isoforms were also
assembled and composed a small fraction of the functional receptors on
the cell surface.
Changes in GABAR subtype expression with differentiation of
NT2 cells
The changes in the expression of GABAR subunit subtypes as a
function of retinoic acid treatment were similar to the changes in
subunit expression in the developing rat brain. Expression of 2,
3, 5, 3, and 3 subtype mRNAs, whose genes are located within the chromosome 4, 15, and X clusters, was highest in perinatal rat brain and decreased in the adult brain (Zhang et al., 1991; Laurie
et al., 1992; Poulter et al., 1992). In contrast, the level of
expression for 1, 6, 2, and 2 subtypes, whose genes are located in the chromosome 15 gene cluster, was minimally expressed early in development and reached maximal expression in the adult brain
(Zhang et al., 1991; Laurie et al., 1992; Wisden et al., 1992). The
changes in the expression of GABAR subunits during the differentiation
of NT2 cells were similar to developmental changes in the rat brain.
Although the NT2 cells undoubtedly did not follow the developmental
pathway of any specific human neuron when grown in isolated cultures,
the study of NT2 cells may provide useful information about the
mechanisms underlying developmental regulation of GABAR subunits.
| |
FOOTNOTES |
Received Sept. 21, 1998; revised May 13, 1999; accepted June 2, 1999.
This research was supported by National Institutes of Health Grant RO1
NS33300 to R.L.M. We thank Dr. Ewen Kirkness for providing the subunit cDNA.
Correspondence should be addressed to Dr. Robert L. Macdonald,
Neuroscience Laboratory Building, 1103 East Huron Street, Ann Arbor, MI
48104-1687.
Dr. Neelands's present address: Vollum Institute, Oregon Health
Sciences University, Portland, OR 97201.
| |
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Copyright © 1999 Society for Neuroscience 0270-6474/99/19167057-09$05.00/0
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TOP
ABSTRACT
INTRODUCTION
