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Previous Article | Next Article
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
ABSTRACT
Top
Abstract
Introduction
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, and
subtypes. The combined pharmacological and RT-PCR results are most consistent with a single or predominant GABAR isoform composed of an
Key words: GABA; electrophysiology; patch clamp; Ntera2; barbiturate; benzodiazepine; neurosteroid; single channel
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 (
) have been studied extensively (Macdonald and Olsen, 1994
), and two new subunit families,
(Davies et al., 1997
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
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
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
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
Compounds were applied to cells using a modified U-tube "multipuffer" application system (Greenfield et al., 1996
Electrophysiology. Whole-cell voltage-clamp and single-channel recordings using the patch-clamp technique were obtained as described previously (Hamill et al., 1981
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
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:
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
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).
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
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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 =
Voltage dependence of GABAR currents
The degree and direction of GABAR current rectification depended on the GABAR isoform (Burgard et al., 1996
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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
Voltage-dependent desensitization to high concentrations of GABA was also dependent on the GABAR isoform expressed (Burgard et al., 1996
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
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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.
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
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Table 1. Single-channel properties of NT2-N neurons
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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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Table 2. Open and closed time constants and relative proportions 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
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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Table 3. Summary of NT2-N cell GABAR pharmacology 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.
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
Zolpidem
The imidazopyridine zolpidem is another GABAR benzodiazepine site agonist sensitive to theDMCM
DMCM is aLoreclezole
The novel anticonvulsant drug loreclezole interacts with a site on the
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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.
Neurosteroids
Neurosteroids have been shown to differentially modulate GABAR currents. Alphaxalone (5
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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%.
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
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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.
Furosemide
Furosemide, an anthranilic acid derivative, inhibits recombinant GABAR currents with IC50s in the micromolar range, but only when either anPicrotoxin
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 ofThe trivalent cation lanthanum (La3+), when coapplied with a GABA concentration near the EC50, inhibits
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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.
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
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Table 4. RT-PCR primers
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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;
DISCUSSION
NT2-N cells express primarily
High mRNA levels for the
, the incomplete phosphorylation state of high molecular weight neurofilament protein, and no MAP2b expression (Pleasure et al., 1992
NT2-N cells express GABARs likely composed primarily of
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
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
NT2-N GABARs likely contain a
Enhancement of GABAR currents by benzodiazepine site ligands requires the presence of aNT2-N GABARs likely contain
Recombinant GABARs with high affinity for benzodiazepines contain theNT2-N GABARs likely contain the
Loreclezole enhancement of recombinant rat GABARs acts at a specific modulatory site on theNT2-N GABAR biophysical properties were consistent with an
Single GABAR channels pulled from NT2-N cells opened in bursts to three different conductance levels: 19, 23, and 27 pS. These conductance levels were similar to those observed in native GABAR isolated in membrane patches from cultured mouse spinal neurons (Bormann et al., 1987
Kinetic analysis of the main conductance level openings suggested the presence of three open states and five closed states, similar to those determined in our laboratory for native GABAR channels in outside-out patches obtained from mouse spinal cord neurons in primary cell culture (Macdonald et al., 1989
Although studies of recombinant rat or bovine GABAR single-channel currents have not demonstrated a particular conductance level or kinetic "fingerprint" for specific isoforms, isoforms containing only an
FOOTNOTES Received Nov. 11, 1997; revised April 14, 1998; accepted April 17, 1998.
We thank Nadia Esmaeil and Yunning Yang for their technical support in differentiating and maintaining the NT2-N cells and Dr. Ewen Kirkness for contribution of
Correspondence should be addressed to Dr. Robert L. Macdonald, Neuroscience Laboratory Building, 1103 East Huron Street, Ann Arbor, Michigan, 48104-1687.
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