Introduction

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-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain, and fast inhibitory postsynaptic potentials are mediated by GABA_A receptors (GABARs). GABARs belong to the superfamily of ligand-gated ion channels that includes the glycine, nicotinic cholinergic (nAChR), and 5-hydroxytryptamine receptors. Four GABAR subunit families (1-6, 1-3, 1-3, and ) have been studied extensively (Macdonald and Olsen, 1994). The majority of native GABARs are thought to be heteropentamers composed of combinations of subunits from three different families (e.g., , , and ) that form a chloride-selective ion channel. The potential diversity of GABAR isoforms has increased with the addition of two recently described subunits:  (Hedblom and Kirkness, 1997) and  (Davies et al., 1997). The amino acid sequence of the  subunit is most closely related to the  subunits, having between 38 and 43% identical residues (Davies et al., 1997). Electrophysiological (Chang et al., 1996) and biochemical studies (Tretter et al., 1997) have suggested that the pentamer may be composed of two  subunits, two  subunits, and a single  subunit. It is thought that the  subunit, like the  subunit (Saxena and Macdonald, 1994), is capable of replacing the  subunit in the GABAR pentamer to form functional channels with distinct pharmacological and biophysical properties. Most GABAR isoforms require binding of GABA to initiate entry into open states. However, spontaneous channel activity has been reported in recombinant 41 receptors as well as 1 or 3 homomeric receptors, although these isoforms are insensitive to activation by GABA.

Naturally occurring GABAR isoforms are determined (or restricted) by the distinct regional expression of each subunit (Wisden et al., 1992). The mRNA encoding the  subunit was found to be restricted to the amygdala, thalamus, and subthalamic nuclei of the human brain using Northern blot analysis (Davies et al., 1997). In contrast, in situ hybridization studies in the squirrel monkey brain localized mRNA expression in the arcuate-ventromedial area of the hypothalamus and the hilus of the hippocampus but found no detectable expression in either the amygdala or subthalamic nucleus (Whiting et al. 1997). The  subunit gene was independently identified and mapped to a cluster of GABAR genes, including 3 and putative 4 subunit genes, on the human X chromosome (Xq28; Wilke et al., 1997). Interestingly, this location on the X chromosome is a candidate region for two neurological disorders: early-onset Parkinson's disease (Laxova et al., 1985) and X-linked mental retardation (Gedeon et al., 1991). Whether the  subunit is associated with either of these disorders has yet to be determined.

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) and zinc (Draguhn et al., 1990). Originally, it was reported that  subunit-containing receptors were unique among GABARs in their insensitivity to the general anesthetic agents pentobarbital and propofol (Davies et al. 1997). A more recent study, however, reported that GABAR isoforms containing the  subunit were directly activated by pentobarbital and that GABAR currents were enhanced by coapplication of pentobarbital (Whiting et al., 1997). Both groups showed enhancement of  subunit-containing GABAR currents by the neurosteroid 5-pregnan-3-ol-20-one, whereas Whiting et al. (1997) also showed inhibition by zinc with a moderate affinity and rapid apparent desensitization of whole-cell currents.

The goal of this study was to characterize further the pharmacological properties and to determine the biophysical properties of GABARs containing the  subunit. We found enhancement of whole-cell GABAR currents by loreclezole, lanthanum, and pentobarbital, as well as inhibition by zinc and furosemide. Whole-cell recordings consistently had large holding currents with noisy baseline values similar to that reported for spontaneously active  homopentamers (Wooltorton et al., 1997). Application of GABAR positive allosteric modulators and GABAR antagonists to the holding current produced inward and outward currents, respectively, in the absence of applied GABA. Opening frequency of single-channel currents increased during application of GABA, whereas single-channel currents with conductances of ~24 pS were recorded in the absence and presence of applied GABA. Robust effects of GABAR modulators on the holding current and the presence of single-channel openings in the absence of GABA suggested that the  subunit permits spontaneous channel activity while preserving the structural information required for GABA-gated openings.

	Materials and Methods

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Transfection. Full-length cDNAs for rat GABAR 1, 1 (Dr. A. Tobin, University of California, Los Angeles, CA), and 3 (Dr. D. Pritchett, University of Pennsylvania) subtypes were subcloned into the pCMVNeo expression vector for transfection studies. The human  cDNA was received in the pCDM8 expression vector (Dr. E. Kirkness, The Institute for Genomic Research, Rockville, MD), which was then used for transfection studies. For selection of transfected cells, the plasmid pHook-1 (InVitrogen, San Diego, CA) containing cDNA encoding the surface antibody sFv was also transfected into the cells. L929 cells were maintained in Dulbecco's modified Eagle's medium plus 10% heat-inactivated horse serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were passaged by a 5-min incubation with 0.5% trypsin/0.2% EDTA solution in phosphate-buffered saline (10 mM Na₂HPO₄, 0.15 mM NaCl, pH 7.3).

Recording Solutions and Techniques. For both whole-cell and outside-out patch recording, the external solution consisted of 142 mM NaCl, 8.1 mM KCl, 6 mM MgCl₂, 1 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4, and osmolarity adjusted to 295 to 305 mOsm. Recording electrodes were filled with an internal solution of 153 mM KCl, 1 mM MgCl₂, 5 mM K-EGTA, 10 mM HEPES, and 2 mM MgATP, pH 7.4, and osmolarity adjusted to 295 to 305 mOsm. These solutions provided equilibrium potential for Cl near 0 mV. Patch pipettes for whole-cell recordings were pulled from either borosilicate glass (Fisher Scientific Co., Pittsburgh, PA) or Labcraft microhematocrit capillary tubes (Curtin Matheson Sci., Houston, TX) on a P-87 Flaming Brown puller (Sutter Instrument Co., Novato, CA) to a resistance of 8 to 12 M. For single-channel recording, patch pipettes were pulled from thick-walled borosilicate glass with an internal filament (World Precision Instruments, Sarasota, FL), fire polished to a resistance of 5 to 10 M, and coated with Q-dope (GC Electronics, Rockford, IL) to reduce capacitance.

Data Analysis. Whole-cell currents were analyzed off-line using the programs Axotape and Prism (GraphPAD, San Diego, CA). Statistical tests were performed using the Instat program (GraphPAD). For initial single-population fits, the normalized concentration-response data for the different isoforms were fit with a four-parameter logistic equation: Current = maximum current/{1 + [10 (log EC₅₀  log [drug])*n]}, where n is a slope factor. All four parameters were "floating," and therefore, the maximum effect observed was not necessarily the upper limit of the fit (e.g., see Figs. 2A and 7B). The application of either 0.3 or 1 µM GABA (for agonists and antagonists, respectively) was repeated until the peak currents had stabilized and functioned as controls for each cell. Coapplication of the same concentration of GABA with increasing concentrations of individual modulators was performed to determine the maximal effect and potency of each modulator on -containing GABARs. Fits were made to normalized data with the current expressed as a percentage of the maximum current elicited for each cell.

Analysis of Single-Channel Currents. Single-channel recordings were digitized using Axoscope and analyzed using pClamp6 (Axon Instruments) and Interval5 (Dr. Barry S. Pallotta, University of North Carolina at Chapel Hill). For analysis, the data were digitized at 20 kHz and filtered at 2 kHz. Intervals were measured with a 50% threshold detection method. Subconductance levels were occasionally observed (5% of openings) but were not included in the analysis. To reduce errors due to multichannel patches, recordings were included in the kinetic analysis only if overlaps of simultaneous openings occurred for less than 1% of the openings. Overlapped openings and bursts were not included in the kinetic analysis. The presence of multiple channels would decrease the apparent duration of the longer closed components but would have no effect on the open state or burst properties. Duration histograms were constructed and fit by a maximum likelihood method. The number of exponential functions required to fit the distribution was increased until additional components did not significantly improve the fit as determined by the log-likelihood ratio test. Intervals with durations less than 1.5 times the system dead-time were displayed in the histograms but were not included in the fit. For the definition of bursts, the two shortest closed components were considered as intraburst closures. A burst terminator for each patch was calculated from the closed interval distribution to equalize the proportion of misclassified events (see Fisher and Macdonald, 1997).

	Results

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Pharmacological Properties of the Spontaneous (Holding) Current

L929 cells were transfected with combinations of cDNAs encoding the 1, 1, 3, or  subtypes and voltage-clamped in the whole-cell configuration of the patch-clamp technique. After formation of a high resistance seal and patch rupture, it was noted that cells transfected with 1x subtype cDNAs required an unusually large holding current to maintain a potential of -75 mV and that the resulting baseline value was relatively unstable. The holding current reversed polarity at the chloride equilibrium potential and increased in a linear fashion as the membrane potential was made more negative (data not shown). To determine the source and specificity of the holding current, positive and negative allosteric modulators of GABARs were applied to cells transfected with 13-2L, 13, 13, 13, 1, or 3 subtypes or the  subunit alone (Fig. 1). Drugs including picrotoxin (30 µM), loreclezole (3 µM), pentobarbital (300 µM), zinc (100 µM), and GABA (30 µM) were applied for 5 to 8 s to cells held at a membrane potential of -75 mV (see Materials and Methods) (Fig. 1). Holding currents in cells expressing 13 subtypes (Fig. 1b), but not the other combinations (Fig. 1, c-h), were sensitive to picrotoxin, loreclezole, and zinc. This holding current was not altered by the application of glycine (data not shown). The application of pentobarbital and GABA evoked inward currents in cells expressing all of the subunit combinations except 1 and  alone (Fig. 1, b-h). Cells that expressed the 3 subtypes were slightly activated by the application of pentobarbital, but no inward current was evoked by 30 µM GABA.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Pharmacology of holding currents for various recombinant GABAA receptors. After establishing a seal and breaking into L929 cells expressing 13 channels, a large current was required to hold the membrane potential at -75 mV. To test the hypothesis that spontaneous channels were responsible for these consistently large holding currents, we examined the effects of direct drug application in the absence of GABA. a, L929 cells expressing recombinant GABAA receptors were voltage clamped to -75 mV. b-h, responses to direct applications of 30 µM picrotoxin (Pic), 3 µM loreclezole (Lor), 300 µM pentobarbital (PB), 100 µM zinc (Zn++), and 30 µM GABA are shown for multiple receptor isoforms. Only 13 (b) receptors exhibited significant holding currents that were sensitive to direct application of picrotoxin, loreclezole, and zinc. Cells transfected with 13 (b), 132L (c), 13 (d), or 13 (e) were activated by the application of both 300 µM pentobarbital and 30 µM GABA. Cells expressing 1 (f) and  alone (h) were insensitive to all modulators, but the application of pentobarbital to 3-containing cells produced a small current (g). Bars, 6-s drug applications. *, application of 10 µM GABA for 132L trace.

L929 cells coexpressing either 11 or 13 subtypes were activated directly by pentobarbital (10-300 µM) in a concentration-dependent manner (Figs. 1b and 2A). Peak currents evoked by 300 µM barbiturate were 214.1 ± 39.8 pA for 13 and 136.6 ± 53.8 pA for 11. Averaged peak currents evoked by pentobarbital (1-300 µM) revealed similar EC₅₀ values for these two isoforms. The 11 isoform had a pentobarbital EC₅₀ of 211 µM (n_H = 0.9, n = 3), whereas the pentobarbital EC₅₀ for the 13 isoform was 112 µM (n_H = 1.3, n = 3) (Fig. 2A).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Direct activation of GABA receptors by pentobarbital and loreclezole. A, concentration-response relationships for direct activation of GABA receptors by pentobarbital. , Responses of 13 receptors (n = 3). , 11 responses (n = 3). B, concentration-response relationship of loreclezole on the holding current (n = 6). Data are mean ± S.E.M. The EC50 values and nH were derived from fitting a four-parameter logistic equation but did not include the highest loreclezole concentration (30 µM; hatched line).

The application of loreclezole (100 nM to 30 µM) alone to cells expressing 13 receptors evoked inward currents (Fig. 1b) in a concentration-dependent manner (Fig. 2B). Maximal currents of ~105 pA were evoked by concentrations of loreclezole greater than 3 µM (n = 6). Higher concentrations of loreclezole produced less current, likely due to open channel block (Donnelly and Macdonald, 1996). The loreclezole concentration-response curve was fit with a logistic equation with an EC₅₀ of 1.0 µM and a Hill slope (n_H) of 3.2 (Fig. 2B).

Picrotoxin produced a concentration-dependent reduction of the holding current in cells transfected with 13 subtypes, with an IC₅₀ of 1.8 µM (Fig. 1b). Maximal outward currents (70.7 ± 23.1 pA, n = 4) were produced by the application of 10 µM picrotoxin, which represented 80% to 90% reduction of the holding current (Fig. 3A). Bicuculline, another GABAR antagonist, also reduced the holding current but with less efficacy than picrotoxin (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Inhibition of 13 holding currents by picrotoxin and zinc. A, concentration-response relationship for picrotoxin inhibition of the holding current (n = 4), normalized in each cell to the maximum amplitude block of holding current and plotted as the percent picrotoxin-sensitive current remaining after each application. B, concentration-response relationship of zinc on the holding current (n = 3), normalized to the maximum amplitude zinc block of holding current in each cell and plotted as percent of zinc-sensitive current remaining after zinc application. Data are mean ± S.E.M.

Zinc also reduced the holding current (Fig. 1b). As increasing concentrations of zinc were applied to the cell, the total holding current diminished in amplitude (n = 3). To control for zinc-induced current rundown, we measured the ability of each concentration of zinc to inhibit the holding current recorded just before its application. The averaged percent block calculated by this method had an IC₅₀ of 22.3 µM with an n_H of -0.9 (n = 3) (Fig. 3B).

It should be noted that the benzodiazepine site agonist, diazepam (1 µM), did not enhance GABAR current or alter the holding current (data not shown). This was consistent with reports that benzodiazepine sensitivity required inclusion of a  subunit in association with an 1, 2, 3, or 5 subtype and a  subunit.

Characterization of GABAR Currents in -Containing GABARs

Inward currents evoked by GABA (10 nM to 100 µM) in cells expressing 13 receptors increased in a concentration-dependent manner, with faster apparent activation rates and greater apparent desensitization with higher GABA concentrations (Fig. 4A). Currents were normalized to the maximal current for each cell, averaged, and fit with a logistic equation with an EC₅₀ = 0.8 µM and n_H = 0.9 (n = 9) (Fig. 4B). The half-maximal response to GABA was lower, and the n_H was reduced compared with cells transfected with 13 subtypes as previously reported by our laboratory.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   GABA concentration-response profile for 13 receptors. A, representative current traces from application of 0.1, 3, and 30 µM GABA to a single cell. Horizontal bar, drug application. B, normalized concentration-response curve for GABA-evoked currents (n = 9). Data are mean ± S.E.M.

The current-voltage (I-V) relation for 13 receptors was obtained by repeated applications of 1 µM GABA at holding potentials ranging from 100 to +75 mV in 25-mV increments (n = 4) (Fig. 5A). The I-V relation was linear at negative holding potentials, whereas potentials above +25 mV revealed inward rectification for all cells tested (Fig. 5B). To verify that current rundown was not responsible for this apparent rectification, GABA was reapplied at -75 mV following each I-V protocol. Rundown was not detected in any cells during this protocol, suggesting this rectification reflected an intrinsic property of the channel.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Voltage-related properties of GABA-evoked 13 currents. A, representative current traces evoked by 1 µM GABA in a single cell clamped at -75, 0, and +75 mV. Subsequent reapplication of 1 µM GABA at -75 mV demonstrates the absence of current rundown. Horizontal bars, drug application. B, peak currents evoked by 1 µM GABA are plotted versus membrane holding potential (n = 4). Data are mean ± S.E.M.

Pharmacological Properties of -Containing GABARs

Barbiturate Sensitivity of  Subunit-Containing GABARs. The effect of the barbiturate pentobarbital on  subunit-containing GABARs has been controversial. Pentobarbital had no effect on GABAR currents when 13 receptors were expressed in human embryonic kidney 293 cells (Davies et al., 1997), whereas 11 receptors expressed in Xenopus laevis oocytes were potentiated by pentobarbital (Whiting et al., 1997). Effects of pentobarbital on GABAR currents have not been shown to depend on either  subunit subtype or expression system, so the basis for the different results was unclear. In our study, pentobarbital enhanced both 11 and 13 currents (Fig. 6, A and B) by 150.5 ± 23.8% (n = 4) and 83.7 ± 25.1%, respectively, with EC₅₀ values of ~40 µM (n = 6) (Fig. 6C). A normalized, averaged concentration-response curve for each isoform was fit with a logistic equation with an EC₅₀ of 40 µM and an n_H between 1.8 and 1.9 (Fig. 6C).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Enhancement of GABA-evoked currents by pentobarbital. A and B, superimposed current traces of successive application of 0.3 µM GABA (upper trace in A and B) and 0.3 µM GABA plus 30 µM pentobarbital (lower trace in A and B) for 11 receptors (A) and 13 receptors (B). Horizontal bars, drug application. C, concentration-response relationship for pentobarbital enhancement of currents evoked by 0.3 µM GABA. , 11 currents (n = 5). , 13 currents (n = 4). Data are mean ± S.E.M.

Loreclezole Sensitivity of  Subunit-Containing GABARs. Loreclezole enhancement of GABAR currents depends on the presence of 2 or 3 subtypes (Wingrove et al., 1994). Loreclezole (30 nM to 30 µM) enhanced 13 currents evoked by 0.3 µM GABA in a concentration-dependent manner, with a maximal enhancement of 55.6 ± 18.6% at 3 µM (n = 8) (Fig. 7A). With higher loreclezole concentrations, an apparent inhibition of currents to below control levels was observed. The range over which enhancement occurred was fit with a logistic equation with EC₅₀ = 0.9 µM and n_H = 8.8. This inhibition was more dramatic than that seen with receptor currents, in which loreclezole produced only slight inhibition at 30 µM after maximal enhancement at 10 µM (Donnelly and Macdonald, 1996). Inclusion of the  subunit in the GABAR appeared to increase the ability of high concentrations of loreclezole to inhibit GABAR currents.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Modulation of GABA-evoked 13 currents by loreclezole and lanthanum. A, concentration-response relationship for loreclezole modulation of currents evoked by 0.3 µM GABA (n = 8). EC50 value for loreclezole enhancement (0.03-10 µM loreclezole) was derived independent of the apparent inhibition seen at higher concentrations (hatched line). Inset, superimposed current traces of successive application of 0.3 µM GABA current (upper trace) and 0.3 µM GABA plus 1 µM loreclezole (lower trace) are shown. Horizontal bar, drug application. B, concentration-response relationship for lanthanum enhancement of 0.3 µM GABA-evoked currents (n = 5). Inset, superimposed current traces of successive application of 0.3 µM GABA (upper trace) and 0.3 µM GABA plus 300 µM lanthanum (lower trace). Horizontal bar, drug application. Data are mean ± S.E.M.

Lanthanum Sensitivity of  Subunit-Containing GABARs. The trivalent cation lanthanum has been shown to have subunit-specific effects on recombinant GABARs. GABAR currents from receptors containing the 6 subtype were inhibited by lanthanum, whereas 1 subtype-containing receptor currents were enhanced (Saxena et al., 1997). Lanthanum (1 µM to 3 mM) produced a concentration-dependent increase in 13 currents evoked by 0.3 µM GABA. Significant enhancement of the GABAR current was first observed at 100 µM lanthanum and increased through 3 mM lanthanum (148.1 ± 5.9%) (n = 5) (Fig. 7B). Normalized data were fit with a logistic equation (see Materials and Methods) with an EC₅₀ value of 500 µM and an n_H of 0.9 (Fig. 7B).

Zinc Sensitivity of  Subunit-Containing GABARs. The inhibitory effects of the divalent cation zinc have been well characterized for recombinant and native GABARs (Draguhn et al., 1990; Kapur and Macdonald, 1996). Control 13 currents evoked by 1 µM GABA were inhibited in a concentration-dependent manner by zinc. Zinc maximally inhibited 96 ± 2.6% (n = 7) of GABAR currents, with an IC₅₀ of 32.5 µM and an n_H of -1.0 (Fig. 8A), similar to that reported by Whiting et al. (41.9 µM).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Inhibition of GABA-evoked 13 currents by zinc and furosemide. A, concentration-response relationship for zinc inhibition of currents evoked by 1 µM GABA (n = 7). Inset, superimposed current traces of successive application of 1 µM GABA (lower trace) and 1 µM GABA plus 30 µM zinc (upper trace). Horizontal bar, drug application. B, concentration-response relationship for furosemide inhibition of currents evoked by 1 µM GABA (n = 5). Inset, superimposed current traces of successive application of 1 µM GABA (lower trace) and 1 µM GABA plus 100 µM furosemide (upper trace). Horizontal bar, drug application. Data are mean ± S.E.M.

Furosemide Sensitivity of  Subunit-Containing GABARs. The anthranilic acid derivative furosemide inhibited recombinant GABAR currents with IC₅₀ values in the micromolar range only when an 4 or 6 subtype was expressed (Wafford et al., 1996). However, in this study, furosemide potently inhibited 13 GABAR currents evoked by 1 µM GABA with an apparent IC₅₀ of 167 µM (n_H = 0.7, n = 5) (Fig. 8B). Maximal inhibition of control currents was 83.9 ± 4.4% at 3 mM furosemide, which was the solubility limit of furosemide in 0.1% dimethylsulfoxide. The affinity and efficacy of furosemide block for this isoform were much greater than those reported for 1x2L receptors but was similar to the 162 µM IC₅₀ reported for the 432L isoform (Wafford et al., 1996).

Single-Channel Properties of -Containing GABARs

To examine the single-channel properties of these receptors, 1 µM GABA was applied to outside-out patches pulled from transfected fibroblasts. 13 single-channel openings were relatively long in duration and were separated into closely grouped bursts of openings (Fig. 9A, top trace). Openings occurred primarily to a single conductance level (average amplitude = 1.6 pA at -70 mV). The amplitudes of single-channel openings were measured at holding potentials ranging from 80 to +80 mV and were fit with linear regression analysis to determine single-channel conductance (Fig. 9B, ). From fits of individual patches, the average conductance (±S.E.M.) of 13 channel openings was 23.7 ± 0.13 pS (n = 4). This was similar to the main conductance level reported for both 132L and 13 channels (27 pS) (Fisher and Macdonald, 1997) and was larger than the main conductance level reported for 11, 12, or 13 channels (11-13 pS) (Verdoorn et al., 1990; Angelotti and Macdonald, 1993; Fisher and Macdonald, 1997). The average reversal potential for the individual patches was near 0 mV (2.9 ± 0.8 mV), as predicted for a chloride ion-selective channel.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Single-channel GABA-evoked 13 currents A, representative single-channel current traces evoked by 1 µM GABA applied to an outside-out patch (upper trace) or spontaneous openings recorded in the absence of applied GABA (lower trace). B, single-channel current-voltage relationships are shown for GABA-evoked openings (, n = 4) or spontaneous openings (, n = 2). Data are mean ± S.E.M.

To confirm that single-channel openings of the -containing GABARs were responsible for the spontaneous current, outside-out patches were pulled from fibroblasts transfected with 13 subunits. Brief, infrequent single-channel openings were recorded in the absence of applied GABA (Fig. 9A, bottom trace). The amplitudes of single-channel openings were measured at holding potentials ranging from 80 to +80 mV and were fit with linear regression analysis to determine single-channel conductance (Fig. 9B, ). From fits of individual patches, the average conductance of 13 channel openings was 22.2 pS (n = 2). The average reversal potential for the two patches was near 0 mV (2.6 ± 2.3 mV) as predicted.

The kinetic properties of GABAR single-channel openings and closings were examined by constructing open and closed duration histograms from data obtained during long (5-10 min) applications of 1 µM GABA. Openings occurred with low frequency with an average percent open time of 1.04 ± 0.33% (n = 4). Open interval histograms were fitted best with the sum of two exponential functions with nearly equal relative proportions (Fig. 10A). The time constants and relative areas (±S.E.M.) averaged 0.388 ± 0.057 ms (57.4 ± 5.9%) and 2.24 ± 0.08 ms (42.6 ± 5.9%) with an average mean open time of 1.18 ± 0.08 ms (n = 4 patches) (Table 1). Closed duration histograms were fitted best with the sum of five exponential functions (Fig. 10B). The average durations of the longer closed components were relatively variable, probably due to differences in the number of channels in the patches. The short-duration closed times, however, represented intraburst channel closings and therefore were not affected by multiple-channel patches. The averages for time constants (and relative areas) of the components for the four patches were 0.128 ± 0.004 ms (0.538 ± 0.021), 1.123 ± 0.07 ms (0.194 ± 0.024), 12.63 ± 1.55 ms (0.113 ± 0.005), 138.7 ± 41.3 ms (0.083 ± 0.007), and 1596.3 ± 481.6 ms (0.072 ± 0.014) (Fig. 10B; Table 1). The average mean shut time was 133 ± 55 ms. The relatively high proportion of the shortest closed components was consistent with the bursting behavior of the channels, whereas the long duration of the longer components was consistent with entry into desensitized states.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Open- and closed-duration histograms of single-channel 13 currents evoked by 1 µM GABA. A, open-time histograms were best fit by two exponential distributions with means (±S.E.M.) and relative areas (±S.E.M.) of 0.388 ± 0.057 ms (57.4 ± 5.9%) and 2.24 ± 0.08 ms (42.6 ± 5.9%). B, closed times were best fit with five exponential distributions, with means and relative areas of 0.128 ± 0.004 ms (53.8 ± 2.1%), 1.123 ± 0.07 ms (19.4 ± 2.4%), 12.63 ± 1.55 ms (11.3 ± 0.5%), 138.7 ± 41.3 ms (8.3 ± 0.78%), and 1596.3 ± 481.6 ms (7.2 ± 1.47%).

The activity of ligand-gated channels often occurs in bursts of closely grouped openings. We examined the burst properties by defining a critical gap between the closed components C2 and C3 that represented the termination of a burst of openings. This assigned the two shortest components as intraburst closures. Distributions of burst durations and number of openings per burst were constructed and fit with the sum of two or three exponential or geometric functions. The average burst duration was 3.72 ± 0.39 ms with an average number of openings per burst of 2.94 ± 0.13 (n = 3 patches).

	Discussion

Top Summary Introduction Materials & Methods Results Discussion References

Incorporation of the  Subunit Produced a Spontaneously Active GABAR Channel. L929 cells expressing the 13 GABAR isoform consistently produced a large holding current when voltage clamped at -75 mV that was sensitive to both positive and negative allosteric modulators of GABARs but was not affected by the application of glycine. Other subunit combinations, including 132L, 13, 13, 1, and 3, and  alone failed to produce significant holding currents that were sensitive to enhancement by loreclezole or block by picrotoxin or zinc. Specifically, the negative results obtained with transfections of 1 or 3 subtypes or the  subunit alone strongly suggested that channels potentially composed of only one or two of the three transfected subunits were not contributing to the observed holding current.

Effects of Pentobarbital on -Containing GABARs. Pentobarbital and other general anesthetic agents have been shown to have three actions on GABARs: 1) GABAR currents were potentiated by coapplication of pentobarbital (Schulz and Macdonald, 1981); 2) pentobarbital directly activated GABARs (Schulz and Macdonald, 1981); and 3) application of high concentrations of pentobarbital alone or in combination with GABA resulted in an open-channel block of the ion pore (Schwartz et al., 1986).

Additional Pharmacological Properties of GABARs Containing the  Subunit. Functional GABAR isoforms had pharmacological properties that were different from those of , , and GABARs (Table 2). Notably, benzodiazepine insensitivity corroborates the proposed requirement of a  subunit to form a benzodiazepine site. Zinc inhibited both the holding and GABA-evoked 13 currents with similar IC₅₀ values, suggesting that a single GABAR isoform was responsible for both currents. Furosemide affinity, which depended on the  subtype in previous experiments, was dramatically altered. Further experiments are required to determine whether the  subunit increased the affinity of furosemide for all  subunits or changed the rank order of potency across different -containing isoforms. Lanthanum potentiated 13 currents with a lower affinity than it potentiated 132L currents. Finally, the  subunit conferred a greater sensitivity to the inhibitory effects of high concentrations of loreclezole. These data highlight the notion that the context of intersubunit interactions can significantly affect "subunit-specific" properties.

Biophysical Properties of GABARs Containing the  Subunit. Spontaneous and evoked single-channel currents recorded from cells expressing the 13 isoform were not significantly different from each other but had properties that distinguished them from other isoforms. The single-channel conductance of 13 channels was similar to conductances of isoforms containing the 2L and  subunits (Angelotti et al., 1993; Fisher et al., 1997) but were larger than single-channel conductances (Verdoorn et al., 1990; Angelotti and Macdonald, 1993; Fisher and Macdonald, 1997). Unlike isoforms that had three open states and five closed states (Macdonald et al., 1989), the 13 isoform had only two open states. The second open state was similar to the O2 state recorded from native neurons in that it showed long bursts of multiple openings (Twyman et al., 1990) but was longer and more frequent than those seen with 13 or 13 isoforms (Fisher and Macdonald, 1997). Interestingly, three open states, as seen in spinal cord, were observed in recombinant channels only when a  subunit was present, raising the possibility that the O3 open state may be a  subunit-dependent property.

Summary. In this study, we established the existence of robust spontaneous channel activity and extend the pharmacological characterization of GABARs containing the  subunit. This is the first example of a heterotrimeric GABAR that exhibits both spontaneous and GABA-gated channel openings. It is apparent that the  subunit can influence the contributions of other subunits to GABAR pharmacology, yet the specific mechanisms remain obscure. Structure-function studies might provide insight into the relationship between ligand binding and gating and its partial uncoupling in this isoform. Although spontaneous chloride conductances present an attractive potential mechanism to modulate the basal excitability of neurons, further studies in vivo are necessary to establish the role of the  subunit in native systems.