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We have investigated the role of the α subunit in the modulation of γ-aminobutyric acid type A (GABAA) receptors by the general anesthetic propofol, using whole-cell patch clamp recordings made from distinct stable fibroblast cell lines which expressed only α1β3γ2 or α6β3γ2 GABAA receptors. At clinically relevant anesthetic concentrations, propofol potentiated submaximal GABA currents in α1β3γ2 receptors to a far greater degree than those in α6β3γ2 receptors. The α subunit influenced the efficacy of propofol for modulation, but not its potency. In contrast, direct gating of the ion channel by propofol, in the absence of GABA, was significantly larger in the α6 than the α1 containing receptors. The potentiation of submaximal GABA by trichloroethanol, and the potentiation and direct gating by methohexital, was also studied, and showed the same relative trends as propofol.
GABAA (γ-aminobutyric acid type A) receptors are the major receptors for inhibitory neurotransmission in the mammalian brain. The GABAA receptor is a pentameric complex formed by different glycoprotein subunits (α1–6,β1–4, γ1–4,δ) which combine to form a chloride channel (reviewed by Burt and Kamatchi, 1991; Olsen and Tobin, 1990; Rabow et al., 1995). GABAA receptor subunit expression in the central nervous system is heterogeneous (Laurie et al., 1992a,b; Wisden et al., 1991). For example, the GABAA α6 subunit isoform is localized solely in cerebellar granule cells, whereas α1 is widely expressed throughout the brain (Luddens et al., 1990; Kato, 1990; Mertens et al., 1993; Wisden et al., 1992).
The GABAA receptor complex is modulated allosterically by a wide range of compounds which act at discrete but unknown sites on the receptor (Macdonald and Olsen, 1994). One major group of GABAA receptor modulators is the class of general anesthetics, many of which have been demonstrated to augment GABAA receptor chloride currents at clinically relevant concentrations (e.g. Zimmerman et al., 1994), and are thought to elicit anesthesia by enhancing inhibitory synaptic transmission (Nicoll et al., 1975; Gage and Robertson, 1985). General anesthetics known to potentiate the actions of GABA (γ-aminobutyric acid) include propofol [2,6-diisopropylphenol (PRO); Hales and Lambert (1991); Hara et al. (1994)], steroid anesthetics (Harrison and Simmonds, 1984; Harrison et al., 1987), barbiturates (Study and Barker, 1981), chlormethiazole (Hales and Lambert, 1992), halogenated volatile anesthetics (Wakamori et al., 1991; Jones et al., 1992), and trichloroethanol (TCEt, the active metabolite of chloral hydrate; Lovinger et al., 1993; Peoples and Weight, 1994).
Different combinations of GABAA receptor subunits show variable sensitivity to allosteric modulators (e.g. Horne et al., 1993). The best documented role of specific subunits is for benzodiazepine modulation, where the presence of a γ subunit is required (Pritchett et al., 1989), but benzodiazepine agonist sensitivity is also influenced critically by the type of α subunit isoform. Specifically, the exchange of a single amino acid confers benzodiazepine sensitivity on the normally benzodiazepine-insensitive α6-containing GABAA receptor (Kleingoor et al., 1993).
The role of GABAA receptor subunits in general anesthetic modulation is less clear. For example, modulation of GABAA receptors by PRO is qualitatively independent of the γ subunit (Jones et al., 1995) and can be observed in heteromeric αβ or even in homomeric β1 receptors (Sanna et al., 1995a,b). It was recently shown that the α subunit influences modulation by pentobarbital (Thompson et al., 1996). This study was therefore designed to compare the effects of PRO on GABA-induced chloride currents in receptors containing two different α subunits against a common αβ background. The α6 subunit was chosen as it shares the least homology [along with α4, see Wafford et al. (1996)] with the other α subunit isoforms (Tyndale et al., 1995), and contributes to the differential pharmacology of benzodiazepines (Hadingham et al., 1993). We hypothesized that if differences in PRO modulation were to exist among α subunit isoforms, they might be most apparent in comparisons with α6-containing receptors. Portions of this work were previously presented in abstract form (O’Shea et al., 1996).
An Ltk− mouse fibroblast cell line from ATCC (Rockville, MD, U.S.A.) was stably transfected with dexamethasone-inducible GABAA receptor α1β3γ2 or α6β3γ2 cDNA, as previously described in detail (Hadingham et al., 1992). The resultant cell lines were cultured in supplemented Eagle’s minimum essential medium (Sigma, St. Louis, MO, U.S.A.) as previously described.
Electrophysiological recordings were performed at room temperature using the whole-cell patch clamp technique. The electrode solution contained (in mM): 147, N-methyl-D-glucamine hydrochloride; 5, CsCl; 5, K2ATP; 5, 4-(2-hydroxyethyl)-l-piperazine ethane sulfonic acid (HEPES); 1, MgCl2; 0.1, CaCl2; and 1.1, ethylene glycol bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), pH 7.2, osmolarity 315 mosmol. Pipette-to-bath resistance was 4–7 MΩ. During an experiment, cells were constantly perfused with extracellular solution containing (in mM) 145 NaCl, 3 KCl, 1.5 CaC12, 1 MgCl2, 5.5 D-glucose and 10 HEPES, pH 7.4, osmolarity 320–330 mosmol. Cells were voltage-clamped at −60 mV. Since the intracellular and extracellular solutions contained symmetrical chloride concentrations, the chloride reversal potential was ca 0 mV.
GABA was applied to the cell under study by brief pressure ejection (2–6 p.s.i., 10–100 msec) from low-resistance micropipettes filled with 20 μM GABA for cells containing α1β3γ2 receptors and 2 μM GABA for cells containing α6β3γ2 receptors. This produced transient inward currents which were standardized by varying the duration of the pressure pipette pulse. For both cell lines, a maximal response was elicited by a 1 sec pulse of GABA from the pressure pipette, which was then decreased to 25–50 msec to achieve transient chloride currents that were ca 20% of the maximum current obtainable by 20 or 2 μM (test response; 19.3 ± 0.89% and 19.2 ± 0.94% for the α1β3γ2 and α6β3γ2 cells, respectively). GABA was applied every 20 sec. Anesthetic agents were applied to the bath until a stable, maximal potentiation of the GABA response was achieved. Drugs were then washed out until the pre-drug current was regained. Using this method, anesthetic equilibrium and recovery typically took 5–10 min. Current responses were low-pass filtered at 5 kHz (−3 dB, Bessel filter 902; Frequency Devices, Inc, MA, U.S.A.), digitized (TLl-125 interface; Axon Instruments, Foster City, CA, U.S.A.), and stored for off-line analysis (AXOBASIC, Axon Instruments).
GABA and/or anesthetics were rapidly applied to the cell by local perfusion [as described in Koltchine et al. (1996)] using a motor-driven solution exchange device (Bio Logic Rapid Solution Changer RSC-100; Molecular Kinetics, Pullman, WA, U.S.A.). An approximate EC10 GABA test dose was used as the control value (α1β3γ2/PRO: 9.0 ± 2.2% of maximal current; α1β3γ2/methohexital (MTX): 8.4 ± 0.7%; α6β3γ2/PRO: 9.2 ± 0.2%; α6β3γ2/MTX: 10.8 ± 1.4%) for the potentiation experiments. The solution changer was controlled by protocols in the acquisition programs AXOBASIC or pCLAMP5 (Axon Instruments). Laminar flow was maintained by applying all solutions at identical flow rates via a multi-channel infusion pump (Smelting, Wood Dale, IL, U.S.A.). Prior to recording, a blue dye (FD and C Blue No. 1 and FD and C Red No. 40, McCormick, Baltimore, MD, U.S.A.) was used to check the alignment of the solution streams from the rapid solution changer.
Drug-induced potentiation of a GABA-induced current was defined as the percentage increase of the control GABA response (defined as the average of the pre-drug and post-drug GABA induced currents). Concentration-response data were fitted (KaleidaGraph; Reading, PA, U.S.A.) with the logistic equation: I/Imax = 100*[drug]N/([drug]N+(EC50)N), where I/Imax is the percentage of the maximum obtainable GABA response, EC50 is the concentration producing a half-maximal response, and N is the Hill coefficient. Pooled data are presented as mean ± SEM. Statistical significance was determined by Student’s two-tailed, unpaired t-test.
Binding was performed on homogenized membranes prepared from cells cultured as above. Cells were harvested in ice-cold binding buffer containing (in mM): 20, Tris; 2, ethylenediamine tetraacetic acid (EDTA); 150, KCl, pH 7.4, then centrifuged at 2700g for 10 min at 4°C. The cell pellet was hypotonically lysed in deionized water and centrifuged at 48 000g for 20 min at 4°C. Finally, the pellet was rinsed with binding buffer and stored at −80°C for up to 2 months. Prior to experiments, membranes were thawed on ice and resuspended in assay buffer containing (in mM): 124, NaCl; 1.3, MgS04; 25, HEPES; 2.9, KCl; 1.2, KH2P04; D-glucose, 5.2, pH 7.4. The pellet was then homogenized with a Brinkman polytron (final protein concentration 0.3–0.5 mg/ml by Bradford assay, BSA standard). Membrane homogenates, along with eight concentrations of [3H]muscimol (saturation curve) or GABA plus a fixed concentration of [3H]muscimol (inhibition curve), were incubated in triplicate. After equilibration at room temperature, the assay mixtures were vacuum filtered through GF/B filter paper (Whatman, Clifton, NJ, U.S.A.) using a cell harvester (Brandel model MB-48, Gaithersburg, MD, U.S.A.), and quickly washed 10 times with 0.25 ml assay buffer. Specific binding was determined by subtracting the radioactive counts in the presence of 10 mM GABA from the radioactive counts in buffer alone. Similar results were obtained with a centrifugation assay using binding buffer (data not shown).
Data points are taken from specific binding in individual experiments, and represent the mean of triplicate assays±SEM. Saturation binding data were fit (KaleidaGraph; Reading, PA, U.S.A.) with the equation: B = (Bmax * [L])/([[L] + Kd), where B is the specific binding, [L] is [muscimol], and Kd is the dissociation constant for muscimol. Inhibition data were fit with the equation:
where Bmin is the lowest specific binding, [drug] is the final concentration of GABA, IC50 is the concentration producing a half-maximal displacement of [3H]muscimol. The Ki value for GABA was calculated from: Ki = IC50/(1 + ([L]/Kd).
Stock solutions of GABA, bicuculline methiodide, TCEt (Sigma), PRO (Aldrich, Milwaukee, WI, U.S.A), MTX sodium (Brevital®, Eli Lilly, Indianapolis, IN, U.S.A.), and midazolam hydrochloride (Versed®, intravenous/intramuscular solution preparation; Roche Pharmaceuticals, Manati, PR, U.S.A.) were diluted into extracellular solution daily before use. PRO was first dissolved into dimethyl sulfoxide (DMSO; Sigma) to form a stock solution of 10 mM PRO which was then dissolved into the extracellular solution to form the final PRO solutions (maximum final concentration of DMSO was 0.05% for a 50 μM PRO solution). Carrier controls were performed with 0.05% DMSO in extracellular medium. No significant effects of this DMSO solution were observed on GABA-induced currents in cells expressing either α1β3γ2 or α6β3γ2 receptors. [3H]muscimol (sp. act. 14.9 and 19.1 Ci/mmol) was obtained from NEN (Wilmington, DE, U.S.A.).
We initially characterized the electrophysiological responses to GABA of the α1β3γ2 and α6β3γ2 combinations. Application of GABA to either cell line by the rapid solution changer elicited concentration-dependent inward currents [Fig. 1(A, B)]. Analysis of the concentration-response data [Fig. 1(C) and Table 1] shows that the α6β3γ2 receptor has a higher apparent affinity for GABA (EC50 0.8 ± 0.1 μM) and a lower Hill coefficient (N= 0.9 ± 0.2) than the α1β3γ2 line (EC50 2.2 ± 0.2 μM, N = 1.9 ± 0.2, respectively; p < 0.05 for both comparisons). These results are in accord with other investigations that compared GABA responses in α1 and α6-containing GABAA receptor combinations (Ducic et al., 1995; Thompson et al., 1996). Also, the α1β3γ2 line produced larger maximal GABA currents than the α6β3γ2 line (2690 ± 400 pA vs 1186 ± 448 PA, p < 0.05). Midazolam (1 μM) potentiated GABA currents in cells containing the α1 subunit, but not in cells containing the α6 subunit, and GABA currents in both receptor subtypes were blocked by 20 μM bicuculline (data not shown).
As shown in Fig. 2(A) and Table 2, the α6β3γ2 receptor shows a higher affinity for [3H]muscimol than α1β3γ2 (α6β3γ2 Kd 28 ± 5 nM versus α1β3γ2 Kd 90 ± 23, p <0.05). A Scatchard plot of the data (Fig. 2(B)) illustrates that binding at both receptors was appropriately fit by a one-site model. In displacement assays (Fig. 2(C)), GABA binding affinity to α6 was also higher than at α1-containing receptors, as shown by its lower IC50 (Table 2: α6β3γ2: 45 ± 14 nM vs α1β3γ2: 370 ± 78, p <0.05), and lower Ki value (α6β3γ2: 34 ± 10 versus α1β3γ2: 337 ± 71, p < 0.05).
GABA potentiation by PRO was measured electrophysiologically using two separate methods: the picospritzer and rapid solution changer. The picospritzer was used from the initial set of experiments (Fig. 3, Table 3), since this method allowed for a wide range of modulator concentrations to be tested rapidly. However, once the appropriate PRO concentration range was established, later experiments with the rapid solution changer (Fig. 4) provided the means to apply known concentrations of GABA.
Significant potentiation of submaximal GABA-induced chloride currents by PRO was first observed at 0.2 μM and 0.5 μM PRO for the α1β3γ2 and α6β3γ2 GABAA receptors, respectively (Fig. 3(A), p < 0.05 for each). Table 3 shows that the magnitude of the maximal potentiation of GABA-induced currents by PRO in these picospritzer experiments was significantly greater in cells expressing the α1 isoform relative to the α6 isoform (maximum: 4. l-fold greater efficacy; p < 0.05). Statistically significant differences between the efficacy for PRO modulation of α1 and α6 receptors were observed at all concentrations > 0.2 μM PRO.
The rapid solution changer was then used to investigate GABAA receptor modulation in greater detail. As shown in Fig. 4, and Table 4, the efficacy of PRO potentiation applied at 10 μM was higher in α1 than at α6-ontaining receptors (1024 ± 203% vs 120 ± 39%; percent enhancement of an EC10 GABA test concentration by α1β3γ2 and α6β3γ2-containing receptors, respectively; p < 0.05).
As Fig. 4 illustrates, some of the single cell responses to PRO show an inward current during pre-equilibration of the anesthetic, prior to co-application with GABA. This direct channel gating by 10 μM PRO was much more pronounced in α6β3γ2 than α1β3γ2-containing receptor (Fig. 4(C): 24.0 ± 6.4% versus 5.3 ± 1.9% of maximal current, respectively; p< 0.05).
To determine whether these findings applied to other GABA modulators, the barbiturate MTX was also studied (Fig. 5 and Table 4). As with PRO, the efficacy of MTX enhancement of an EC10 GABA test concentration was higher in α1 than α6-containing receptors (Table 4; 816 ± 63% vs 309 ± 86%, respectively; p < 0.05). Figure 5 shows that, in agreement with previously published data studying pentobarbital modulation of GABAA receptors expressed in Xenopus oocytes (Thompson et al., 1996), 10 μM MTX also elicits significantly larger direct current in α6β3γ2 than α1β3γ2 containing receptors (16.7 ± 3.3% versus 2.4 ± 0.8% of maximal current, respectively; p < 0.05). Trichloroethanol was also studied, using picospritzer applied GABA (Fig. 3(B), Table 3). This drug also potentiated submaximal GABA currents, although it was less efficacious than PRO (2.2-fold less efficacious at both receptor subtypes). In a similar fashion to PRO, the efficacy of TCEt potentiation was significantly greater in α1β3γ2 than α6β3γ2-containing receptors p < 0.05).
The concentrations of PRO, TCEt, and MTX that caused enhancement of GABA-induced currents in this study correlate with clinically relevant anesthetic concentration ranges determined in vivo. PRO induces general anesthesia in rats and dogs with an estimated EC50 of 0.4 μM free PRO (Franks and Lieb, 1994), and TCEt anesthetizes canines and humans at concentrations in the range of 0.2–2 mM (Breimer, 1977; Garrett and Lambert, 1973). Threshold anesthetic concentrations for MTX in humans are estimated to range from 12 to 37 μM (Lauven et al., 1987).
The results of this study underline the importance of subunit composition in the modulatory effects of PRO. As summarized in Tables 3 and and4,4, replacing an α6 with an α1 subunit in GABAA receptors with an identical β3γ2 subunit background markedly increased the modulatory action of PRO. Although the larger maximal current (as shown in Table 1) and higher maximal binding [Bmax; Fig. 2(A and B)] suggest that more α1-containing receptors are being expressed, normalization of the enhancement against parallel GABA test concentrations demonstrates that the differential enhancement cannot be explained by differences in receptor expression levels.
Our data with MTX contrast slightly with other investigations of the role of the α subunit in modulation by barbiturates. Our finding that direct activation by MTX is greater in α6 than α1-containing receptors is in agreement with two studies assessing pentobarbital modulation in GABAA receptors expressed in Xenopus oocytes (Wafford et al., 1996; Thompson et al., 1996). However, in contrast to our findings, those two studies found that pentobarbital potentiated submaximal GABA currents to a slightly greater degree in α6 than α1-icontaining receptors. These differences may be accounted for by the use of different expression systems, barbiturates, and βγ backgrounds between those studies and ours. Use of the rapid solution changer allows for rapid equilibration of the applied GABA concentration, a condition which may be more difficult to achieve in whole oocytes. Nevertheless, from these studies, as well as our own, it is clear that the α subunit plays a significant role in both PRO and barbiturate modulation.
Inspection of Table 3 reveals a difference in the maximal potentiation (efficacy), but not the EC50, for GABA potentiation by PRO between α1β3γ2 or α6β3γ2 GABAA receptors. Thus, differences between the structures of the α1 and α6 subunit isoforms may be important in the extent of allosteric modulation by PRO, but perhaps not direct binding per se, of the drug to the GABAA receptor. Previous work has already shown that modulation by PRO does not require the γ subunit (Jones et al., 1995), and in fact, is even seen in β1 homomers expressed in Xenopus oocytes (Sanna et al., 1995b). Our finding that the α subunit isoform type does not affect the apparent affinity of PRO is consistent with the primary determinants of PRO binding being located on the β subunit, or on components of the α subunit which are highly conserved.
Research support was provided by NIH grants GM45129 and GM00623 (NLH), NIDA grant DA07255 (SMO), and NIMH grant MH11504 (MDK).