Anesth Analg 2005;101:401-406
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000155264.67729.E4
ANESTHETIC PHARMACOLOGY
The 
-Subunit Governs the Susceptibility of Recombinant -Aminobutyric Acid Type A Receptors to Block by the Nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6, 2N)
Ewa D. Zarnowska, PhD,
Robert A. Pearce, MD, PhD,
Abdallah A. Saad, BS, and
Misha Perouansky, MD
Department of Anesthesiology, University of Wisconsin, Madison
Address correspondence and reprint requests to Misha Perouansky, MD, 601 Science Dr., Madison, WI 53719. Address e-mail to mperouansky{at}wisc.edu.
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Abstract
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To identify anesthetic effects that produce the different components of the complex anesthetic state, the so-called nonanesthetics/nonimmobilizer classes of compounds have been introduced. Because ionotropic 
-aminobutyric acid type A (GABAA) receptors play an important role in the mediation of the central nervous system (CNS) effects of general anesthetics, and their susceptibility to modulation by various drugs depends on subunit composition, we have compared the effect of the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6) on GABAA receptors expressed in human embryonic kidney 293 cells transfected with 
1ß2 versus 1ß22s subunits. Using rapid perfusion and whole-cell recording techniques, we found that, like isoflurane, F6 blocked GABA-induced currents through 1ß2 receptors but, unlike isoflurane, the presence of the 2s subunit conferred complete resistance to block by F6. Also, in contrast to isoflurane, F6 had no effect on deactivation kinetics of GABA-induced currents in either type of receptor. We conclude that modulation of ß receptors plays little or no role in the actions of F6, but the block of ß receptors may contribute to its effects on the CNS.
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Introduction
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Anesthesia is a complex state whose characteristic behavioral effects are immobility, hypnosis, and amnesia. Inhaled drugs (fluorinated hydrocarbons) are often used to induce and maintain anesthesia, but the cellular mechanisms underlying these behavioral effects are largely unknown. To advance our understanding of anesthetic mechanisms, a number of compounds similar to general anesthetics in their physicochemical characteristics were introduced into the experimental paradigm (1). These drugs are interesting because at relative concentrations comparable to those of classic inhaled anesthetics predicted by lipophilicity to produce anesthesia, they either cause none or only some of the behavioral effects of anesthetics. Those that cause amnesia without immobility have been termed nonimmobilizers (2). The best studied of these is 1,2-dichlorohexafluorocyclobutane (F6 or 2N). Two properties of F6 are of interest in the context of the interaction of anesthetic drugs and -aminobutyric acid type A (GABAA) receptors: it impairs learning (2) and induces seizures (1,3).
The GABAA receptor is a prominent target of many general anesthetics and all volatile anesthetics, and it has been hypothesized that enhancement of its function contributes to the anesthetic state (4). However, the susceptibility of the GABAA receptor to various neurotropic drugs is strongly affected by its subunit composition. The -subunit, in particular, determines the susceptibility of the receptor to modulation by benzodiazepines (5), and we found that the -subunit also influences modulation by the inhaled anesthetic isoflurane (6). Therefore, we have now investigated whether the -subunit also influences the effects of F6 on the GABAA receptor. To this aim, we compared the effect of F6 on GABA-induced chloride currents through 1ß2 receptors with its effect on 1ß22s receptors expressed in human embryonic kidney 293 (HEK 293) cells.
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Methods
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HEK 293 cells (CRL 1573; American Type Culture Collection, Manassas, VA) were grown under standard culture conditions (37°C; 5% CO2). The culture medium consisted of Minimum Essential Medium (MEM) with l-glutamine, supplemented with MEM amino acids solution (0.1 mM), MEM sodium pyruvate (1 mM), penicillin/streptomycin 1% (vol/vol), and 10% fetal bovine serum (Harlan, Indianapolis, IN). Complementary DNA (cDNA) encoding the rat GABAA receptor subunits 1 (FLAG tagged) (7), ß2, or 2s were inserted into the multiple cloning site of the mammalian expression vector pCEP4 (Invitrogen, Carlsbad, CA). Cells were co-transfected with 1ß2 (1:1) or 1ß22s (1:1:10 or 1:1:1, 1:1:0.5, and 1:1:0.1) subunits using lipofectamine 2000. The total cDNA transferred into the HEK cells for 1ß2 was 1 µg and for 1ß22s ranged from 0.5 µg to 3 µg.
Recordings were performed at room temperature (22°C–24°C) on the stage of an Olympus BX50WI upright microscope. Transfected cells were identified using a bead immunolabeling technique (8). Dynabeads (Dynal, Oslo, Norway) were coated with sheep anti-rat antibodies, which were attached to anti-FLAG rat antibodies directed against the flag epitope sequence inserted into the 1 subunit (7). Ten minutes before initiating an experiment, labeled beads were added to the culture dish at a 1:500 dilution. After stable whole-cell access was obtained, slight negative pressure was applied, and the cell was lifted from the coverslip and positioned in front of the rapid application pipette. Recording pipettes were pulled from borosilicate glass (1.7 mm OD, 1.1 mm ID; KG-33, Garner Glass, Claremont, CA) using a two-stage puller (Flaming-Brown model P-87; Sutter Instruments, Novato, CA). Pipette tips were fire polished and had open-tip resistances of 2.5–4 M
when filled with the recording solution. All whole-cell recordings were obtained at a holding potential of –40 mV using an Axopatch 200A patch-clamp amplifier (Axon Instruments, Foster City, CA) and pClamp 8.0 software (Axon Instruments). Access resistances were typically 8–15 M and were then compensated by 50%–80% using amplifier circuitry. Data were low-pass filtered at 5 kHz, sampled at 10 kHz (Digidata 1200; Axon Instruments), and stored online on a computer hard disk.
Solutions were applied using a tetra-barrel square glass application pipette, which was connected to a piezoelectric stacked translator (model P-245.50; Physik Instrumente, Costa Mesa, CA). The voltage input to the high voltage amplifier (model P-270; Physik Instrumente) used to drive the stacked translator was filtered (50–100 Hz) using an 8-pole Bessel filter (model 902LPF; Frequency Devices, Haverhill, MA) to reduce oscillations arising from rapid acceleration of the pipette. Open-tip exchange rates of 1–2 ms (10%–90% rise time) were achieved.
The recording chamber was perfused continuously with a solution containing (in mM): NaCl 145, KCl 5, HEPES-FA 10, MgCl2 1, and CaCl2 1.8, with a pH value 7.3. Pipettes were filled with intracellular solutions containing (in mM): CsCl 140 or KCl 140, Na-HEPES 10, BAPTA 10, and MgATP 2, with a pH value of 7.2. Solutions containing GABA were prepared daily from powder. Diazepam was prepared as a stock solution (10 mM in DMSO) stored at –20°C and diluted in saline the day of an experiment. Solutions containing F6 (Lancaster Synthesis Inc., Pelham, NH) were prepared, as previously described (9). Briefly, a F6 stock gas was prepared in a Chemware Teflon FEP gas sampling bag (North Safety Products, Cranston, RI) by first evacuating air from the bag and then adding 100 mL of distilled water, 1–2 mL of liquid F6, and 300 mL of room air. This yielded humid air saturated with F6. Appropriate amounts of F6-containing air, as calculated using a saline/gas partition coefficient for F6 of 0.026 (9), and fresh air were then transferred to a second saline-filled Teflon bag, which was placed on a shaker tray for at least 1 h to equilibrate gas and aqueous phases. The concentration of F6 was determined in each experiment using gas chromatography.
We used Origin software (Microcal Software Inc., Northampton, MA) for analysis. The deactivation time course was fitted with a sum of two exponentials y(t) = 
Ai x exp (–t/
i), where i and Ai are the time constants and relative fractions of respective components, using the Levenberg-Marquardt algorithm. The weighted time constant was calculated as: w = (iAi)/Ai. Dose-response data were fitted using the logistic equation y = A[1 – 1/1+(x/x0)p], where A is the maximal block, x0 is the half-maximal inhibitory concentration (IC50), and p is the Hill coefficient. Data are presented as mean ± sem. Student’s t-test was used to determine statistically significant differences (P < 0.05).
The potency of volatile anesthetics is expressed as the minimum alveolar anesthetic concentration (MAC; expressed as fractional concentration of the drug in exhaled gas) that prevents movement in response to noxious stimulation in 50% of subjects. MAC can be predicted from the lipid solubility of a drug based on the Meyer-Overton correlation (MAC = 1.82 atm/oil/gas partition coefficient) (10). The predicted MAC (MACpred) of F6, i.e., the concentration at which it would produce immobility according to the Meyer-Overton correlation, is 4.2% atm (1), corresponding to a concentration of 16 µM in aqueous solutions at room temperature (11).
All chemicals were obtained from Sigma (St. Louis, MO), and cell culture reagents were obtained from Gibco Life Technologies, Inc. (Grand Island, NY) unless otherwise indicated.
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Results
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In cells co-transfected with 1 and ß2 subunits, F6 reduced responses to 10-ms pulses of 1 mM of GABA (Fig. 1A), and the result was reversible. By contrast, in cells co-transfected with 1, ß2, and 2s subunits, F6 had no such effect (Fig. 1B). A summary of results from 9 cells co-transfected with 1 and ß2 subunits (1:1) and 22 cells co-transfected with 1, ß2, and 2s subunits (transfection ratios ranging from 1:1:0.1 to 1:1:10) is shown in Figure 1C. For 1ß2 receptors, the extent of block by F6 was concentration dependent, with an IC50 of 7.7 ± 2.6 µM (approximately 0.5 x MACpred). However, F6 did not block the current through 1ß22s receptors for any of the transfection ratios, even at the largest concentrations tested (100 µM; approximately 6 x MACpred).
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Figure 1. The 2s subunit prevents block of chloride currents by 1,2-dichlorohexafluorocyclobutane (F6). (A) Inward current generated by brief (10 ms) application of 1 mM of -aminobutyric acid (GABA) is reversibly blocked by F6 (2.9 x predicted mean alveolar anesthetic concentration [MAC]pred) by 71% in a cell transfected with 1 and ß2 subunits. (B) In a cell transfected with 1, ß2, and 2s subunits, by contrast, F6 (4.2 x MACpred) had no effect on peak current amplitude. (C) Summary of the results from 31 cells. Filled circles represent cells transfected with 1 and ß2 subunits, open symbols represent cells transfected with 1, ß2, and 2s subunits, using transfection ratios of 1:1:0.1 (squares), 1:1:0.5 (circles), 1:1:1 (triangles), and 1:1:10 (diamonds). GABA currents in 1ß2 transfected cells were blocked by F6 by up to 93% with a 50% effective concentration of 7.7 µM and a Hill coefficient of 1.0. 2s-containing cells were resistant to block independent of the transfection ratio. For the logistic fit, P = 1.0 ± 0.8, A = 93 ± 21, x0 = 7.7 ± 2.6.
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Volatile anesthetics affect not only the amplitude of GABA-evoked currents, but also slow the rate of deactivation after a brief pulse of GABA. Indeed, this effect is thought to contribute importantly to their clinical effects. Therefore, we also tested whether F6 affects deactivation of 1ß2 and 1ß22s receptors. From the experiments shown in Figure 1, we fitted the current decay after a 10-ms pulse of 1 mM of GABA to a biexponential function and calculated the weighted time constant of decay. As previously reported (12), deactivation was faster under control conditions for 1ß22 compared to 1ß2 receptors, and the larger the amount of 2s co-transfected with 1 and ß2, the faster the deactivation (Table 1). In contrast to isoflurane, F6 did not alter deactivation of either 1ß2 (Fig. 2A) or 1ß22s receptors (Fig. 2B). A summary of experiments with F6 at concentrations from 8 to 100 µM is presented in Figure 2C, together with effects of isoflurane at equivalent concentrations for comparison. It is evident that over a broad range of concentrations, extending from amnestic (
0.5 x MACpred) to convulsive (1.2 x MACpred) and beyond (up to 6 x MACpred), F6 did not alter deactivation of either subunit combination.
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Discussion
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The primary finding of this study is that GABAA receptors lacking the -subunit are potently blocked by the nonimmobilizer F6. We also found that F6 does not alter deactivation of either 1ß2 or 1ß22s receptors. Our finding that receptors lacking the -subunit are inhibited at concentrations of F6 that induce amnesia and convulsions is the first demonstration of an interaction between this drug and the principal inhibitory transmitter system in the mammalian brain. Because the modulation of GABAA receptors by other drugs affects memory and seizures, this suggests that F6 may cause at least some of its effects by this mechanism. The finding that F6 inhibits GABAA receptors without prolonging their deactivation points to a dissociation between the molecular events underlying its blocking and prolonging effects.
In testing the influence of the 2-subunit on modulation by F6, we used different transfection ratios of and ß versus the -subunit. In agreement with previously published work (12), we found that over-expression of the -subunit accelerated the deactivation of chloride currents in response to brief GABA pulses (Table 1). However, as opposed to the sensitivity of the current decay to transfection ratio, we found that the presence of even small ratios of the -subunit to and ß conferred resistance to block by F6 (Fig. 1C). We have no ready explanation for this puzzling result, but we have also observed a similar phenomenon when zinc is applied (Boileau et al., unpublished data). Possible explanations might include effects of transfection ratio on receptor trafficking, secondary modifications, or the production of some excess receptors that are nonfunctional with regard to current generation but that can influence the decay rate of functional receptors. In any case, the clear blocking effect of F6 on receptors that was produced by transfecting only and ß subunits demonstrates that receptors that lack a -subunit are susceptible to block at concentrations that produce amnesia and convulsions.
The effect of F6 on GABAA receptors has been investigated (13). Responses of recombinant 1ß22s receptors expressed in Xenopus oocytes to small GABA concentrations (5 µM) were found to be unaffected by F6 at concentrations up to 80 µM. These results are consistent with our present results obtained in HEK cells expressing the same subunits but tested with near-saturating GABA concentrations (1 mM). By using a rapid perfusion system, we were able to extend these findings to demonstrate that F6 also does not affect the deactivation of 2-subunit containing receptors. These results with recombinant receptors emulate our recent findings with native synaptic receptors of hippocampal CA1 pyramidal neurons, which are also neither blocked nor prolonged by F6 (14). Taken together, these findings thus suggest that the 2-subunit, which influences receptor targeting (15), also makes them insensitive to F6.
By contrast, receptors that incorporated only 1 and ß2 subunits were blocked by up to 80% (Fig. 1C), with a half-maximal effect occurring at a concentration of approximately one-half MACpred. Although most receptors in the brain do contain the 2-subunit, up to 40% of all GABAA receptors detected by [3H]muscimol binding do not (16). Many of these contain an alternate third subunit (i.e., 1, 3, or 
), but some native receptors may consist solely of and ß subunits (16). Whether all receptors that lack the 2-subunit are blocked by F6 is not known. However, the presence of F6-susceptible native receptors, located probably at extrasynaptic sites on the somata of a subpopulation of interneurons (17), indicates that the ability of F6 to block some types of GABAA receptors may thereby contribute to its behavioral actions (2). Our present results suggest that this susceptibility may derive from the absence of a 2-subunit. Because the net effect of anesthetics in the hippocampal circuit is to enhance inhibition (18), whereas F6 reduces inhibition for some receptor combinations, it is also possible that these opposing actions contribute to the behaviorally antagonistic effects on memory that were recently reported (19).
The role played by the -subunit in the interaction of anesthetics with GABAA receptors is not as well defined as those of the and ß subunits. For the latter two, point mutations have been identified that profoundly affect the sensitivity of receptors to modulation by inhaled and injected anesthetics (20–22). The effects of analogous mutations in the 2s subunit have not been described. However, it is clear that the presence of the -subunit does affect the interaction of the GABA receptor with volatile anesthetics (6,23,24). When activated by the 30% effective concentration of GABA, the current through 2-containing receptors is enhanced (23). However, when exposed to saturating GABA concentrations, receptors that contain a 2-subunit are less susceptible to block by isoflurane, and the net charge transfer is enhanced by isoflurane in ß receptors but decreased in ß receptors (6,23). The finding that the 2-subunit influences receptor block by F6, as it does by isoflurane, suggests that a common mechanism may underlie this effect of the two drugs.
If the similar effects of the -subunit on the susceptibility to block by F6 and isoflurane does reflect a common site of action for these two drugs, then the ability of F6 to block currents in 1ß2 receptors without substantially prolonging their deactivation supports the hypothesis that separate binding sites underlie the blocking and prolonging effects of volatile anesthetics observed in native (25) and expressed (26) GABAA receptors. Several arguments that support this hypothesis have been advanced previously, including a difference in concentration dependence of the two effects (25), the presence of a surge current that may reflect the rapid dissociation of anesthetic from a blocking site within the channel pore (26,27), and quantitative differences between the relative strengths of these effects for different anesthetics (25). Our present finding is an extreme example of this latter argument. It may prove useful to exploit this difference in future experiments that aim to identify binding sites that produce the different actions, such as via the use of chimeric receptors (20).
We are grateful to Drs. Michael Laster and Edmond Eger II (University of California, San Francisco) for providing gas phase calibration standards for isoflurane and F6 and to Mark Perkins (University of Wisconsin, Madison) for performing gas chromatographic analysis.
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Footnotes
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Presented, in part, at the 12th Neuropharmacology Conference, Orlando, FL, November 2002, and at the 33rd Annual Meeting of Society for Neuroscience, New Orleans, LA, November 2003.
Supported, in part, by grants GM 55719 and GM 47818 (to RAP) and by the Department of Anesthesiology, University of Wisconsin—Madison.
Accepted for publication December 17, 2004.
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Abstract
Introduction
