Gamma-aminobutyric acid type A (GABAA) receptors, the major inhibitory receptors in the brain, are important targets of many drugs, including general anesthetics. These compounds exert multiple effects on GABAA receptors, including direct activation, prolongation of deactivation kinetics, and reduction of inhibitory postsynaptic current amplitudes. However, the degree to which these actions occur differs for different agents and synapses, possibly because of subunit-specific effects on postsynaptic receptors. In contrast to benzodiazepines and intravenous anesthetics, there is little information available about the subunit dependency of actions of volatile anesthetics. Therefore, the authors studied in detail the effects of isoflurane on recombinant GABAA receptors composed of several different subunit combinations.
Human embryonic kidney 293 cells were transiently transfected with rat complementary DNAs of alpha1beta2, alpha1beta2gamma2L, alpha1beta2gamma2S, alpha5beta3, or alpha5beta3gamma2S subunits. Using rapid application and whole cell patch clamp techniques, cells were exposed to 10- and 2,000-ms pulses of gamma-aminobutyric acid (1 mm) in the presence or absence of isoflurane (0.25, 0.5, 1.0 mm). Anesthetic effects on decay kinetics, peak amplitude, net charge transfer and rise time were measured. Statistical significance was assessed using the Student t test or one-way analysis of variance followed by the Tukey post hoc test.
Under control conditions, incorporation of a gamma2 subunit conferred faster deactivation kinetics and reduced desensitization. Isoflurane slowed deactivation, enhanced desensitization, and reduced peak current amplitude in alphabeta receptors. Coexpression with a gamma2 subunit caused these effects of isoflurane to be substantially reduced or abolished. Although the two gamma2 splice variants imparted qualitatively similar macroscopic kinetic properties, there were significant quantitative differences between effects of isoflurane on deactivation and peak current amplitude in gamma2S- versus gamma2L-containing receptors. The net charge transfer resulting from brief pulses of gamma-aminobutyric acid was decreased by isoflurane in alphabeta but increased in alphabetagamma receptors.
The results indicate that subunit composition does substantially influence modulation of GABAA receptors by isoflurane. Specifically, the presence of a gamma2 subunit and the identity of its splice variant are important factors in determining physiologic and pharmacologic properties. These results may have functional implications in understanding how anesthetic effects on specific types of GABAA receptors in the brain contribute to changes in brain function and behavior.
γ-AMINOBUTYRIC acid type A (GABAA) receptors are the major inhibitory neurotransmitter receptors in the brain. They are modulated by a wide variety of drugs, including sedatives, hypnotics, anxiolytics, and anticonvulsants. Because their activity is enhanced by a wide variety of intravenous and volatile agents, they are also considered a prime target site for general anesthetics.1–6A striking feature of general anesthetic action on the GABAAreceptor is the multitude of effects that are observed. These include a slowing of deactivation, altered rate and extent of desensitization, increases or decreases in peak current amplitude, and an increased apparent affinity for γ-aminobutyric acid (GABA).7–16In addition, at high concentrations, general anesthetics can directly activate GABAAreceptors.3
γ-Aminobutyric acid type A receptors are heteropentamers, and 19 subunits (α1–6, β1–3, γ1–3, Δ, ε, π, θ, ρ1–3) have been identified in the mammalian brain. Most receptors are thought to incorporate two α subunits, two β subunits, and one γ subunit. Substantial structural diversity arises from different combinations of subunits, and for some subunits, including the γ2subtype, additional diversity is achieved by alternative splicing.17,18The identity of the constituent subunits determines the physiologic characteristics and pharmacologic sensitivities of receptors.1,18–20Presumably, these differences account for the different spectra of behavioral effects caused by different drugs.21–24
For many intravenous agents, including propofol, etomidate, and benzodiazepines, modulation requires the presence of specific subunits or combinations of subunits.25–29In contrast, most studies of recombinant GABAAreceptors have revealed little influence of subunit composition on modulation by volatile anesthetics.30–35Nevertheless, heterogeneous effects of volatile agents on distinct types of inhibitory synapses in the brain have been observed, including differences in anesthetic effects on the amplitudes and time courses of inhibitory postsynaptic currents (IPSCs) in the hippocampus9,10,14and the cerebellum.13Because the γ2subunit plays an important role in synaptic targeting or clustering of receptors36and because it was reported recently that incorporation of a γ2Ssubunit renders expressed receptors relatively insensitive to block by isoflurane,37we examined in detail the influence of the γ2subunit and its two splice variants on modulation by the prototypical volatile anesthetic isoflurane. We did so by coexpressing the γ2subunit splice variants together with α1and β2or α5and β3, because these represent two of the most prevalent native subunit combinations found in the brain, and particularly in the hippocampus17,18
Materials and Methods
Cell Culture and Receptor Expression
Human embryonic kidney 293 cells (American Type Culture Collection CRL 1573, Manassas, VA) were cultured in minimum essential medium with l-glutamine, supplemented with minimum essential medium amino acids solution (0.1 mm), sodium pyruvate (1 mm), 1% (volume/volume) streptomycin, and 10% fetal bovine serum (Harlan, Indianapolis, IN). Cells were grown at 37°C in a humidified 5% CO2–95% air atmosphere and subcultured twice weekly using trypsin (0.25%)–EDTA (1 mm). Twenty-four to 72 h before transfection, cells were plated on 60-mm culture dishes. Complementary DNAs (cDNAs) encoding the rat GABAAreceptor subunits α1(“flag”epitope tagged38), β2, γ2L, γ2S, α5, and β3were inserted into the multiple cloning site of the mammalian expression vector pCEP4 (Invitrogen, Carlsbad, CA) as was the cDNA for enhanced green fluorescent protein (EGFP) from pEGFP-N1 (BD Biosciences Clontech, Palo Alto, CA). After reaching 80–90% confluence, cells were cotransfected with α1β2(1:1), α1β2γ2L(1:1:10), α1β2γ2S(1:1:10), α5β3(1:1), or α5β3γ2S(1:1:10) subunits using Lipofectamine 2000 (Gibco Life Technologies, Inc., Grand Island, NY). Overexpression of the γ2subunits was used to bias expression toward receptors containing all three subunits.39,40cDNA quantities were adjusted to obtain maximum peak currents of 200 pA to 8 nA. Usually, 250–500 ng α1, β2, α5, or β3and 2.5–5 μg γ2Sor γ2LcDNA was cotransfected with 180 ng EGFP cDNA. One to 3 h before performing electrophysiologic recordings, cells were replated onto 12-mm glass coverslips (Fisher Scientific, Pittsburgh, PA).
Whole Cell Patch Clamp Recordings
Experiments were performed at room temperature (22°–24°C) on the stage of an upright microscope (BX50WI; Olympus, Melville, NY) equipped with a long-working distance water-immersion objective (Achroplan 40×; 0.75 numerical aperture; Carl Zeiss, Thornwood, NY) and differential interference contrast (Nomarski) optics. For visual identification of individual transfected cells, we used a bead immunolabeling technique.41Dynabeads (Dynal, Oslo, Norway) were coated with sheep anti-rat antibodies, incubated with anti-flag rat antibodies directed against an N-terminus “flag” epitope fused to the α1subunit, and added to the culture dish at a 1:500 dilution. For some experiments, cells that had been cotransfected with EGFP were also visualized using a mercury arc lamp (Mercury 100; Chiu Technical Corp., Kings Park, NY) and an EGFP filter set (Chroma Technology Corp., Rockingham, VT). Whole cell recordings were made from only the smallest cells to maximize mechanical stability and to minimize solution exchange time. After gaining stable whole cell access, negative pressure was applied, and the cell was lifted from the coverslip and positioned in front of the rapid application pipette.
Pipettes were fabricated 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) and coated with Sylgard 184 (Dow Corning Company, Midland, MI) to reduce pipette capacitance. Pipette tips were fire polished and had open-tip resistances of 2–5 MΩ when filled with recording solution.
All whole cell recordings were obtained at a holding potential of −40 mV using an Axopatch 200B patch clamp amplifier (Axon Instruments, Union City, CA) and pClamp 8.0 software (Axon Instruments). Access resistances were typically 4–11 MΩ and were compensated by 75–90%. Access resistance and cell capacitance were monitored throughout the course of the experiment. Recordings were terminated if these became unstable (> 15% change). Data were low-pass filtered at 5 kHz using internal amplifier circuitry, sampled at 10 kHz (Digidata 1200; Axon Instruments), and stored on the hard disk of a Pentium-based computer.
Rapid Solution Exchange Technique
Whole cells lifted from the coverslip were exposed to solutions using a two-barrel “theta” application pipette mounted to a piezoelectric stacked translator (model P-245.50; Physik Instrumente, Costa Mesa, CA). The application pipette was fabricated from “thin theta” glass (Sutter Instruments, Novato, CA) and connected to solution reservoirs using polyimide and Teflon tubing (Cole Parmer Instruments, Vernon Hills, IL). The voltage input to the high-voltage amplifier (model P-270; Physik Instrumente) used to drive the stacked translator was filtered at 100 Hz using an 8-pole Bessel filter (model 902LPF; Frequency devices, Haverhill, MA) to reduce oscillations arising from rapid acceleration of the pipette. Solution exchange rates were estimated by measuring open-tip junction currents with a diluted solution at the end of each experiment. The exchange speed was adjusted by changing the height of the reservoirs so that, typically, open-tip exchange times of 500 μs (10–90% rise time) were achieved. For whole cells positioned near the interface of the two flowing solutions (approximately 100 μm from the tip of the pipette), solution exchange rates were slightly slower (approximately 2 ms) than indicated by the open-tip rate.42
γ-Aminobutyric acid–evoked currents were elicited by stepping between a “control” extracellular solution and one containing GABA (1 mm). To measure the effects of isoflurane on GABA-evoked responses, both solutions flowing from the application pipette were switched to solutions containing isoflurane using low-volume, manually controlled Teflon valves (model 1126; Omnifit Limited, Cambridge, United Kingdom). Cells were typically exposed to isoflurane for longer than 1 min before responses to GABA plus isoflurane were obtained.
Solutions and Drugs
The recording chamber was perfused continuously with HEPES-buffered “extracellular saline” consisting of 130 mm NaCl, 3.1 mm KCl, 10.9 mm Na-HEPES, 1.44 mm MgCl2, and 2.17 mm CaCl2at a pH of 7.3. The same standard saline served as “control” solution in the rapid-application pipette. Patch pipettes were filled with 140 mm CsCl, 10 mm Na-HEPES, 10 mm EGTA, 5 mm QX-314, and 2 mm MgATP at a pH of 7.2 and 280–290 mOsm. GABA was dissolved in extracellular saline to obtain a 1 mm solution. GABA solutions were prepared daily from powder and not used for longer than 5 h. ZnCl2was prepared as a 10 mm stock solution in H2O and diluted with extracellular saline to achieve the desired concentration.
Isoflurane solutions were prepared from saturated stock solutions (15 mm) in extracellular saline and diluted to the final concentration in gas-tight Teflon bags (Chemware, Portage, WI). Concentrations in the reservoir were confirmed using gas chromatography. All tubing and connectors were made of Teflon to minimize loss of the anesthetic. Based on previous data, the minimal alveolar concentration for isoflurane in the rat corresponds to an aqueous concentration of 0.31 mm.10,43
All chemicals were obtained from Sigma (St. Louis, MO). Cell culture reagents were from Gibco (Life Technologies, Inc.) unless indicated otherwise. Distilled water was used to prepare all solutions.
Data were analyzed using ClampFit 8.0 (Axon Instruments, Union City, CA), Origin 6.1 (MicroCal, Northampton, MA), Excel (Microsoft, Redmond, WA), and Prism 3.0 (GraphPad, San Diego, CA). Decay kinetics (deactivation and desensitization) were analyzed by fitting current traces to the exponential function y = y0+Σ Anexp[−t/τn], where y0is the steady state current during desensitization (0 for deactivation), and Anand τnare the amplitude and the time constant of the nth component of a multiexponential fit. Goodness of fit was evaluated by visual inspection. Percent contribution of each component (An%) was calculated as An/ΣAn× 100. In most cases, deactivation and desensitization were well fitted by biexponential functions. However, some of the responses were best fitted with only one or more than two time constants. To facilitate comparison of responses with differing numbers of decay constants, we calculated a weighted time constant τwt=Σ(Anτn)/ΣAn. To analyze the effect of isoflurane on deactivation and desensitization kinetics, currents were normalized to the peak amplitude and calculated as the ratio τwt(iso)/τwt(ctrl) with at least three to five traces averaged for each. Isoflurane effects on net charge transfer were determined by integrating the area under the curve of the averaged raw current traces under control conditions and in the presence of isoflurane.
Results are reported as mean ± SD. Statistical comparisons of decay rates and peak amplitudes between data sets were made using the Student t test or one-way analysis of variance followed by the Tukey post hoc test, as appropriate. Differences were considered significant at P < 0.05.
Effects of the γ2Subunit on Macroscopic Kinetics
We first examined the effect of the γ2subunit itself on macroscopic kinetics. We showed previously that when the γ2Ssubunit is coexpressed with the α1and β2subunits, deactivation after a brief pulse of GABA is accelerated, and desensitization, the decline in current that occurs during the continued presence of a high concentration of GABA, is reduced.39To determine whether the γ2Ssubunit causes these same effects when it is coexpressed with different α and β subunits and whether the γ2Land γ2Ssubunits produce similar effects, we expressed γ2Swith α5and β3as well as α1and β2, and expressed both γ2Sand γ2Lwith α1and β2subunits.
Examples of responses to brief pulses (10 ms) and to prolonged application (2 s) of a high concentration of GABA (1 mm) are shown in figure 1. The results of fitting current deactivation and desensitization to exponential functions are presented in tables 1 and 2. As observed previously by other investigators, deactivation and desensitization were in most cases fitted best by biexponential functions. Similar to our previous results, coexpression of γ2Swith α1and β2accelerated deactivation (fig. 1Aand table 1) and reduced desensitization by causing either a loss or a significant reduction of the faster component of desensitization so that a large portion of γ2S-containing receptors exhibited monophasic desensitization (fig. 1Band table 2). This also held true for coexpression of γ2Swith α5and β3subunits. In contrast, coexpression of γ2Lwith α1and β2did not significantly accelerate deactivation, whether after a brief (fig. 1A) or a long (fig. 1B) pulse of GABA (table 1; P > 0.05). This was not due to lack of incorporation of the γ2Lsubunit, because these receptors were insensitive to block by ZnCl2(100 μm)44(fig. 2). Like γ2S, coexpression of γ2Lwith α1and β2subunits reduced the rate and extent of desensitization (table 2). Deactivation was significantly slower for α5β3than α1β2receptors, whether GABA was applied for 10 ms (Student t test, P < 0.001) or 2 s (P < 0.001). Therefore, the effects of γ2Sand γ2Ldiffered for deactivation but not desensitization, and the identity of α and β subunits did not seem to be a determining factor for the impact of γ2Son deactivation or desensitization.
Modulation by Isoflurane: Responses to Brief Pulses of GABA
Similar to its effects on IPSCs,8–10,13isoflurane prolonged the decay and decreased the amplitude of currents evoked by brief pulses of GABA (1 mm, 10 ms). These effects were concentration dependent, were fully reversible, and were seen for all subunit combinations tested (figs. 3 and 4). However, there were striking differences in the degree of changes observed, depending on the presence or absence of a γ2subunit and on the γ2splice variant that was expressed. Effects on both deactivation and amplitude were most pronounced in receptors composed of only α and β subunits: α1β2receptors exhibited a nearly 10-fold increase in the weighted time constant of deactivation accompanied by greater than 90% reduction in amplitude at an isoflurane concentration of 1 mm (figs. 3A, 4B, and 4D), and α5β3receptors were sixfold slower to deactivate and nearly 90% reduced in amplitude with 0.5 mm isoflurane (figs. 3C, 4B, and 4D). Coexpression of γ2Lwith α1and β2subunits substantially reduced the effect of isoflurane on amplitude (figs. 3B, 4C, and 4D) but not deactivation (figs. 4A and B). Coexpression of the γ2Ssubunit with either α1β2or α5β3receptors similarly reduced the effect of isoflurane on amplitude (figs. 3C and 4D), and it also reduced its effect on deactivation (figs. 3C and 4B). Therefore, just as γ2Sand γ2Ldifferently affected deactivation under control conditions, these two splice variants also differed in their influence on modulation by isoflurane.
An additional difference between the two γ2splice variants was a substantial variability in response to isoflurane with γ2Sbut not γ2L(compare error bars in fig. 4D, γ2Sversus γ2L, and fig. 5). Whereas in many cells expressing γ2Sthe peak current amplitude was reduced by isoflurane, the degree of reduction was quite variable, and in some cells, there was even an increase in amplitude (fig. 5A) so that, on average, there was no change (fig. 4D; P > 0.05). Figure 5Bsummarizes the diversity of isoflurane effects on peak amplitude observed for α1β2γ2Sreceptors (n = 28). Responses ranged from 81% reduction to 52% enhancement. Cells labeled cell 1 or cell 2 at either end of the spectrum correspond to current traces labeled cell 1 or cell 2 in figure 5A. Accompanying the variable effects on amplitude were variable effects on deactivation. Cells in which peak current amplitude was most strongly reduced by isoflurane also exhibited the most pronounced effects on decay (figs. 5A and C, cell 1). Conversely, increased amplitude was associated with modest prolongation (figs. 5A and C, cell 2).
This correspondence applied not only to effects of isoflurane on amplitude and deactivation, but also to differences in baseline kinetics of different subunit combinations: Those that displayed the greatest reduction in amplitude in the presence of isoflurane (fig. 5A) were slower to deactivate in the absence of the anesthetic (fig. 5C) and displayed a greater degree of desensitization (fig. 5D). The strong correlations between amplitude reduction and decay prolongation by isoflurane and degree of desensitization under control conditions in α1β2γ2Sare summarized in figure 5E.
Comparison of isoflurane effects on receptors of different subunit composition revealed that the variability of isoflurane effects on α1β2γ2Sreceptors paralleled a general difference between αβ and αβγ receptors: Those most strongly blocked also were most prolonged by isoflurane (fig. 6A) and showed slower deactivation (fig. 6B) and faster desensitization (fig. 6C) in the absence of the anesthetic.
In previous studies, anesthetic-induced enhancement of peak currents occurred when low (subsaturating) concentrations of GABA were used but not when near-saturating concentrations were applied,33–35,45as in this study. Because slow solution exchange resulting in exposure to only subsaturating concentrations of GABA during brief (10-ms) pulses represents a possible explanation for the observed increases in peak current amplitude in α1β2γ2Sreceptors in the presence of isoflurane,16we therefore examined the 10–90% rise time (t10–90) for different subunit combinations. However, in all cases, t10–90values were 1.5–4 ms, which is substantially briefer than the pulse (duration 10 ms). Only for α1β2receptors was there a significant decrease in rise time, and for these receptors, the effect was modest (t10–90= 84% of control at 0.5 mm isoflurane, P < 0.05; t10–90= 71% of control at 1 mm isoflurane, P < 0.01, n = 16). For other subunit combinations, effects were not significant (P > 0.05), and there was no correlation between the effect of isoflurane on the amplitude and rise time (r =−0.11, P > 0.05, n = 47). These findings argue against slow solution exchange as a cause for the increased amplitudes in α1β2γ2Sreceptors.
Although in many cases it may be useful to distinguish anesthetic effects on amplitude and deactivation, in some situations, net charge transfer may be the important parameter. The two major effects of isoflurane that we observed, reduced amplitude and slowed deactivation, have opposing influences in this regard. We assessed the net effect of isoflurane on charge transfer for different subunit combinations and for a range of isoflurane concentrations (fig. 7). Net charge transfer was significantly increased by isoflurane for all receptors containing a γ2subunit but was significantly reduced for α1β2and α5β3receptors. Therefore, for αβ receptors, the slowed deactivation induced by isoflurane, although dramatic, was not sufficient to fully compensate for its profound blocking effect.
Modulation by Isoflurane: Desensitization
Desensitization is a prominent feature of all types of GABAAreceptors. It has been suggested that modulation of desensitization by anesthetics may contribute to their net effect,16,45–47and desensitization of GABA receptors may have profound effects on the shape of IPSCs.48,49We tested the impact of the two γ2splice variants on modulation of desensitization by isoflurane by applying a high concentration of GABA (1 mm) for 2 s in the presence and absence of the anesthetic. For α1β2receptors (n = 4), in addition to reducing peak amplitude, isoflurane increased significantly the rate and extent of desensitization (fig. 8). In contrast, even when peak amplitude was reduced, desensitization was not significantly altered in receptors containing either a γ2Sor γ2Lsubunit (P > 0.05, n = 7; figs. 8B–D).
“Coapplication” of GABA and isoflurane together resulted in a significantly smaller reduction in peak current amplitude compared with the “continuous” application of isoflurane (fig. 9). In receptors lacking a γ2subunit, coapplication also produced “rebound currents” after termination of a 2-s pulse (figs. 9A and C).
Given the extensive structural diversity that exists for GABAAreceptors in the brain and previous reports that subunit composition influences physiologic18properties and pharmacologic sensitivity to a number of drugs that target GABAAreceptors,25–29we considered whether modulation by isoflurane depends on the constituent subunits or splice variants. To investigate this issue, we tested the influence of the two splice variants of the γ2subunit on macroscopic kinetics and on modulation by isoflurane, comparing the influence of γ2Sand γ2Lsplice variants when they were coexpressed with a single αβ combination (α1β2). Further, to determine whether the effect of the γ2subunit depends on the identity of α and β subunits with which it combines to form functional receptors, we compared effects of the γ2Ssubunit when it was coexpressed with two different αβ combinations (α1β2and α5β3).17,18We found that although there were quantitative differences between the two splice variants in the extent of their influence on baseline kinetics and on isoflurane modulation, in most regards, they imparted qualitatively similar effects: Receptors that incorporated a γ2subunit displayed less desensitization and faster deactivation under control conditions, effects of isoflurane on peak amplitude and on kinetics of deactivation and desensitization were substantially reduced in γ2-containing receptors, and net charge transfer was increased by isoflurane for αβγ but decreased for αβ receptors. We conclude that subunit composition does influence modulation of GABAAreceptors by isoflurane and, more specifically, that the presence of the γ2subunit and the identity of its splice variants are important factors in determining physiologic properties and pharmacologic sensitivity.
Although the influence of GABAAreceptor subunit composition on drug modulation has not been examined as extensively or systematically for volatile anesthetics as it has been for many drugs, existing studies have documented that a number of subunit combinations are modulated by a variety of volatile agents. In general, it has been found that responses to GABA are augmented, as reflected by an increase in response amplitude when low concentrations of agonists are applied, or a leftward shift when full GABA concentration–response relations are compared.30–33,35Because qualitatively similar results were observed for αβ- and αβγ-containing receptors, it was suggested that the identity of constituent subunits has little influence on susceptibility to modulation by volatile agents.30By contrast, our current results indicate that the γ2subunit strongly influences anesthetic modulation of peak amplitude and kinetic properties. How can we reconcile these apparently discordant findings? A likely explanation lies with the methods by which channels were activated in the different experiments. Many previous studies were performed using subsaturating concentrations of GABA and drug application techniques that had low temporal resolution, e.g. , bath application to Xenopus oocytes.30,32,33,35In this case, several processes that influence net current amplitude occur simultaneously as agonist is applied. These include activation, desensitization, and possibly receptor block. Also, the rates of desensitization and deactivation are often not resolved unless techniques that permit rapid solution exchange are used. Anesthetic-induced shifts in the GABA concentration–response33,34,45may well overcome any coexisting block, thus obscuring a reduction in peak amplitude, particularly when low agonist concentrations are tested. Those studies that showed that isoflurane can reduce peak current amplitude used techniques that produced solution exchanges in the range of tens of milliseconds, approaching the rapid solution exchanges in our current study.37,50The suggestion that concentration and time course of GABA exposure to the receptor play a crucial role in the different effects of anesthetics on GABA-mediated currents may also help to explain the results of Jones et al. ,7,8who found that IPSC amplitudes were decreased by volatile anesthetics, although peak current amplitudes evoked by exogenous GABA application were increased in hippocampal neurons.
Effects of the γ2Subunit on Baseline Kinetics
Consistent with previous results from our group39and others,51we found that incorporation of a γ2subunit had a major impact on kinetic properties of receptors. αβ-containing receptors showed significantly faster desensitization and slower deactivation than αβγ receptors. This difference was most prominent for α5β3compared with α5β3γ2Sreceptors (fig. 1and tables 1 and 2) but was seen for α1β2receptors and for γ2Land γ2Sas well. The slower deactivation of αβ receptors might be a consequence of the enhanced desensitization, because desensitized receptors are thought to reenter the “open” state before agonist unbinding occurs, thereby prolonging deactivation.48Prolonged deactivation of αβ receptors could also reflect slower agonist unbinding or channel closing,16both of which would lead to lower GABA EC50in αβ- compared with αβγ-containing receptors.39
Multiple Actions of Isoflurane
As demonstrated previously for recombinant receptors37,50,52and for native receptors at synapses,8–10,13our data show that isoflurane exerts two opposing actions: reduction of amplitude and slowing of deactivation (figs. 3 and 4). Incorporation of a γ2subunit significantly affected both of these actions, reducing the effect on prolongation (figs. 3B, 3D, and 4B) and reducing the blocking effect of the anesthetic (figs. 3B, 3D, and 4D). Because we explored only a limited range of isoflurane concentrations, we cannot distinguish between differences in potency versus efficacy in any of the drug actions. Also, overlapping ranges of concentrations that modulate, activate, and block receptors do complicate interpretation of such experiments. Differences in the relative concentrations of isoflurane versus enflurane that slowed IPSC decay and reduced IPSC amplitude in hippocampal pyramidal cells suggested that the two effects are produced by anesthetics binding to two different sites.10Similarly, the different relations between block and prolongation for αβversus αβγ receptors (fig. 6A) may again reflect actions at two different sites, with the γ2subunit influencing the two sites differently. Although the presence of two anesthetic-sensitive sites of action seems to us to be the most likely explanation, it is also possible that differences in gating properties of αβ and αβγ receptors could lead to these differences. Until binding sites that underlie the two effects are identified, it will remain difficult to make stronger conclusions. In this regard, several amino acids near the extracellular ends of the transmembrane domains have been identified that influence the ability of isoflurane to enhance responses.53It has been proposed that these residues line an anesthetic-binding pocket.54Whether mutations of these residues also influence the susceptibility to block is not known.
Kinetic Mechanisms of Receptor Modulation
It has been suggested that “open channel block” underlies the reduction of GABA-evoked peak responses by several volatile anesthetics.8,50,55,56This hypothesis is based in part on the occurrence of rebound (“hump”) currents that follow coapplication of GABA with anesthetic and that have been interpreted as the reversal of an open channel block due to faster unbinding rates of the anesthetic compared to the agonist. We also observed rebound currents after 2-s pulses of isoflurane together with GABA, but only in α1β2and α5β3receptors, not in receptors that contained a γ2subunit (fig. 9). Although this could reflect a different mechanism of block, it could also reflect differences in unbinding rates. If so, a slower unbinding rate for γ2-containing receptors would have to be more than compensated by an even slower binding rate, because the degree of block is less for γ2-containing receptors. Alternatively, isoflurane might act as a negative allosteric modulator rather than an open channel blocker. This would be consistent with the observation that there is not a strong use dependence of block (fig. 4C).
Several different kinetic mechanisms might account for the effect of isoflurane on deactivation, including a decrease in agonist unbinding rate16or a direct change in gating.57Whereas peak responses of recombinant and native GABAAreceptors are usually increased by anesthetics only when low concentrations of GABA are applied,3we found that in γ2S-containing receptors, peak currents were at times increased even when a near-saturating agonist concentration was tested. This is most consistent with the hypothesis that isoflurane alters gating characteristics, e.g. , increases the opening rate or decreases the closing rate, so that peak open probability is increased. If this is not accompanied by block, it would lead to an increase in the peak current.
Volatile anesthetics have been reported to enhance receptor desensitization in some studies45,58,59but not others.16We show here that this occurs only for receptors that lack a γ2subunit. For these receptors, accelerated transition into a fast desensitized state could contribute to the reduction in peak current and, at the same time, prolong deactivation as receptors reenter the open state before unbinding occurs.48Considering the strong correlations between block and prolongation and between block and desensitization (fig. 6), enhanced desensitization by isoflurane represents a promising mechanism that might account for or contribute to the observed blocking and prolonging actions for αβ receptors, as proposed for neurosteroids.60However, an open channel block mechanism has been described that simultaneously prolongs agonist-induced responses and decreases response amplitude.61Also, it may be that distinct mechanisms produce to the two effects of isoflurane, as suggested for pentobarbitone.11
Compared with α1β2, α5β3, and α1β2γ2Lreceptors, coexpression of a γ2Ssubunit resulted in a large degree of variability of responses to isoflurane (figs. 4 and 5). Although we can only speculate on the mechanisms underlying the observed differences between γ2Land γ2Ssplice variants, one intriguing possibility is that the variability is related to a difference in phosphorylation. The long splice variant (γ2L) differs from the short variant (γ2S) by the inclusion of eight additional amino acids in the intracellular loop between TM3 and TM4. These amino acids contain an additional consensus site for phosphorylation.62This additional site in γ2Lmight increase the possibility that at least one of the TM3–4 loop sites is phosphorylated. Although it does seem somewhat counterintuitive to suggest that more sites for phosphorylation could lead to less heterogeneity, this might happen if phosphorylation of any one site were sufficient to alter responses to isoflurane. Therefore, the more variable responses seen in γ2Scontaining receptors might reflect a more variable fraction of phosphorylated versus dephosphorylated γ2Ssubunits in a given cell.
To what extent do differences in γ subunit and splice variant–specific modulation influence responses in the brain or in the whole animal? Potentially, the impact could be substantial, considering the large and even qualitatively different effects that we describe here. However, because there have been relatively few detailed studies with volatile agents and different types of inhibitory synapses and receptors in situ , particularly in preparations in which subunit compositions of receptors have been established, this question is difficult to answer. The γ2subunit plays a role in targeting and clustering of receptors to synapses,36so effects may depend on whether responses of synaptic or extrasynaptic receptors are examined and on brain region and cell type. There is evidence that receptors containing only α and β subunits do exist in the brain, and they are present not only during development but also postnatally.17If they are located primarily at extrasynaptic sites, the transmitter concentration and temporal profile may be quite different than the brief, high concentrations that we used in our current studies. Therefore, it may not be appropriate to extrapolate our current findings to this situation or, in general, to expected effects of anesthetics on “tonic” inhibitory currents.63
Although both γ subunit splice variants are expressed throughout the brain, γ2Sis relatively more abundant in neocortical layer VI, the hippocampus, and the olfactory bulb, whereas cerebellar Purkinje cells, the medulla, and the pons express higher levels of γ2L.64,In vitro studies that use expressed receptors and low concentrations of GABA have generally been consistent in demonstrating increased responses with a variety of volatile agents, but results using intact tissues and synaptic circuits have sometimes been contradictory and confusing.10,65–67Even within an individual neuron, striking differences in responses of distinct types of IPSCs to the volatile agent enflurane have been observed.9It seems likely that at least some of this variability reflects subunit-specific drug effects. Subunit-specific modulation also may account for our observation that receptors excised from the soma of CA1 pyramidal neurons, which have different kinetic properties than synaptic receptors,68are potently blocked by isoflurane at concentrations that do not alter the amplitude of synaptic responses (M. I. B. and R. A. P., unpublished data, 1997).
In summary, our current results indicate that the subunit composition of GABAAreceptors, specifically the presence or identity of the γ2subunit, influences the response to isoflurane. This may contribute to the heterogeneity of synaptic and behavioral responses induced by volatile anesthetics.
The authors thank Cynthia Czajkowski, Ph.D. (Associate Professor), Andrew Boileau, Ph.D. (Associate Scientist), and Mathew Jones, Ph.D. (Assistant Professor) (all from the Department of Physiology, University of Wisconsin, Madison, Wisconsin), for helpful discussions and comments on the manuscript and Mark Perkins, B.S. (Research Specialist, Department of Anesthesiology, University of Wisconsin), for excellent technical assistance.