A major action of volatile anesthetics is enhancement of gamma-aminobutyric acid receptor type A (GABA(A)R) currents. In recombinant GABA(A)Rs consisting of several subunit mixtures, mutating the alpha1 subunit serine at position 270 to isoleucine [alpha1(S270I)] was reported to eliminate anesthetic-induced enhancement at low GABA concentrations. In the absence of studies at high GABA concentrations, it remains unclear whether alpha1(S270I) affects enhancement versus inhibition by volatile anesthetics. Furthermore, the majority of GABA(A)Rs in mammalian brain are thought to consist of alpha1, beta2, and gamma2 subunits, and the alpha1(S270I) mutation has not been studied in the context of this combination.
Recombinant GABA(A)Rs composed of alpha1beta2 or alpha1beta2gamma2L subunit mixtures were studied electrophysiologically in whole Xenopus oocytes in the voltage clamp configuration. Currents elicited by GABA (0.03 microM to 1 mM) were measured in the absence and presence of isoflurane or halothane. Anesthetic effects on GABA concentration responses were evaluated for individual oocytes.
In wild-type alpha1beta2gamma2L GABA(A), anesthetics at approximately 2 minimum alveolar concentration (MAC) shifted GABA concentration response curves to the left approximately threefold, decreased the Hill coefficient, and enhanced currents at all GABA concentrations. The alpha1(S270I) mutation itself rendered the GABA(A)R more sensitive to GABA and reduced the Hill coefficient. At low GABA concentrations (EC5), anesthetic enhancement of peak current was much smaller in alpha1(S270I)beta2gamma2L versus wild-type channels. Paradoxically, the leftward shift of the whole GABA concentration-response relation by anesthetics was the same in both mutant and wild-type channels. At high GABA concentrations, volatile anesthetics reduced currents in alpha1(S270I)beta2gammaL GABA(A)Rs. In parallel studies on alpha1beta2 (gamma-less) GABA(A)Rs, anesthetic-induced leftward shifts in wild-type receptors were more than eightfold at 2 MAC, and the alpha1(S270I) mutation nearly eliminated anesthetic-induced leftward shift.
The results support a role for alpha1S270 in alpha1beta2gamma2L GABA(A)R gating and sensitivity to inhibition by volatile anesthetics. The alpha1S270 locus also modulates anesthetic enhancement in alpha1beta2 GABA(A)R. The presence of the gamma2L subunit reduces anesthetic-induced left shift of wild-type GABA(A)R and nullifies the impact of the alpha1(S2701) mutation on anesthetic modulation. Thus, the gamma2L subunit plays a significant role in GABA(A)R modulation by volatile anesthetic compounds.
THE γ-aminobutyric acid receptor type A (GABAAR) is a member of the Cys-loop superfamily of ligand-gated ion channels 1and a major inhibitory neurotransmitter receptor in the central nervous system. GABAAR is a pentameric complex formed by different subunits (α1–6, β1–4, γ1–4, δ, ε, ρ1–2) that encircle a central chloride permeable pore. Each subunit consists of four transmembrane domains, the second of which (TM2) lines the pore of the channel and is thought to form a α-helical structure (fig. 1). 2The largest population of GABAARs in the mammalian brain contains the α1subunit in combination with β2and γ2with a stoichiometry of 2α:2β:1γ. 3–5A ternary subunit composition is favored if α, β, and γ subunits are present, and is maintained to the exclusion of binary combinations. 6
GABA type A receptors are a major site of action of volatile anesthetics. Volatile anesthetics enhance GABAAR function, and this is likely associated with diminished neuronal excitability. Experimentally, anesthetic-induced enhancement (usually of an electrophysiologic response) is directly observed when GABAARs are activated with subsaturating (low) GABA concentrations, resulting in a leftward shift in GABA concentration–response relations. Anesthetic enhancement is also observed as a prolongation of inhibitory postsynaptic potentials in neurons. 7,8In addition, volatile anesthetics are known to inhibit GABAAR function when stimulated with high GABA concentrations. 9–11
The enhancing actions of volatile anesthetics may be mediated by a site formed by parts of the TM2 domains on the GABAAR protein. Chimera and mutagenesis studies in human GABAARs identified a highly conserved serine near the extracellular end of TM2 (S270 on α1;fig. 1) that influences anesthetic-induced enhancement at low GABA concentrations. 12Mutation of this serine to isoleucine in either α[e.g. , α1(S270I)] or β[e.g. , β1(S265I)] subunits reduced or eliminated volatile anesthetic–induced enhancement in recombinant GABAARs formed of two (α1β1and α2β1) or three (α2β1γ2L) subunits. 12,13The αS270 locus has been studied in most detail, and this residue has been proposed to form part of an anesthetic binding cavity that mediates enhancement. 13,14
The impact of αS270 mutations on volatile anesthetic actions at high GABA concentrations has not been reported. Thus, it remains unclear whether the α(S270I) mutations weaken anesthetic-induced enhancement or, alternatively, strengthen anesthetic inhibition of GABAARs. A combination of these two effects might also occur. Furthermore, αS270 mutations have not been studied in GABAARs consisting of α1β2γ2subunits. Because this combination represents the majority of GABAARs in mammalian brain, studies in this subunit mixture are most relevant to humans and critical before introducing mutations into mammalian animal models.
We investigated the role of α1S270 by studying the α1(S270I) mutation in recombinant GABAARs consisting of α1β2and α1β2γ2Lsubunit mixtures expressed in Xenopus oocytes. We electrophysiologically measured the effects of isoflurane and halothane at a wide range of GABA concentrations. Anesthetic-induced enhancement was quantified as the leftward shift in GABA concentration responses, a value that is unaffected by anesthetic inhibition. Our analysis reveals a marked difference in how the α1(S270I) mutation affects anesthetic-induced leftward shift in GABAARs, depending on the presence or absence of the γ2Lsubunit.
Materials and Methods
Molecular Biology Procedures
cDNAs for the α1, β2, and γ2Lsubunits of human GABAAR in pCDM8 vectors were provided by Dr. Paul J. Whiting (Merck Sharp & Dohme Research Labs, Essex, United Kingdom). The S270I mutation was introduced into wild-type α1cDNA by high-fidelity polymerase chain reaction oligonucleotide-directed mutagenesis. The presence of the mutation and absence of stray mutations in the cDNA were confirmed by dideoxynucleotide sequencing.
mRNAs were transcribed in vitro from linearized cDNA templates using T7 RNA polymerase (Ambion, Austin, TX). mRNAs were isolated from the transcription reaction using affinity beads (BIO-101, Vista, CA) and stored at −80°C.
Xenopus Oocyte Expression
Wild-type and mutant GABAARs consisting of α1β2γ2L, α1β2(γ-less), or α1γ2L(β-less) subunit mixtures were expressed in Xenopus laevis oocytes. Detailed methods of oocyte preparation and injection have been described previously. 15Animal maintenance and oocyte harvest procedures were approved by the Massachusetts General Hospital Committee on Animal Research. Selected stage IV and V oocytes were injected with mixtures of GABAAR subunit mRNAs (total, 20–100 ng in 50–75 nl) in ratios of 1α:1β:1γ, 1α:1β, or 1α:1γ. Oocytes were incubated at 17°C and used for electrophysiologic experiments on days 1–3 after injection.
Whole Oocyte Two-microelectrode Electrophysiology
All experiments were performed at room temperature (20–22°C). Oocytes were placed in a 0.1-ml flow chamber and rested in a small depression in the recording chamber with the animal pole facing upward. They were impaled with two capillary glass electrodes with open tip resistances of 0.2–2 MΩ containing 3 m KCl and were voltage clamped (model OC-725; Warner Instrument Corp., Hamden, CT) at −50 mV. Extracellular solution containing 96 mm NaCl, 2 mm KCl, 1 mm CaCl2, 0.8 mm MgCl2, 10 mm HEPES, pH 7.6, was used to continuously superfuse oocytes at 2 ml/min.
Drugs were directly added to extracellular solution shortly before the experiments. GABA alone or in combination with volatile anesthetic was applied for 20–50 s to elicit a peak current response, and a 5–15-min interval was used between GABA applications for recovery of channel activity from desensitization. Anesthetics were not preapplied to oocytes before coapplication with the agonist. Currents were digitized at 50–200 Hz, recorded on a personal computer, and analyzed offline.
GABA Concentration–Response Studies.
Effects of anesthetics on GABA-activated currents (0.03 μm to 1 mm GABA) were established in individual oocytes. Responses to low GABA concentrations were alternated with control measurements at 1 mm GABA (a maximal-activating concentration) to control for run-up or run-down of currents. GABA concentration responses in the presence of 0.75 mm isoflurane or 0.85 mm halothane were studied in the same oocyte, again using 1 mm GABA (without anesthetic) as a control. For each condition (wild-type or mutant form of the α1β2γ2Lor α1β2GABAAR), four to eight individual oocytes were studied.
Studies at EC5.
For EC5studies, the GABA concentration eliciting a peak current of 5% of control peak at 1 mm GABA was determined for individual oocytes, and responses to the EC5concentration with and without anesthetic were measured in triplicate. Each measurement was paired with a control measurement using 1 mm GABA. Data were obtained from oocytes isolated from at least two different frogs.
Volatile Anesthetic Application
Before use, halothane (Halocarbon Laboratories, River Edge, NJ) was passed over an activated aluminum oxide (Aldrich Chemical, Milwaukee, WI) column to remove preservatives. Saturated solutions of halothane or isoflurane (Ohmeda, Liberty Corner, NJ) were prepared by adding a surplus of the volatile anesthetic to extracellular solution and by stirring in airtight glass bottles for at least 3 h before use. The concentrations of these stocks were taken to be 15 mm for isoflurane and 17 mm for halothane, as determined by Firestone et al . 16Based on Bunsen water–gas partition coefficients of 1.08 for isoflurane and of 1.2 for halothane at 25°C, concentrations of 0.48 mm isoflurane or 0.54 mm halothane correspond to approximately 1 vol%. 16Saturated stocks were diluted immediately before use and transferred to a glass syringe reservoir. All parts of the oocyte superfusion system consist of glass or polytetrafluoroethylene, and the oocyte was positioned within 1 mm of the end of the tubing to minimize adsorptive and evaporative loss of anesthetics. Gas chromatographic measurements have shown that the loss of volatile anesthetics is less than 15% in this system. 17
GABA-activated whole oocyte currents were normalized to the average of the preceding and following control currents. For concentration–response experiments, normalized responses from individual oocytes were fitted with logistic equations of the form: where Imaxis the maximum current, EC50is the GABA concentration eliciting a half-maximal response, and nH is the Hill coefficient of activation. Nonlinear least-squares fits were performed using Origin software (Microcal Inc., Northampton, MA). Fitted values are reported as mean ± SD.
For leftward shift analysis, the ratios of fitted EC50values in the absence of anesthetic to the EC50values in the presence of anesthetic (EC500/EC50Anes) were calculated for individual oocytes from logistic fits. To compare volatile anesthetic effects in wild-type versus mutant GABAARs, two-tailed Student t tests were used to compare sets of EC50ratios (leftward shift) and nH values. Data from oocytes expressing the α1β2γ2Land α1β2subunits were analyzed independently.
Volatile Anesthetic Effects on Wild-type α1β2γ2LGABAAR Function
Among oocytes expressing α1β2γ2LGABAARs, EC5GABA concentrations ranged from 2 to 9 μm. This variation necessitated evaluation of anesthetic effects at EC5in individual oocytes. At GABA EC5, both isoflurane and halothane markedly and reversibly enhanced currents (figs. 2A and 2C). Enhancement ratios in the presence of isoflurane were 8.0 ± 1.3 at approximately 2 minimum alveolar concentration (MAC) and 4.6 ± 0.7 at approximately 1 MAC. Similar enhancement ratios were observed with halothane (7.8 ± 0.9 at approximately 2 MAC and 4.7 ± 0.4 at approximately 1 MAC). In the absence of GABA, no direct activation of currents was observed at these anesthetic concentrations.
GABA concentration–response relations were measured in the absence and presence of a fixed concentration of isoflurane or halothane. Concentrations near 2 MAC were chosen for each drug because a pronounced leftward shift could be observed, improving sensitivity to mutant associated changes.
In individual oocytes expressing wild-type α1β2γ2LGABAARs, currents elicited by different concentrations of GABA were fit with EC50values ranging from approximately 40 to 70 μm, and nH values ranged from 1.3 to 1.5 (table 1). Because of the variation of EC50values among different oocytes, effects of anesthetics on individual cells were assessed. When isoflurane or halothane was added to GABA solutions, a leftward shift of the concentration–response curves was observed (isoflurane, fig. 3A; halothane, fig. 3C). The average ratio of control EC50without anesthetic to that in the presence of isoflurane was 3.4 ± 0.7 (n = 4;table 1) and to that in the presence of halothane was 2.9 ± 0.6 (n = 4;table 1). In the presence of halothane or isoflurane, activation nH values were reduced (to 1.0 ± 0.05 with isoflurane and to 1.1 ± 0.07 with halothane, n = 4;table 1). These values were significantly less than the control nH (P ≤ 0.01 for both anesthetics).
Both isoflurane and halothane enhanced currents at all GABA concentrations, and the enhancement decreased with increasing GABA concentration. The maximal peak current at 1 mm GABA increased as much as 15% (isoflurane, figs. 3A and 4A; halothane, figs. 3C and 4C). At 10 mm GABA, peak currents were slightly higher than those elicited at 1 mm, and volatile anesthetic enhancement was still present. After discontinuation of stimulation with high GABA in the presence of anesthetic, a brief rebound increase in current occurred, reflecting recovery of channels from anesthetic inhibition during deactivation (figs. 4A and 4C, center traces).
Volatile Anesthetic Effects on α1(S270I)β2γ2LGABAAR Function
Incorporation of the α1(S270I) mutation in α1β2γ2LGABAARs resulted in significant changes in the behavior of GABA-induced currents. The mutation rendered GABAARs more sensitive to GABA, with average EC50values of the mutant channels approximately fourfold lower than wild-type (table 1). EC50values varied markedly among individual oocytes (table 1). In addition, the nH values were significantly lower in α1(S270I)β2γ2Lreceptors (0.9 ± 0.1, n = 8) compared with those of wild-type receptors.
With EC5GABA (0.1–0.9 μm) activation of α1(S270I)β2γ2Lreceptors, both isoflurane and halothane enhanced currents, but the degree of enhancement was significantly less than that observed in wild-type α1β2γ2Lreceptors (P ≤ 0.01;figs. 2B and 2D). Anesthetic effects were reversible after wash, and no direct activation by anesthetics was observed in the absence of GABA.
With addition of volatile anesthetics, GABA concentration–response curves measured in α1(S270I)β2γ2Lreceptors were consistently shifted leftward (figs. 3B and 3D). Although the magnitude of the leftward shifts appeared smaller than that observed in wild-type GABAARs, the EC500/EC50Anes ratios derived from logistic fits were statistically indistinguishable from wild-type values using both isoflurane (P = 0.2;table 1) and halothane (P = 0.8). nH values were not altered by the anesthetics. At high GABA concentrations, both drugs slightly reduced the peak currents up to 5% (figs. 3B, 3D, 4B, and 4D). Again, after removal of GABA and anesthetic, rebound currents were observed (figs. 4B and 4D, center traces).
Anesthetic Effects on Wild-type α1β2GABAAR Function
Leftward shift analysis revealed previously unknown details about the impact of the α1(S270I) mutation on anesthetic modulation in α1β2γ2LGABAARs. We extended the leftward shift analysis to both α1β2(γ-less) and α1γ2L(β-less) GABAARs in oocytes containing the α1(S270I) mutation to see if non-α subunits influenced our results.
Oocytes (n = 31 for wild-type and mutant channels) injected with mRNA for α1and γ2Lsubunits produced no measurable currents within 96 h. Connor et al. 18also reported that the GABAAR α1subunit requires a β2subunit for receptor assembly and functional surface expression in Xenopus oocytes. The α1β2combination produced functional GABAARs.
The gating behavior of wild-type α1β2GABAARs was distinct from that of α1β2γ2Lreceptors in that the average EC50was nearly threefold lower (18 vs. 52 μm;table 2), and the average nH was reduced (0.9 vs. 1.4). GABA concentration–response studies in oocytes expressing α1β2GABAARs in the absence and presence of 0.75 mm isoflurane showed large leftward shifts (fig. 5A). The average EC500/EC50Iso ratio (8.4 ± 1.4, n = 3;table 2) was much larger than that for wild-type α1β2γ2Lreceptors (3.4 ± 0.7, n = 4;table 1). The low nH was not altered by isoflurane. Currents at all GABA concentrations were enhanced by isoflurane, and the effect was more pronounced at low GABA concentrations (fig. 5A). Rebound currents were observed at the termination of anesthetic application at high GABA concentrations (fig. 5E).
Anesthetic Effects on α1(S270I)β2GABAAR Function
Compared with wild-type α1β2receptors, α1(S270I)β2receptors showed a sixfold decrease in the average GABA EC50and significantly lower nH values (table 2). However, isoflurane caused only very small leftward shifts in GABA concentration–response curves, with an average EC500/EC50Iso that was not significantly different from 1.0 (1.6 ± 0.6). At GABA concentrations greater than 3 μm, the dominant effect of isoflurane on α1(S270I)β2receptors was inhibition, whereas only weak enhancement was observed at GABA concentrations less than 1 μm. Evidence of isoflurane inhibition was present in the form of rebound currents at all GABA concentrations (figs. 5D and 5F). Anesthetic effects were fully reversible after wash.
Investigation of the α1(S270I) mutation in α1β2γ2LGABAARs demonstrates several important findings that point to a complex role for the α1S270 residue. Our data show that the α1(S270I) mutation causes significant changes in GABAAR gating behavior and sensitivity to the inhibitory effects of volatile anesthetics. In contrast to results obtained at single low GABA concentrations (EC5), the analysis of GABA concentration–response curves suggests that the α1(S270I) mutation causes almost no change in sensitivity to volatile anesthetic–induced leftward shift of α1β2γ2LGABAARs. However, this finding is not simply a result of using a different analytic methodology, because the α1(S270I) mutation profoundly affects anesthetic-induced enhancement in α1β2GABAARs. Indeed, we found that GABAAR sensitivity to anesthetic modulation and the impact of the α1(S270I) mutation are significantly influenced by the γ subunit.
α1(S270I) Mutation Alters GABAAR Gating Behavior and Sensitivity to Volatile Anesthetic Inhibition
In comparing the GABA-dependent function of wild-type recombinant GABAARs with those containing the α1(S270I) mutation, we observed several consistent changes. Receptors containing the α1(S270I) mutation had lower GABA EC50values, in agreement with previous reports. 13,14In α1β2γ2LGABAARs, the mutation reduced the average EC50approximately fourfold (table 1), whereas in α1β2GABAARs, the mutation reduced average EC50sixfold (table 2). Because of the variability of EC50values derived from studies on different oocytes, these two ratios are not significantly different, indicating that the mutation has about the same impact on gating whether or not a γ subunit is present in the receptors. On a molecular level, reduced EC50can be caused by higher affinity of GABAARs for GABA (e.g. , by slowing GABA unbinding) 19or by stabilization of the active open-channel state by the mutation.
A second effect of the α1(S270I) mutation on gating is to reduce the nH of GABA activation. In α1β2γ2LGABAARs, the mutation reduced the average nH from 1.4 to 0.9 (table 1), whereas in α1β2GABAARs, the mutation reduced average nH from 0.9 to 0.6 (table 2). The reduction of nH in whole oocyte experiments could be caused by an increase in receptor desensitization rates, which cannot be measured using these methods, or the result of a decrease in cooperativity in GABA-dependent gating.
Third, both α1(S270I)β2γ2Land α1(S270I)β2GABAARs are inhibited by volatile anesthetics in the presence of high GABA, whereas the corresponding wild-type receptors are not. In traces from oocytes expressing mutant receptors, rebound currents observed after discontinuation of the superfusion of GABA plus anesthetics were larger than those in wild-type currents (figs. 4 and 5). Rebound currents may represent reopening of anesthetic-blocked open channels or recovery of anesthetic-stabilized, agonist-bound closed states (e.g. , desensitized) via the open state. These results suggest that the α1(S270I) mutation sensitizes activated GABAARs to inhibition by volatile anesthetics, accounting for up to a 20% difference in anesthetic effects at maximal GABA concentrations.
Analysis of Agonist Concentration–Response Curves Shows that the α1(S270I) Mutation Has Little Impact on Anesthetic Enhancement in α1β2γ2LGABAARs
We examined the effects of volatile anesthetics on GABA-induced gating enhancement in both wild-type α1β2γ2Land mutant α1(S270I)β2γ2LGABAARs. Results with both isoflurane and halothane at approximately equipotent concentrations were essentially the same, suggesting a common mechanism for GABAAR enhancement by halogenated alkane and ether anesthetics. We analyzed anesthetic enhancement and the impact of the α1(S270I) mutation using two different approaches. One approach to assess enhancement, which has been used by a number of researchers, is to measure anesthetic-induced change in currents at a single, low, equipotent GABA concentration (e.g. , EC5). At GABA EC5, enhancement of currents by anesthetics was significantly lower in oocytes expressing mutant versus wild-type channels (fig. 2). Thus, based on EC5results alone, the α1(S270I) mutation apparently reduces, but does not abolish, anesthetic enhancement in α1β2γ2LGABAARs, as reported in previous studies of α1β1, α2β1, or α2β1γ2LGABAARs. 12,13
Our second approach to quantifying anesthetic enhancement of GABAAR function was leftward shift analysis. Compared with studies using a single “equipotent” GABA concentration, leftward shift analysis requires accumulation of significantly more data, but it is more informative in assessing both inhibiting and enhancing anesthetic actions and how they are affected by mutations. For example, the reduced anesthetic enhancement observed in α1(S270I)β2γ2LGABAARs at EC5might be explained by a number of changes associated with the mutation: (1) weakened anesthetic enhancement; (2) increased anesthetic inhibition; or (3) reduced cooperativity of gating. Characterization of receptors containing the α1(S270I) mutation over a wide range of GABA concentrations showed that both GABA gating and sensitivity to anesthetic inhibition were, in fact, altered.
Unexpectedly, the EC500/EC50Anes ratios were the same in oocytes expressing wild-type α1β2γ2Land α1(S270I)β2γ2Lchannels (fig. 4and table 1). On casual inspection, data in figure 4appear to show smaller anesthetic-induced leftward shifts in α1(S270I)β2γ2Lthan in α1β2γ2Lreceptors, but this appearance is actually the result of the mutant-associated change in anesthetic inhibition. After renormalization to correct for the different inhibitory effects of anesthetics in wild-type and mutant receptors at high GABA concentrations, the similarity of the isoflurane-induced leftward shifts becomes apparent (fig. 6, horizontal arrows). Thus, based on leftward shift analysis, the α1(S270I) mutation does not alter anesthetic-induced enhancement of α1β2γ2LGABAARs.
These leftward shift results in α1β2γ2LGABAAare not in direct conflict with previous reports of α(S270I) mutations, 12–14because this is the first study using this subunit combination. Indeed, other investigators have reported that subunit composition can significantly impact anesthetic modulation of GABAARs. Anesthetic actions in GABAARs have been shown to be dependent on the β-subunit isoform 20–22as well as on receptor subunit mixture. 23We therefore experimentally addressed whether the impact of the α1(S270I) mutation on GABAAR modulation by anesthetics could be dependent on non-α subunits.
α1(S270I) Mutation Profoundly Reduces Anesthetic Enhancement in α1β2GABAARs
In GABAARs expressing only α1and β2subunits, isoflurane at approximately 2 MAC caused very strong enhancement at EC5as well as a large leftward shift in GABA concentration–response curves. Introduction of the α1(S270I) mutation in these receptors nearly eliminated isoflurane-induced enhancement by either measure (fig. 5and table 2). Thus, for α1β2GABAARs, both EC5and leftward shift analyses of our data agree completely with previous studies of the α1(S270I) mutation. 12,13
Presence of the γ2LSubunit Modifies Anesthetic Sensitivity and the Impact of the α1(S270I) Mutation
Our results demonstrate that incorporation of the γ subunit into wild-type GABAARs reduces isoflurane-induced left shift at 2 MAC isoflurane by almost a factor of three (8.4-fold in α1β2vs. 3.4-fold in α1β2γ2L;tables 1 and 2). Furthermore, the presence of the γ subunit nearly nullifies the effect of the α1(S270I) mutation on anesthetic modulation, as shown by the contrasting results of our studies on the α1(S270I) mutation in α1β2γ2Lversus α1β2GABAARs. Other investigators previously noted that GABAARs containing the γ subunit show less anesthetic enhancement than those formed of only α- and β-subunit types. 24In addition, incorporation of either δ or ε subunits as replacement for γ further suppresses anesthetic modulation. 25,26
On the other hand, several reports have suggested that the γ subunit plays a negligible role in anesthetic actions. For example, the γ subunit apparently has little impact on GABAAR enhancement by propofol. 27,28Two studies have suggested a minor role for the γ subunit in the presence of αS270 mutations. The α2(S270H) mutation reduced isoflurane enhancement in both α2β1and α2β1γ2SGABAARs at low GABA concentrations. 29In GABAARs containing α2and β1subunits, the α2(S270I) mutation reduced ethanol enhancement at low GABA concentrations, and addition of the γ2Lsubunit slightly reduced the effects of the mutation. Moreover, the homologous S280I mutation in the γ2Lsubunit TM2 domain only weakly altered ethanol sensitivity in α2β1γ2LGABAARs. 13It is noteworthy that these studies drew their conclusions based on results obtained at single low GABA concentrations, because our EC5results in wild-type and mutant α1β2γ2LGABAAalso show a significant impact of the α1(S270I) mutation (fig. 2). It is only by quantifying anesthetic-induced leftward shifts that we demonstrated the magnitude of the influence of the γ subunit.
Do Volatile Anesthetics Act at GABAAR Subunit Interfaces?
From studies of GABAAR mutants, Mihic et al. 12proposed that anesthetics bind at an intrasubunit pocket formed by residues on TM2 and TM3. Other investigators have extended this model by suggesting that the α1S270 sidechain makes direct contact with anesthetic molecules, and that when the size of this sidechain is increased, it occupies the space where anesthetics bind, mimicking anesthetic-induced gating changes. 14Evidence for interactions between anesthetics and the α1S270 sidechain is based on cysteine mutagenesis and modification studies. 30Notably, incorporation of a photo-activatable general anesthetic into nicotinic receptors from Torpedo , which are close structural homologs of GABAARs, also identify a residue at the outer end of a TM2 domain. 31Our studies partially conform to the idea that α1S270 contributes to an anesthetic binding site, because the decreased EC50and nH caused by the α1(S270I) mutation are similar to those observed in the presence of anesthetics. However, a detail that emerges from our data is that changes in gating behavior associated with the α1(S270I) mutation are unaffected by the γ subunit, whereas changes in anesthetic-induced leftward shift caused by the mutation in α1β2receptors are nearly abolished by incorporation of the γ subunit. This dissociation of two effects of the mutation indicate that, although α1S270 influences anesthetic modulation of GABAARs, it is unlikely to be in direct contact with anesthetic molecules, at least when the γ subunit is present.
Previous studies have emphasized the roles of α and β subunits in channel gating and anesthetic modulation of GABAARs, whereas our data show that incorporation of the γ subunit in wild-type GABAAsignificantly influences EC50, activation nH, and anesthetic-induced leftward shift, while nullifying the impact of the α1(S270I) mutation on anesthetic modulation. Thus, agonist gating and anesthetic enhancement are significantly influenced by all three types of subunits in the dominant GABAAR isoform from mammalian brain. Furthermore, changes caused by mutation in one subunit (α1) can be modified via interactions with another subunit (γ2L). Our results do not rule out an intrasubunit site for anesthetics, but we speculate that volatile anesthetics interact with sites formed at GABAAR subunit interfaces, causing their effects by altering the coordinated interactions between subunits that are necessary for agonist-activated gating. Indeed, other families of drugs that influence receptor gating, such as GABA receptor agonists and benzodiazepine compounds, bind at the interfaces between GABAAR subunits. 32This model may explain how subunit composition and mutations influence both GABAAR gating behavior and anesthetic modulation.
The authors thank Carol Gelb, M.A. (Senior Laboratory Technician, Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, MA), for expert technical assistance, and Paul J. Whiting, Ph.D. (Merck Sharp & Dohme Research Labs, Essex, United Kingdom), for generously supplying the cDNAs for the GABAAR subunits. We greatly appreciate the helpful advice and comments from Keith W. Miller, D.Phil. (Professor, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Boston, MA).