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Anesth Analg 2004;98:1297-1304
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000111108.78745.AD

ANESTHETIC PHARMACOLOGY

The Effects of Isoflurane on Desensitized Wild-Type and {alpha}1(S270H) {gamma}-Aminobutyric Acid Type A Receptors

Adam C. Hall, PhD*, Kathleen C. Rowan, MA*, Renna J. N. Stevens*, Jill C. Kelley{dagger}, and Neil L. Harrison, PhD{dagger} Section Editor

*Department of Biological Sciences, Neuroscience Program, Smith College, Northampton, Massachusetts, and the {dagger}Department of Anesthesiology, Weill Medical College, Cornell University, New York, New York

Address correspondence and reprint requests to Adam C. Hall, PhD, Department of Biological Sciences, Clark Science Center, Smith College, Northampton, MA 01063. Address email to ahall{at}science.smith.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
{gamma}-aminobutyric acid type A receptors (GABAA-R) mediate synaptic inhibition and meet many pharmacological criteria required of important general anesthetic targets. During synaptic transmission GABA release is sufficient to saturate, maximally activate, and transiently desensitize postsynaptic GABAA-Rs. The resulting inhibitory postsynaptic currents (IPSCs) are prolonged by volatile anesthetics like isoflurane. We investigated the effects of isoflurane on maximally activated and desensitized GABAA-R currents expressed in Xenopus oocytes. Wild-type {alpha}1ß2 and {alpha}1ß2{gamma}2s receptors were exposed to 600 µM GABA until currents reached a steady-state desensitized level. At clinical concentrations (0.02–0.3 mM), isoflurane produced a dose-dependent enhancement of steady-state desensitized current in {alpha}1ß2 receptors, an effect that was less apparent in receptors including a {gamma}2s-subunit. When serine at position 270 is mutated to histidine ({alpha}1(S270H)) in the second transmembrane segment of the {alpha}1-subunit, the currents evoked by sub-saturating concentrations of GABA became less sensitive to isoflurane enhancement. In addition, isoflurane enhancements of desensitized currents were greatly attenuated by this mutation and were undetectable in {alpha}1(S270H)ß2{gamma}2s receptors. In conclusion, isoflurane enhancement of GABAA-R currents evoked by saturating concentrations of agonist is subunit-dependent. The effects of isoflurane on desensitized receptors may be partly responsible for the prolongation of IPSCs during anesthesia.

IMPLICATIONS: Isoflurane enhances desensitized {gamma}-aminobutyric acid type A receptor (GABAA-R) currents, an effect that is subunit-dependent and attenuated by a mutation in an {alpha}1-subunit pore residue of the GABAA-R. As GABA release at inhibitory synapses is typically saturating, isoflurane modulation of desensitized receptors may be partly responsible for prolongation of inhibitory postsynaptic currents during anesthesia.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
{gamma}-aminobutyric acid type A receptors (GABAA-R) are the principal receptors for fast inhibitory neurotransmission in the mammalian central nervous system (CNS). GABAA-R are members of the ionotropic ligand-gated ion channel superfamily. Their pentameric structure can be composed of different subunits ({alpha}1–6, ß1–4, {gamma}1–4, {delta}, {epsilon}, and {pi}) forming membrane-spanning Cl–-selective ion channel complexes activated by the binding of GABA (1). In mammalian neurons, the predominant GABAA-R isoform appears to be {alpha}1ß2{gamma}2 with a proposed subunit stoichiometry of 2:2:1, respectively (1,2). By analogy with the nicotinic acetylcholine receptor (3), each subunit is predicted to include four {alpha}-helical transmembrane segments (TM1–4), with TM2 lining the pore.

GABAA-R are recognized as molecular targets for the effects of both inhaled and IV general anesthetics in the mammalian CNS (4). At clinically relevant concentrations (1 MAC, minimum alveolar concentration), volatile anesthetics enhance GABAA-R responses to sub-saturating concentrations of agonist (5,6), resulting in leftward shifts in GABA dose-response curves (7). At the synapse, where concentrations of transmitter are typically saturating (~500 µM-1 mM) (8), clinical concentrations of volatile anesthetics prolong GABA-mediated inhibitory postsynaptic currents (IPSCs) (9,10). After the release and binding of neurotransmitter, receptors enter desensitized states (11) that can influence the duration of IPSCs (8,12–14). Indeed, the prolongation of IPSCs by the IV anesthetic, propofol, may result in part from anesthetic modulation of desensitization (15), although there is little evidence for similar effects of volatile anesthetics (4,5,16).

The effects of IV anesthetics at GABAA-R are often determined by the subunit composition of the receptor, whereas volatile anesthetics typically show little subunit-dependence (17). However, an important amino acid residue, serine 270, at the extracellular end of TM2 in the {alpha}-subunit is critical for volatile anesthetic action on the receptor (18–20). Point mutations that increase the side-chain volume (e.g., serine to histidine, S270H in {alpha}1) abolish or attenuate potentiation of GABA responses by isoflurane in {alpha}ß and {alpha}ß{gamma} receptors expressed in human embryonic kidney 293 cells (19,20). In another study, in which the mutant S270I (serine to isoleucine)-{alpha}1 and ß2 subunits were expressed in Xenopus oocytes, it was concluded that addition of a {gamma}2L-subunit in the GABAA-R nullifies the effect of mutation at S270 on anesthetic modulation (21). This lack of agreement between oocyte and the mammalian cell expression studies has not been resolved.

We investigated the effects of isoflurane on currents evoked by sub-saturating and maximal GABA concentrations in recombinant GABAA-R expressed in Xenopus oocytes and examined four subunit combinations: wild-type {alpha}1ß2 and {alpha}1ß2{gamma}2s and mutant {alpha}1(S270H)ß2 and {alpha}1(S270H)ß2{gamma}2s. Our results clarify the role of the {gamma}-subunit in the modulation of GABAA-R responses by isoflurane and point to potential mechanisms for the prolongation of GABA-mediated IPSCs by volatile anesthetics. The study also revealed a potent subunit-dependent enhancement of steady-state desensitized current by isoflurane that is largely attenuated by the {alpha}1(S270H) mutation.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
cDNAs encoding the {alpha}1, ß2, {gamma}2s subunits of human GABAA receptors were kindly provided by Dr. Paul J. Whiting (Merck, Sharp & Dohme Research Laboratories, Harlow, UK). cDNAs (wild-type and mutant) in the vector pcDNA3.1+ vector were prepared as described previously (19) and stored at –20°C before oocyte injection.

Four combinations of GABAA-R subunits were expressed: wild-type {alpha}1ß2 and {alpha}1ß2{gamma}2s and mutant {alpha}1(S270H)ß2 and {alpha}1(S270H)ß2{gamma}2s. Briefly, Xenopus laevis (Xenopus Express, Plant City, FL) were anesthetized with 2% tricaine and oocytes were harvested through laparotomy. Eggs were treated with 1 mg/mL collagenase D (Roche Diagnostics Corporation, Indianapolis, IN) in (mM) 82 NaCl, 2 KCl, 1 MgCl2, 5 HEPES (pH, 7.6) for up to 2 h at room temperature. Oocytes were then transferred to a solution (ND-96) containing (mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES with 100 U/mL penicillin and 100 µg/mL streptomycin (pH, 7.6). Eggs were defolliculated manually by repetitive rolling on plastic Petri dishes. Plasmid was introduced by nuclear injection using a Nanoject II (Drummond Scientific, Broomall, PA). The injection volume was 32 nL with concentrations of {alpha}1 (or {alpha}1S270H) subunit and ß2 subunit plasmid cDNA at 12 ng/µL and {gamma}2s subunit cDNA at 6 ng/µL (when included). Injected oocytes were incubated in ND-96 at 16°C for up to 4 days during experimentation. Animal maintenance and oocyte harvest procedures were approved by Smith College’s Institutional Animal Care and Use Committee.

Between 1 and 4 days after cDNA injection, oocytes were screened for GABA-evoked currents in a 100-µL oocyte chamber (Warner Instruments Corp., Hamden, CT). All experiments were performed at room temperature (20°C–23°C). Eggs were placed in a small depression, animal-pole face up, and continually superfused at 5 mL/min with ND-96 (without penicillin-streptomycin). Recordings were made using standard two-electrode voltage-clamp technique with an OC-75C clamp (Warner Instruments Corp.). Glass micropipettes (World Precision Instruments, Sarasota, FL) were fabricated using a two-stage pull (Narishige, Tokyo, Japan) and filled with 3M KCl giving typical resistances of 1–3 M{Omega}. Impaled oocytes were voltage-clamped at –50 mV. All drugs were dissolved in ND-96 immediately before use, and solutions were applied via gravity feed (5 mL/min) using an automated switching device (ALA Scientific Instruments, Westbury, NY). Currents were digitized at 200 Hz and recorded and analyzed using pClamp 6.0 software (Axon Instruments, Union City, CA).

For all subunit combinations, we measured peak currents evoked by increasing concentrations of GABA (0.1 µM–1 mM), and plotted the resulting dose-response data. GABA currents were completely blocked by bicuculline and picrotoxin (data not shown). Incorporation of the {gamma}2s-subunit was confirmed by a rightward shift in the GABA dose-response plots (Table 1, Fig. 1) and by loss of sensitivity of the GABA current to block by 10 µM Zn2+. Free aqueous concentrations of the volatile anesthetic isoflurane (Halocarbon Laboratories, River Edge, NY) were determined by diluting saturated anesthetic (15.3 mM) solutions with recording solution (22), considering 0.3 mM to be equivalent to 1 MAC at room temperature. Reservoirs containing isoflurane were loosely sealed with a rigid plastic float, and all tubing and valves were made from polytetrafluoroethylene to avoid loss of volatile anesthetics (6). Isoflurane was preapplied to the oocyte for 3 min before coapplication with GABA, unless stated otherwise. All chemicals were purchased from Sigma Chemical (St. Louis, MO) unless stated otherwise.


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  		Table 1. Control {gamma}-Aminobutyric Acid (GABA) Responses for the Four GABAA Receptor Subunit Combinations
 


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  		Figure 1. {gamma}-aminobutyric acid (GABA) dose-response plots for the four combinations of recombinant receptor. Currents were normalized against the maximum current evoked by any concentration of GABA within the dose-response protocol. Data (means ± SEM) were fitted with the Hill equation (Table 1). Both exclusion of the {gamma}2s subunit and inclusion of the point mutation resulted in leftward shifts in the GABA dose-response curves.

 
Currents were measured using PClamp 6.0 software (Axon Instruments, Union City, CA). Dose-response data for GABA-evoked currents were fitted with the Hill equation using Origin software (OriginLab Corp., Northampton, MA). The Hill equation is


where I is the GABA-evoked current at a given concentration, IGABAmax is the peak current at saturating [GABA], EC50 is the concentration of GABA that elicits a half-maximal response, and nH is the Hill coefficient. GABA EC5 and EC20 values were derived from fitted curves for each individual oocyte. Statistics were performed using Minitab Statistical Software running analysis of variance and multiple regression analyses with significance levels set to P < 0.01. Data are expressed as mean ± SEM and were calculated from a minimum of n = 6 individual oocytes for all experiments.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Isoflurane Enhancement of Sub-Saturating GABA Currents from Wild-Type and {alpha}1(S270H) Mutant GABAA-R
Oocytes were routinely screened for GABAA-R expression by application of a large concentration of GABA (1 mM) that evoked the maximum GABA current (IGABAmax). Comparisons of IGABAmax values for the 4 subunit combinations (wt {alpha}1ß2{gamma}2s = {alpha}1(S270H)ß2{gamma}2s >> wt {alpha}1ß2 = {alpha}1(S270H)ß2, Table 1) indicated that inclusion of the {gamma}2s-subunit resulted in larger maximal currents, independent of the presence of the {alpha}1(S270H) point mutation. Omission of the {gamma}2s subunit or the introduction of the point mutation caused the dose-response curves to shift to the left, indicating increased sensitivity to agonist (Table 1, Fig. 1).

We first investigated the action of isoflurane on the 4 subunit combinations in the presence of sub-saturating concentrations of GABA. Oocytes were pre-exposed for 3 min to isoflurane before coapplication with GABA EC20 (effective concentration that evokes 20% of IGABAmax). In wild-type receptors, 1 MAC isoflurane produced large enhancements (~400%) of EC20 GABA currents that were attenuated, but not abolished, in receptors including the {alpha}1(S270H) mutation (Fig. 2). We explored this further by constructing isoflurane dose-effect curves for modulation of the GABA EC20 currents (Fig. 3A). The relative extent of the anesthetic enhancement of open channel currents was wt {alpha}1ß2 >> wt {alpha}1ß2{gamma}2s > {alpha}1(S270H)ß2{gamma}2s = {alpha}1(S270H)ß2. Close inspection of the dose-effect curves for isoflurane in the wild-type {alpha}1ß2 receptors revealed that the currents elicited by GABA plus 2 and 4 MAC isoflurane were greater in amplitude than the IGABAmax, corresponding to an enhancement of 600%–700% over the control GABA EC20 currents. The maximal potentiation of an EC20 GABA current should by definition be 400%, suggesting to us that our measurements of maximal GABA currents in the oocytes were strongly compromised by desensitization and that the degree of this attenuation might be influenced by isoflurane.



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  		Figure 2. {alpha}1(S270H) mutation attenuates the potentiation of {gamma}-aminobutyric acid (GABA)-evoked currents by isoflurane. Current traces from individual oocytes, expressing 4 different combinations of GABAA-R, demonstrate the relative effects of 1 MAC (0.3 mM) isoflurane on the current elicited by the GABA EC20. For each combination, the left-hand trace shows the current evoked after application of GABA EC20, and the right-hand trace shows the equivalent GABA exposure in the presence of 1 MAC isoflurane (after 3 min pre-exposure to the anesthetic). All currents recovered fully to control levels after washout of the anesthetic (not shown).

 


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  		Figure 3. Addition of the {gamma}2s-subunit does not affect the attenuation of isoflurane potentiation by the {alpha}1(S270H) point mutation. A, dose-effect plots for modulation of {gamma}-aminobutyric acid (GABA) EC20 (effective concentration giving 20% of maximal current) currents by varying [isoflurane]. Data points are mean ± SEM from at least 6 individual oocytes for each concentration and combination. Using multiple regression analyses for all data up to 4 MAC, there was a significant increase in the level of current enhancement with increasing isoflurane concentration (P < 0.01). There was also a significant attenuating effect on the level of enhancement with both addition of the {gamma}2s-subunit and with inclusion of the {alpha}1(S270H) mutation, and an interaction between these two factors (R2 = 71%, P < 0.01). Further regression analyses revealed that isoflurane modulation of GABA EC20 responses in {alpha}1(S270H)ß2 and {alpha}1(S270H)ß2{gamma}2s mutant receptors were not significantly different throughout the concentration range (R2 = 36%, P < 0.01). Note: the extents of the enhancements for wild-type GABA EC20 currents by 2 and 4 MAC isoflurane (608.4 ± 85.7 n = 6, and 634.4 ± 100.5 n = 6, respectively) indicate potentiated currents > IGABAmax. B, dose-effect plots for modulation of GABA EC5 currents by varying [isoflurane]. Data points are means ± SEM from at least 6 individual oocytes for each concentration and combination. Using multiple regression analyses for all data up to 4 MAC, there was a significant increase in the level of current enhancement with increasing isoflurane concentration (R2 = 62%, P < 0.01). There was also a significant attenuating effect on the level of enhancement with inclusion of the {alpha}1(S270H) mutation but not with addition of the {gamma}2s-subunit with no interaction between these two factors (P < 0.01). Further regression analyses revealed that isoflurane modulation of GABA EC5 responses in {alpha}1(S270H)ß2 and {alpha}1(S270H)ß2{gamma}2s mutant receptors were not significantly different at all concentrations up to 4 MAC (R2 = 39%, P < 0.01).

 
To address this issue, further experiments were conducted using GABA at the EC5 concentration (Fig. 3B). Once again, the potentiation of the wild-type {alpha}1ß2 currents by isoflurane was substantial (for example, 1330% ± 60%, n = 6 at 2 MAC), but the difference in anesthetic enhancement of GABA responses between wild-type {alpha}1ß2 and wild-type {alpha}1ß2{gamma}2s responses was now diminished. In both sets of receptors, the level of current enhancement declined at 8 MAC isoflurane (2.4 mM); this is indicative of a secondary inhibitory action of the drug at very high concentrations. Although the maximal degree of potentiation may be unreliable as a result of desensitization and the inhibitory effect of isoflurane, an approximate value for half-maximal enhancement of sub-maximal GABA currents was estimated for wild-type receptors at ~0.3 mM isoflurane.

Effects of Isoflurane on Desensitized GABAA-R Combinations
Given the probable impact of desensitization in GABAA-R responses on the isoflurane dose-response data (Fig. 3), we asked whether isoflurane could indeed modulate desensitized GABA currents. In initial experiments, we observed that isoflurane enhanced IGABAmax for wild-type {alpha}1ß2 by 33.9% ± 8.9% (n = 8) whereas for mutant {alpha}1(S270H)ß2{gamma}2s, there was no effect of the anesthetic (–5.9% ± 9.6%, n = 6) on IGABAmax (Fig. 4). In whole oocyte recordings, accurate measurements of the kinetics of activation and desensitization are impossible due to the slow speed of solution exchange, turbulent flow, and variable positioning of a large cell in the recording chamber. To assess the effects of isoflurane on desensitized GABAA-R more directly, we applied a large concentration of GABA (600 µM) for an extended period (>10 min) until the current declined to a steady-state of level (Iss, Fig. 5). Ratios of Iss to IGABAmax were calculated and did not differ for the four subunit combinations (Table 1).



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  		Figure 4. Apparent enhancement of maximal {gamma}-aminobutyric acid current (IGABAmax) by isoflurane for wild-type {alpha}1ß2 but not mutant {alpha}1(S270H)ß2{gamma}2s receptors. 1 mM GABA was applied for 20s every 3min until peak IGABAmax had reached a consistent amplitude. Oocytes were then pre-exposed for 3 min to 2 MAC isoflurane with subsequent co-application of agonist. A, in oocytes expressing wild-type {alpha}1ß2 receptors, the "IGABAmax" was increased in the presence of 2 MAC (0.6 mM) isoflurane. B, in oocytes expressing mutant {alpha}1(S270H)ß2{gamma}2s receptors, IGABAmax was unaffected in the presence of 2 MAC (0.6 mM) isoflurane.

 


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  		Figure 5. Enhancement of steady-state desensitized current (Iss) by isoflurane. Currents were recorded from oocytes expressing 4 different subunit combinations after exposure to a high concentration of {gamma}-aminobutyric acid (GABA, 600 µM) for at least 10 min to achieve a steady-state level of desensitization. Increasing concentrations of isoflurane (19 µM to 0.3 mM, 1/16 to 1 MAC) were then sequentially co-applied with the agonist to desensitized receptors (as indicated by the bars above the steady-state current). Pronounced potentiation of Iss was observed for the wild-type {alpha}1ß2 combination, an effect that was attenuated in wild-type {alpha}1ß2{gamma}2s and mutant {alpha}1(S270H)ß2 combinations and abolished in recordings from oocytes expressing {alpha}1(S270H)ß2{gamma}2s.

 
In the wild-type {alpha}1ß2, a dose-dependent increase in the steady-state desensitized current was readily observed, between 1/16 and 1 MAC isoflurane (Fig. 5), with the degree of potentiation of Iss at 1 MAC ranging from 41%–204% (n = 11). The extent of this modulation was less significant in both wild-type {alpha}1ß2{gamma}2s and mutant {alpha}1(S270H)ß2 combinations ranging from 13%–51% (n = 21) and 7%–81% (n = 11), respectively. This effect of isoflurane (<=1 MAC) on desensitized currents was essentially absent in the mutant {alpha}1(S270H)ß2{gamma}2s receptor. Dose-effect curves for the modulation of Iss by isoflurane indicated marked enhancement of desensitized current in the wild-type {alpha}1ß2 receptor combination and also revealed an inhibitory effect at large concentrations of anesthetic (Fig. 6). This inhibition was apparent for all receptor combinations. An approximate estimate for the concentration of isoflurane producing half-maximal enhancement of Iss in the wild-type receptors {alpha}1ß2, {alpha}1ß2{gamma}2s and mutant {alpha}1(S270H)ß2 was ~0.1 mM.



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  		Figure 6. Dose-effect plots for isoflurane on steady-state desensitized currents (Iss). Data points are means ± SEM from at least 6 individual oocytes for each concentration and combination. The marked enhancement of Iss in the wild-type {alpha}1ß2 combination was abolished with substitution for the mutant {alpha}1(S270H) subunit and inclusion of the {gamma}2s subunit. From multiple regression analyses for all data up to 1 MAC, there were significant main effects of concentration, and of addition of the {gamma}2s-subunit and inclusion of the {alpha}1(S270H) mutation in attenuating the level of Iss enhancement with no significant interaction between these two factors (R2 = 67%, P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study we explored the action of the volatile anesthetic isoflurane on sub-maximally and maximally activated wild-type and mutant ({alpha}1S270H) GABAA-R currents expressed in Xenopus oocytes. We found that isoflurane induced pronounced enhancement of desensitized currents in wild-type GABAA-R, consistent with a relief from desensitization. The inclusion of a {gamma}2s subunit or the mutation attenuated this effect, which was absent in the mutant {alpha}1(S270H)ß2{gamma}2s receptors. In addition, we have shown that the enhancement of sub-saturating GABA currents by isoflurane in {alpha}1(S270H) mutant receptors was unaffected by the presence or absence of the {gamma}2s subunit, in contrast to earlier suggestions (21).

Although the enhancement of GABA currents by isoflurane was severely attenuated in the mutant receptors, an appreciable effect of isoflurane was observed (Fig. 1) as reported previously for oocyte recordings using the S270I mutation (21). However, the limited enhancement of GABA currents produced by isoflurane did not differ between the {alpha}1(S270H)ß2 and {alpha}1(S270H)ß2{gamma}2s combinations at any of the concentrations of isoflurane tested (75 µM to 2.4 mM). Our data therefore do not support the proposal (21) that the effect of the {alpha} subunit mutation is negated or in any way influenced by the presence of the {gamma} subunit. This is consistent with our recent data (20) from mammalian cell expression experiments, in which the {alpha}1(S270H) mutation was highly effective at abolishing isoflurane potentiation of GABA responses in a background of {alpha}1ß2{gamma}2s.

One possible explanation for the discrepancy between our results and those previously reported (21) is provided by comparisons of the dose-effect curves for isoflurane enhancement of sub-saturating GABA currents (Fig. 3). The potentiation of EC20 GABA currents by isoflurane in wt {alpha}1ß2 receptors (>600%; i.e., > IGABAmax) was obviously strongly influenced by desensitization, a problem inherent to whole oocyte recordings. When we repeated these experiments using EC5 GABA, the contribution from desensitized receptors was presumably reduced – certainly the potentiation by isoflurane was now more comparable for the wt {alpha}1ß2 and wt {alpha}1ß2{gamma}2s combinations (Fig. 3B). At large concentrations of isoflurane (>4 MAC), we observed an inhibitory component to the action of isoflurane (4,6) in all receptor combinations which may also influence measurements of potentiation by the anesthetic.

Our analysis of the enhancement of IGABAmax by isoflurane in wild-type {alpha}1ß2 receptors (Fig. 4) indicated that the volatile anesthetic might modulate desensitization. Relief of steady-state desensitization of GABA currents by the IV anesthetic, propofol, has been described in hippocampal neurons (15). In our experiments, the pronounced enhancement of Iss by sub-clinical doses of isoflurane (Figs. 5 and 6) was suggestive of relief of slow desensitization. We estimated the half-maximal concentration of isoflurane for this effect to be around 0.1 mM (1/3 MAC). Therefore, it is important to note that if the volatile anesthetic is relieving slow desensitization, it may be more potent at mediating this effect than at potentiating sub-saturating GABA responses (EC50 ~0.3 mM).

GABAA-R desensitization is thought to have an influence on the duration of IPSCs (11); in fact, kinetic models predict that desensitization may prolong (8) or curtail the IPSC (14), depending on the relative rate constants for exit from the open state. Relief of desensitization by isoflurane may therefore be a sensitive mechanism for modulation of IPSCs (10). Relief of slow desensitization may be especially relevant to synaptic integration during trains of neuronal firing, when amplitudes of consecutive IPSCs diminish in part because of cumulative GABAA-R desensitization (12). In this case, isoflurane would be expected to help maintain the amplitude of repetitive IPSCs and thereby conserve inhibitory tone during periods of increased excitability.

The enhancement of Iss by isoflurane was subunit-dependent, as it was less apparent when the {gamma}2s subunit was present. In addition, although the {alpha}1(S270H) mutation had no effect on the extent of steady-state GABAA-R desensitization (Table 1) (23), it diminished isoflurane modulation of desensitized current, which was undetectable in {alpha}1(S270H)ß2{gamma}2s. Thus, the inclusion of a {gamma}2s subunit and the serine 270 residue may both influence isoflurane-induced relief of slow desensitization. However, there is an alternative explanation for the enhancement of maximally activated and desensitized currents by isoflurane. The maximal open probability of a {alpha}ß{gamma} GABAA-R in the presence of a saturating concentration of agonist has been shown to be ~0.8 (24), a level that is likely reduced for "{gamma}-less" receptors (25) and increased in a {alpha}1-270 mutation (23). Therefore, after applications of saturating agonist concentrations, the extent of isoflurane modulation of Iss for the different receptor combinations may partly depend on the relative dynamic range for increasing mean channel open duration (i.e., via an effect on gating between closed and open states, rather than on relief of desensitization alone). To differentiate conclusively between these two possibilities we plan to perform additional studies using patch-clamp recording and rapid solution exchange. Regardless of the mechanism, the present study has revealed a novel effect of isoflurane on maximally activated GABAA-R that is attenuated by inclusion of the S270H mutation in the {alpha}1-subunit. This action of the volatile anesthetic may have important consequences in shaping IPSCs after release of saturating concentrations of agonist at the synapse during anesthesia.


   Acknowledgments
 
Supported, in part, by grants from the Arthur Vining Davis Foundation to the Smith College Neuroscience Program, Tomlinson Fund to RJNS, Blakeslee Fund to KCR, and by GM45129 from the National Institutes of Health, Bethesda, MD to NLH.

We thank Dr. Stuart A. Forman for advice on establishing the oocyte electrophysiology, and Dr. Andrew Jenkins and Dr. Mary E. Harrington for helpful comments.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication November 14, 2003.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press