Modulation of Recombination Human γ-Aminobutyric Acid-A Receptors by Isoflurane : Influence of the Delta Subunit

Adult female Xenopus laevis (imported from African Xenopus Facility, Noordhoek, South Africa) were kept in a standard glass tank with a 12:12-h light:dark cycle. The water was recirculated through a filter and thermostatically controlled at [nearly =] 23 [degree sign] Celsius. Feeding was carried out daily and consisted of standard Xenopus pellets. Donor frogs were anesthetized by immersion in 2–3 cm of 0.4% 3-aminobenzoic acid ethyl ester (tricaine) before oophorectomy using standard surgical techniques. Viable donor frogs were not reused within 6 weeks of this procedure. Ovarian lobes were cut and collected into calcium-free saline (OR2), which contained 82 mM of NaCl, 2 mM of KCl, 5 mM of HEPES, and 1 mM of MgCl², titrated to pH 7.5 with NaOH. The lobes were washed in a culture dish using OR2 before immersion in a thawed aliquot (3 ml) of collagenase buffer (2 mg/ml of OR2) and digested for 8–10 min. The collagenase was removed and the oocytes were washed four or five times. Stage V and VI oocytes were manually dissected from their loosened epithelial and thecal layers using a low-powered microscope (12.5x + 2.5x) and fine-tipped forceps. Stripped oocytes were resoaked in collagenase for 3–5 min to remove remaining follicular cells, with the enzyme action quenched by immersion in albumin (15 mg/10 ml of OR2) for 5–10 min. Finally, cells were transferred into filtered (0.2 micro meter; Gelman Sciences, Ann Arbor, MI) Modified Barth's saline containing 88 mM of NaCl, 1 mM of KCl, 0.82 mM of MgSO⁴, 0.33 mM of Ca(NO³)², 0.41 mM of CaCl², 2.5 mM of NaHCO³, and 10 mM of HEPES, titrated to pH 7.4 with NaOH, which had been autoclaved and supplemented with gentamicin (100 mg/l), theophylline (90 mg/l), penicillin (10,000 U/l), and streptomycin (10 mg/l).

Samples of human GABA^Areceptor subunit cDNAs alpha¹.2, beta¹.3, gamma^2Land delta were provided by Dr. Paul Whiting (M.S.D., Harlow, United Kingdom) in the pCDM8 eukaryotic expression vector. Stock concentrates of [nearly =] 1 micro gram/micro liter were made up in autoclaved water and stored at -20 [degree sign] Celsius until required. For injections, 1 micro liter of the required subunits were added to 75 micro liter of a buffer consisting of 88 mM of NaCl, 1 mM of KCl, and 15 mM of HEPES, which was titrated to pH 7.0 and sterilized through 0.2-micro meter filters. [17]These dilutions were stored at 4 [degree sign] Celsius between uses and freshly prepared every 3–4 months or when expression levels decreased.

Autoclaved micropipettes (glass capillaries; Laser, Southampton, United Kingdom) with tip sizes ranging from 10–20 micro meter were back-filled completely with heavy white mineral oil (Sigma Diagnostics, St. Louis, MO) and attached to the automatic Drummond microdispenser (Laser). A 1-micro liter sample of the appropriate subunit combination was placed onto parafilm and drawn up into the tip of the micropipette. Oocyte nuclei were injected blindly (20 nl per oocyte) and transferred to 96-well plates containing filtered, supplemented Modified Barth's saline. Cells were incubated at 18–22 [degree sign] Celsius until required (1–5 days).

Electrodes (GC150F-10; Clark Electromedical Instruments, Berkshire, United Kingdom) were back-filled (4–5 mm) with warmed 1% agar in 2 M KCl solution. When required for use, they were back-filled with liquid 2 M KCl and broken back to 0.5 and 3.0 M Omega (current and voltage electrodes, respectively). Agar bridges (bath ground) were made using 2 M of KCl and connected to the preamplifier via reference wells containing 2 M KCl. Oocytes were placed within a narrow perspex bath (30 micro liter in volume) before impalement. Cells were clamped at -70 or -40 mV, depending on the protocol, using a GeneClamp 500 Amplifier (Axon Instruments, Foster City, CA). Frog Ringer's solution, consisting of 115 mM of NaCl, 2.5 mM of KCl, 10 mM of HEPES, and 1.8 mM of CaCl²(titrated to pH 7.2 with NaOH), constantly perfused the clamped cell (10 ml/min) through 2-mm Teflon tubing by a gravity-feed mechanism. Various drug concentrations were held in 50- or 100-ml syringes and selected to flow, as required. Results were measured either as a current or as change from baseline conductance in response to a -20-mV, 200-ms voltage jump, applied at 2.5 Hz via a stimulus isolator (Harvard Apparatus, Kent, United Kingdom). A digital oscilloscope (RadioSpares, Corby, United Kingdom) was used to maximize clamp gain and stability. Real-time recordings were made on a Graphic 1002 Chart Recorder (Lloyd Instruments, Hampshire, United Kingdom) where traces were inadvertently inverted compared with established electrophysiologic convention. In all traces depicted, outward currents can be seen as downward deflections and inward currents as upward deflections.

Agonist GABA was applied for a duration sufficient to elicit peak responses (5–20 s). Modulatory drug responses were assessed at equilibrium (determined by repeat pulses) and expressed as a percentage of peak control GABA response (i.e., percent change in input conductance). Diazepam and flumazenil were dissolved in dimethylsulfoxide (DMSO), then diluted >or= to 1000-fold into Ringer to yield the experimental solutions. DMSO was present at fixed levels before, during, and after the administration of these compounds. All experiments were conducted at room temperature (22–24 [degree sign] Celsius).

Saturated isoflurane solutions were diluted in Ringer's solution to the stated nominal concentration (confirmed by gas chromatography; n = 6). Isoflurane solutions were delivered from glass reservoirs covered with polyethylene (high density) floats to retard loss by evaporation and perfused through Teflon lines. To ascertain the degree of loss during preparation and handling of anesthetic solutions, gas chromatography was used with a gas-phase assay based on established methods [30]and published gas partitioning coefficients over a range of temperatures. [31]The mean (+/- SEM) loss of isoflurane from float-covered cylinders, over 2–3 h (the maximum duration of anesthetic experiment), was 11.3 +/- 2.8%(n = 6 for each of two different anesthetic concentrations), and 87.7 +/- 3.2% of the stated isoflurane concentration reached the cell up to 2.5 h after start of experimentation (n = 6). In this article, we cite nominal calculated isoflurane concentrations and consider the negligible loss only in the discussion.

Sources of Chemicals. Isoflurane was obtained from Abbott Laboratories (Kent, United Kingdom), flumazenil from Roche (Basel, Switzerland), antibiotics from Gibco/Life Technologies (Paisley, Scotland, United Kingdom), Lanthanum Chloride from BDH Laboratory Supplies (Poole, United Kingdom), and all other chemicals from Sigma Chemical Co. (Dorset, United Kingdom).

Data Analysis. Results, measured as change in baseline conductance (unless otherwise stated), were analyzed using Prism software (Graphpad; San Diego, CA) and expressed as mean +/- SEM. Log dose-response curves were fit to a two-term logistic equation (minimum and maximum values fixed; concentration for 50% effect (EC⁵⁰) and Hill slope variable) by nonlinear regression. Statistical comparisons were done by paired, two-tailed t test or one-way analysis of variance (with Tukey's multiple comparison post hoc test), as indicated.

Attempts were made to express a homooligomeric receptor containing only the delta subunit, but no currents were detected in oocytes up to 5 days after injection (n = 40 from two batches of oocytes). The following combinations of subunits were characterized: alpha¹beta¹, alpha¹beta¹gamma^2L, alpha¹beta¹delta, and alpha¹beta¹gamma^2Ldelta (referred to subsequently as alpha beta, alpha beta gamma, alpha beta delta, and alpha beta gamma delta, respectively). Injection of a combination of subunit cDNAs does not necessarily result in all of them assembling into a unique functional receptor. Therefore, we examined the physiologic signalling properties and pharmacology of the expressed combinations to confirm their distinct signatures. Current-voltage (I-V) plots were constructed from the GABA-induced currents between -100 and 0 mV for three subunit combinations (e.g., Figure 1(A)). Reversal potentials were approximately -23 mV for each, which is consistent with the equilibrium potential for a chloride current in oocytes. As shown in Figure 1(B), alpha beta gamma (part i), alpha beta gamma delta (part i; n = 3 per receptor), and alpha beta delta (part ii; n = 4) all displayed outward rectification at the more positive holding potentials.

Results reported subsequently were obtained from an additional 156 oocytes (from many donors) over the 2–5 days after injection. The four receptor combinations differed in their rate of expression to recordable levels in response to a saturating concentration of GABA (3 mM) and also in their peak currents to maximal GABA (data not shown). alpha beta And alpha beta delta isoforms required 3–4 days when peak responses averaged 140.5 +/- 59 micro S per oocyte (n = 7) and 7.7 +/- 3 micro S (n = 9), respectively, at -40 mV; in contrast, alpha beta gamma and alpha beta gamma delta combinations expressed to measurable levels in 1–2 days and yielded peak conductances of 166.1 +/- 42.5 micro S (n = 9) and 171.95 +/- 45 micro S (n = 13), respectively, at this time. The apparent threshold for an inward current at -40 mV was between 0.9 micro Meter and 1.8 micro Meter of GABA for all combinations. The receptor subtypes alpha beta, alpha beta gamma, and alpha beta gamma delta displayed similar dose-response curves, with saturation at or less than 3,000 micro Meter of GABA. Mean Hill slopes were 2.0 +/- 0.3, 1.6 +/- 0.1, and 1.8 +/- 0.1, respectively, indicating cooperativity of GABA binding. The alpha beta gamma and alpha beta gamma delta receptor subtypes displayed indistinguishable affinity for GABA, with mean EC⁵⁰values 43.85 +/- 1.15 micro Meter and 31.26 +/- 1.23 micro Meter, respectively (n = 9 or 10, P > 0.05 using analysis of variance); however, they differed significantly from the EC⁵⁰value of alpha beta, which was 5.03 +/- 1.32 micro Meter (n = 6, P < 0.05). alpha beta delta Receptors displayed a less steep dose-response curve (n = 6), saturating at or at less than 200 micro Meter of GABA, with a mean EC⁵⁰value of 14.13 +/- 1.19 micro Meter (P < 0.05 vs. all combinations) and a Hill slope of 1.2 +/- 0.2. This Hill slope was significantly different from the Hill slopes of alpha beta and alpha beta gamma delta combinations (P < 0.05) but not from that of alpha beta gamma (P > 0.05).

One micromole per liter of diazepam markedly enhanced responses from alpha beta gamma (Figure 2(A)) and alpha beta gamma delta (Figure 2(B)) receptors (n = 4 or 5, P < 0.05 using t test) at subsaturating concentrations of GABA and shifted dose-response curves to the left. Responses of alpha beta (not shown) and alpha beta delta (Figure 2(C)) were not significantly enhanced by 0.333–1.000 micro Meter of diazepam. In contrast to alpha beta gamma receptors, any small modulatory effects of diazepam on alpha beta delta responses (P > 0.05) were insensitive to 1 micro Meter of flumazenil (Figure 2(C); n = 6). These results confirmed that heteromeric receptors without the gamma subunit do not bear a functional benzodiazepine receptor complex.

It has been reported that Zn²+ inhibits currents that do not contain a gamma subunit and has no significant effect when this subunit is present. [32]In our hands, gamma-containing receptors were weakly antagonized by 20 micro Meter of Zn²+; mean reductions were 11%(P > 0.05 using t test) and 33%(P < 0.05) for alpha beta gamma and alpha beta gamma delta responses, respectively. Effects on alpha beta conductances were unequivocal; responses were reduced by an average of 98%(P < 0.05). We also tested alpha beta delta receptors and saw an 87% decrease (P < 0.05) in control responses. Representative examples and normalized data are shown in Figure 3(A)(parts i and ii) and suggest that delta-containing isoforms exhibit intermediate sensitivity to the divalent cation (n = 5 per receptor).

The trivalent ion, lanthanum (La³+), was used to test subunit expression. This has been shown to potentiate alpha beta and alpha beta gamma [33,34]currents but not those produced by alpha beta delta receptors. [29]We saw a consistent but insignificant increase of GABA-induced conductances in alpha beta receptors (140%; P > 0.05 using t test) and a significant enhancement of alpha beta gamma receptor responses (240%; p < 0.05) in the presence of 600 micro Meter of La³+. When tested on alpha beta delta receptors, 600 micro Meter of La³+ marginally depressed responses (70%; P > 0.05) and with the alpha beta gamma delta combination (previously unreported) there were increases (147%; P < 0.05; n = 5 or 6 per receptor;Figure 3(B)). The addition of delta to heterooligomeric receptors appears to reduce sensitivity to potentiation by La³+, albeit not significantly, and the presence of the gamma subunit is capable of reversing this effect.

Desensitization was studied in the presence of a saturating concentration of GABA, applied for 50 s, at a holding potential of -40 mV (while monitoring input conductance). As the example in Figure 4(A) illustrates, for alpha beta, GABA-induced conductance faded only slightly throughout agonist application (n = 5, P > 0.05 using t test). The receptors alpha beta gamma and alpha beta gamma delta showed a more marked desensitization over the same period, decreasing to 48% and 50% of original levels, respectively (n = 5–7, P < 0.05 for each). alpha beta delta Receptors, in common with alpha beta, were less prone to desensitization, although a significant fading over 50 s was observed (P < 0.05). Compounded data are shown in Figure 4(B). Overall, these results were interpreted as evidence for the functional expression of (delta in the oocyte membrane.

Before looking at the action of isoflurane on each receptor isoform, we examined the effect of various concentrations of the volatile anesthetic at a fixed GABA concentration (within the range EC sub 10–25) for each particular subunit combination. Graphs were constructed over the range 58–2,879 micro Meter of isoflurane, which produced bell-shaped concentration-response profiles (e.g., Figure 5(A)) for each of the receptor subtypes (the illustrated curves in Figure 5(B) depict data up to the maximal only). The isoforms alpha beta (n = 2) and alpha beta gamma (n = 7) exhibited similar responses to the anesthetic over this range of concentrations and differed from alpha beta gamma delta (n = 4); alpha beta and alpha beta gamma enhancement saturated at [nearly =] 440 micro Meter, yielding apparent EC⁵⁰values of 206 +/- 1 micro Meter and 225 +/- 1 micro Meter, respectively, whereas alpha beta gamma delta potentiation peaked at [nearly =] 1,176 micro Meter of isoflurane, with an apparent EC⁵⁰value of 399 +/- 1 micro Meter. alpha beta delta Receptors (n = 4) demonstrated an intermediate affinity for the modulatory anesthetic; peak enhancement occurred at [nearly =] 588 micro Meter with an apparent EC⁵⁰value of 372 +/- 1 micro Meter of isoflurane. The EC⁵⁰values significantly differed (P < 0.05 using one-way analysis of variance); alpha beta was not included in this analysis because of limited replication (n = 2). Time-matched treatment blanks (data not shown) resulted in almost superimposeable curves if pre- and posttreatment responses were averaged, indicating that these responses were entirely attributable to the drug, at least for alpha beta gamma (n = 3) or alpha beta gamma delta (n = 2) receptors. In the two cells expressing alpha beta gamma delta, the blanks indicated a slightly more labile response at the saturating doses of GABA over time, which indicates that we may have marginally underestimated the modulatory efficacy of isoflurane at the higher GABA concentrations on this isoform. Our isoflurane dose-response experiments, however, indicate the alpha beta delta isoform to be modulated profoundly (mean enhancement > 10-fold); the rank order of efficacy was alpha beta delta > alpha beta > alpha beta gamma >or= to alpha beta gamma delta. Isoflurane did not appear to induce any direct agonist effect in the absence of GABA, even at the highest concentration applied.

To probe the modulatory mechanisms further, we then generated concentration-response curves for GABA for three of the subunit combinations before, during, and after exposure to anesthetic at the approximate EC⁵⁰concentrations indicated previously. Attempts to construct meaningful curves before, during, and after anesthetic using the alpha beta receptor were not successful. This was due to the protracted nature of the experiment, marked variation in agonist affinity, and fluctuation in expression levels noted within and between batches of oocytes for this particular receptor. The alpha beta gamma (n = 6) and alpha beta gamma delta (n = 7) conductances were enhanced as a consequence of increased GABA receptor affinity over the range 1–100 micro Meter of GABA (Figure 6(A and B)): There was a parallel leftward shift of this part of the curve, signifying a potentiation of these GABA-activated conductances with no change in Hill slope. Potentiation was most pronounced (up to 600% for alpha beta gamma and 1,100% for alpha beta gamma delta) at low agonist concentrations, and EC⁵⁰values were significantly altered (P < 0.0001 using t test). At higher agonist concentrations (100–5,000 micro Meter of GABA), these receptor subtypes displayed no potentiation or even inhibition of control responses, with alpha beta gamma delta conductances being significantly reduced (P < 0.05) at saturation. When the alpha beta delta receptors were tested with their apparent EC⁵⁰concentration of isoflurane, the maximum responses to saturating concentrations of GABA were significantly enhanced (P < 0.05;Figure 6(C)).

A rat brain delta subunit, which shares approximately 35% sequence identity with cloned alpha and beta subunits, [25,26]has been subjected to only limited physiologic and pharmacologic characterization. [29]These rodent subunits were reported to form functional GABA-activated (glycine-insensitive) chloride channels as homooligomers in transfected cell lines, [25]but this may reflect the presence of endogenous GABA subunits in the human embryonic kidney cells used. [35]Electrophysiologic experiments suggest that homooligomeric beta¹[36]and rho receptors (e.g., [21,23]) can generate functional channel complexes. Our own work with human cDNA, in common with other studies using rodent receptors [28,29](SJ Moss, MRC Laboratory, University College, London, personal communication), in a variety of expression systems, suggests that delta subunits alone do not form functional chloride channels.

The heteromeric receptor combinations all generated unequivocal currents with properties typical of GABA^Areceptors. Three of the heteromeric channels generated outwardly rectifying currents (a notable feature of macroscopic GABA^Acurrents in central nervous system neurones [37]) with a reversal potential characteristic of a chloride current in the oocyte. [38]Our experiments on receptor activation and modulation were conducted at arbitrary times when robust currents could be measured (analysis of the kinetics of channel expression was not systematic). For this reason, our observations on current density should be regarded as semiquantitative, but, as described by others for rat channels, [29]the maximal currents through alpha beta delta isoforms were markedly lower than those for all other isoforms (averaging < 200 nA from Vh-40 mV). Unitary currents in this receptor have a lower conductance (22 pS) than alpha beta gamma delta (33 pS) and alpha beta gamma (30 pS), which alone is insufficient to account for the drastic reduction in peak current density of these macroscopic currents (particularly as their mean open time was longer by 20- to 80-fold [29]). This isoform also exhibited the unique property of noncooperative activation consistent with the shallow slopes previously noted in the rat study [29]and was saturated at relatively low GABA concentrations. All four combinations differed in their affinity for GABA, although addition of delta to alpha beta reduced affinity, whereas addition of delta to alpha beta gamma marginally enhanced affinity. Isoforms containing gamma were associated with lower GABA affinity (as previously reported for rat brain [39]) and generated the most robust currents.

In a previous communication, [40]we reported a paradoxical inverse agonist response to diazepam. By lowering solvent levels and using fixed dimethylsulfoxide concentrations in saline solutions throughout, we found that the alpha beta delta combination is insensitive to benzodiazepine modulation. The sensitivity to zinc and lanthanum, together with effects on maximal current density, affinity, and Hill slope, strongly suggests that delta subunits are functionally co-expressed in oocytes.

One of the unique signatures of delta-containing receptors in recombinant rat receptors was their relative insensitivity to, and rapid recovery from, acute desensitization compared with alpha beta gamma. [29]In our hands, using much longer pulses of saturating GABA and measuring conductance fade, the extent of desensitization observed was alpha beta gamma [nearly =] alpha beta gamma delta ([nearly =] 50%) > alpha beta delta [nearly =] alpha beta. On a cautionary note, our experiments in the oocyte suggest that currents (particularly those large responses in the micro A range) are more labile than conductance measurements, which, for this reason, we used to produce the previous ranking. Although limiting or dissipated ionic gradients in small cells under whole cell clamp are less likely, such a phenomenon might explain the relative insensitivity of tiny currents to fading (almost invariably interpreted as desensitization). [29]Some of the implications of these observations for anesthetic pharmacology are discussed subsequently.

Anesthetic interactions with ionotropic receptors have attracted much recent attention, and the GABA^Areceptor has been proposed as a unifying molecular target. [13]Estimation of the amount of potentiation of GABA^Areceptor isoforms and their affinity for anesthetics is complicated considerably by a strong dependence on the extent of occupation of the GABA site. [22,24]In this study, we endeavored to equalize these factors by studying the modulatory efficacy and affinity at a concentration of GABA strictly between the EC¹⁰and EC²⁵levels (measured in each cell) for each subunit combination. Responses in all subunit combinations were enhanced by isoflurane under these conditions, and no analogies with benzodiazepine subunit dependence can be seen (in accord with previous studies [24]).

No direct agonist effects in the absence of GABA (as have been reported for barbiturates, steroids, and propofol and which are markedly subunit dependent [11,16,41]) were evoked by isoflurane, even at the highest concentrations. Volatile anesthetics have been shown to activate chloride currents directly in expression systems [42]and primary cultures. [43]These divergent results may reflect subunit dependence, the presence of trace amounts of experimental GABA, or different rates of anesthetic administration. The delta subunit does quantitatively regulate anesthetic affinity and efficacy: Both alpha beta delta and alpha beta gamma delta have a lower affinity for isoflurane than the other combinations, but the peak modulatory response is greater by approximately twofold for the former combination. Isoflurane, at or near its EC⁵⁰value, enhances both alpha beta gamma delta and alpha beta gamma currents by increasing GABA receptor affinity, but maximal responses are to some extent depressed. In this respect, alpha beta delta receptors were again exceptional, as even maximal responses at or near saturating concentrations of GABA were enhanced by isoflurane. This result suggests that isoflurane may be increasing the alpha beta delta receptor probability of opening (the slope of the modulated curve was not significantly different from the control, suggesting that cooperative activation was not facilitated by anesthetic) or that the anesthetic is increasing the number of alpha beta delta receptors available for activation. One mechanism for the latter proposal might be through repriming of desensitized receptors or interference with the onset of fast desensitization. The response time in oocytes (large cells, [nearly =] 1 mm in diameter) is undoubtedly constrained by the superfusion method used in this study. Elegant recent work on recombinant nicotinic receptors in cell-free patches [10]demonstrates a fast phase of desensitization that would almost certainly be masked by concurrent activation in our study. The submolecular mechanisms for the low current-carrying capacity of this subunit combination (or its limited expression), together with its unique response to isoflurane, remain unresolved.

The number of theoretical subunit combinations within a pentameric GABA^Areceptor [3]is well in excess of 10,000, but it has been suggested that the number of functional isoforms is more likely to fall between 17 and 850. [2]Most GABA^Areceptors, at least in rat brain, are composed of alpha¹beta²gamma²subunits. [4]For this and other combinations, stoichiometry is uncertain, but one article has suggested that 2alpha, 1beta, and 2gamma may be favored. [44]Despite initial suggestions to the contrary, expression of alpha beta alone results in functional benzodiazepine-insensitive receptors, and their existence in restricted brain regions cannot be excluded (e.g., [2]). Some groups propose that, in the brain, gamma and delta subunits are largely mutually exclusive, [4]although one report suggests that gamma²and delta co-assemble to produce a receptor with novel pharmacology. [27]In a large number of regions, RNA for delta colocalizes with those for alpha¹, alpha⁴, and beta²(principally the thalamic nuclei)[26]; however, mapping of subunit gene expression [45]suggests that alpha¹, beta¹, and gamma²may form a receptor isoform in certain specific areas of rat brain (delta is also distributed in some of these locations [26]). Although no localization studies on human isoforms have been published, on the basis of the these findings we are confident that functionally relevant possibilities were probed in this study.

On the basis of somatosensory-evoked potentials and single-unit recordings in rats, Angel [46]suggested that structurally diverse anesthetics (including the commonly used inhalational agents) first impede transmission of sensory information at the level of the thalamic relay nuclei with further depressant effects on cortical circuits. Ligand-binding experiments also suggest anatomic differences in volatile anesthetic modulation, which may reflect differential subunit sensitivity. [47,48]Subunit dependence of anesthetic action is now acknowledged. [5,24]Our own quantitative experiments extend this database and provide further evidence for selective interaction with proteinaceous targets. Regarding whether delta subunits are important in conferring such differential sensitivity in vivo, we can only speculate. The alpha beta gamma delta isoform had a modulatory profile largely equivalent to the widespread alpha beta gamma [4]; although it had a lower affinity for isoflurane, it was associated with a depression of maximal responses and an increase in GABA receptor affinity. The depression of maximal response may underpin paradoxical reports that inhalational agents do not affect or even depress inhibitory postsynaptic potentials (e.g., [49]) in brain slices. The effects of isoflurane in the this study were seen at concentrations in the clinical range (minimum alveolar concentration-equivalent isoflurane has been cited as 320⁷-510 micro Meter [20]). Throughout, we cite nominal calculated concentrations (based on gas chromatography of saturated and diluted aqueous solutions), which represent an overestimate of up to 12% of isoflurane reaching the cell surface. The alpha beta delta isoform was the least prone of the receptors (excepting alpha beta) to desensitization and, perhaps coincidentally, was associated with the highest intrinsic modulatory efficacy, albeit at relatively high concentrations. The presence of supersensitive/hyperresponsive anesthetic receptors in the brain with a discrete anatomic location is an attractive hypothetical concept. On the basis of the relatively low affinity for isoflurane and their inherently low current-carrying capacity, we do not believe that GABA^Areceptors consisting of alpha beta delta subunits alone represent such a target. Magnetic resonance image-based scanning and the increasing availability of knockout mice for ion channel subunits may contribute to this debate in the foreseeable future.