The gamma-aminobutyric acid (GABA)A receptor/chloride channel has a broad-spectrum anesthetic sensitivity and is a key regulator of arousal. Each receptor/channel complex is an assembly of five protein subunits. Six subunit classes have been identified, each containing one to six members; many combinations are expressed throughout the brain. Benzodiazepines and intravenous anesthetic agents are clearly subunit dependent, but the literature to date suggests that volatile anesthetics are not. The physiological role of the delta subunit remains enigmatic, and it has not been examined as a determinant of anesthetic sensitivity.
Combinations of GABA(A) receptor subunit cDNAs were injected into Xenopus laevis oocytes: alpha1beta1, alpha1beta1gamma2L, alpha1beta1delta, and alpha1beta1gamma2Ldelta. Expression of functional ion channels with distinct signalling and pharmacologic properties was demonstrated within 1-4 days by established electrophysiological methods.
Co-expression of the delta subunit produced changes in receptor affinity; current density; and the modulatory efficacy of diazepam, zinc, and lanthanum; it also produced subtle changes in the rate of desensitization in response to GABA. Isoflurane enhanced GABA-induced responses from all combinations: alphabeta delta (>10-fold) > alphabeta > alphabeta gamma > or = alphabeta gammadelta (approximately 5-fold). Dose-response plots were bell shaped. Compared with alphabeta gamma receptors (EC50 = 225 microM), both alphabeta delta (EC50 = 372 microM) and alphabeta gammadelta (EC50 = 399 microM) had a reduced affinity for isoflurane. Isoflurane (at a concentration close to the EC50 for each subunit) increased the affinity of GABA for its receptor but depressed the maximal response (alphabeta gamma and alphabeta gammadelta). In contrast, the small currents through alphabeta delta receptors were enhanced, even at saturating agonist concentrations.
Delta subunit expression alters GABA(A) receptor function but is not an absolute determinant of anesthetic sensitivity.
Gamma-Aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the mammalian brain, where it is present at up to one third of all synapses. The GABAAreceptor is a member of the ligand-gated ion channel superfamily composed of an aggregation of five subunits around an integral fast chloride channel. Six classes (alpha, beta, gamma, delta, rho, and epsilon) of GABA receptor subunits have been cloned to date, with as many as six distinct proteins in each. This allows for many different combinations and physiologic or pharmacologic isomerism. [4,5]Subunits are expressed differentially throughout the central nervous system.
Anesthetics exert their actions on the central nervous system by modulating neuronal excitability. At least three mechanisms have been proposed to account for such depressant effects:(1) Anesthetics dissolve in the bulk lipid matrix of the biologic membrane to alter fluidity or volume ;(2) The molecules interact directly with hydrophobic binding sites on signalling proteins within the plasma membrane ; or (3) Target proteins are affected indirectly by second messenger systems. Members of the ligand-gated ion channel superfamily are now recognized as stereoselective targets for these drugs and are modulated by clinically relevant concentrations (e.g., [7,9]). Several laboratories are currently attempting to shed light on the submolecular identity and location of domains conferring such sensitivity on ligand-[10,11]or voltage-gated channels. 
A broad range of drugs with widely differing structures have been shown to act at or on the GABAAreceptor, including sedative, hypnotic, anticonvulsant, and anesthetic agents (reviewed in ). Benzodiazepine agonists only produce their characteristic potentiating effect if a gamma subunit is co-expressed with alpha and beta. The alpha subunit has been seen to subtly alter sensitivity to benzodiazepines and to steroids. Sedative concentrations of ethanol (5–30 mM) require the gamma subunit and, more specifically, the extra eight amino acids in the longer splice variant of the gamma subunit (gamma2L), for receptor sensitivity. In the early 1990s, several groups reported the enhancement of GABAAcurrents by inhalational anesthetics. [18–20]Isoflurane and enflurane appeared to cause an increase independently of the presence of alpha, beta, or gamma, although the latter study did suggest that there was a qualitative difference in modulation due to subunit composition. Also, pentobarbital and propofol action have been shown to be markedly dependent on the presence of the newly identified epsilon subunit. Five rho subunits alone (a homooligomeric pentamer) can form a functional receptor complex; to date this is the only chloride channel activated by GABA that is either completely insensitive or depressed by volatile anesthetics. The alpha6subunit strongly increases the affinity and efficacy of direct activation of recombinant human GABAAreceptors by pentobarbital (in the absence of neurotransmitter). The subunit dependence of anesthetic action is topical and has been comprehensively reviewed. 
The delta subunit was cloned relatively recently, and distribution of the rat delta mRNA has been mapped using in situ hybridization studies. The most prominent expression was noted in the cerebellum, with significant levels in the thalamic nuclei, dentate gyrus, olfactory bulb and tubercle, cerebral cortex, and nucleus accumbens. Immunohistochemistry broadly confirms this distribution pattern, and immunoprecipitation experiments suggest that [nearly =] 21% of solubilized rat brain receptors contain the delta subunit. Despite this widespread distribution and the pivotal role of GABAAreceptors in neuronal inhibition, few articles on physiologic signalling roles for delta have been published (e.g., ). Rat brain isoforms were characterized in some detail, but modulation by pentobarbital was not notably dependent on co-expression of delta subunits. The aims of the current study were to seek evidence for the functional expression of delta in recombinant GABAAreceptors and then to examine the influence of this subunit on modulation by isoflurane.
Materials and Methods
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 GABAAreceptor subunit cDNAs alpha1.2, beta1.3, gamma2Land 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. 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).
Anesthetic Formulation and Quantitative Analysis
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 and published gas partitioning coefficients over a range of temperatures. 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, alpha1beta1gamma2L, alpha1beta1delta, and alpha1beta1gamma2Ldelta (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).
Pharmacologic Strategies to Demonstrate Functional Expression of Receptor Isoforms
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. 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. 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)).
Homooligomeric delta Receptors
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. These rodent subunits were reported to form functional GABA-activated (glycine-insensitive) chloride channels as homooligomers in transfected cell lines, but this may reflect the presence of endogenous GABA subunits in the human embryonic kidney cells used. Electrophysiologic experiments suggest that homooligomeric beta1and 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.
Passive Properties of Receptor Isoforms
The heteromeric receptor combinations all generated unequivocal currents with properties typical of GABAAreceptors. Three of the heteromeric channels generated outwardly rectifying currents (a notable feature of macroscopic GABAAcurrents in central nervous system neurones ) with a reversal potential characteristic of a chloride current in the oocyte. 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, 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 ). This isoform also exhibited the unique property of noncooperative activation consistent with the shallow slopes previously noted in the rat study 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 ) and generated the most robust currents.
In a previous communication, 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. 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). Some of the implications of these observations for anesthetic pharmacology are discussed subsequently.
Anesthetic Subunit Dependence
Anesthetic interactions with ionotropic receptors have attracted much recent attention, and the GABAAreceptor has been proposed as a unifying molecular target. Estimation of the amount of potentiation of GABAAreceptor 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 ).
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 and primary cultures. 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 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.
Anesthetics in the Brain
The number of theoretical subunit combinations within a pentameric GABAAreceptor 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. Most GABAAreceptors, at least in rat brain, are composed of alpha1beta2gamma2subunits. For this and other combinations, stoichiometry is uncertain, but one article has suggested that 2alpha, 1beta, and 2gamma may be favored. 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., ). Some groups propose that, in the brain, gamma and delta subunits are largely mutually exclusive, although one report suggests that gamma2and delta co-assemble to produce a receptor with novel pharmacology. In a large number of regions, RNA for delta colocalizes with those for alpha1, alpha4, and beta2(principally the thalamic nuclei); however, mapping of subunit gene expression 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 ). 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 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 ; 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., ) 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 ). 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 GABAAreceptors 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.