Volatile anesthetics prolong inhibitory postsynaptic potentials in central neurons an allosteric action on the gamma-aminobutyric acid type A (GABA(A)) receptor, an effect that may underlie the hypnotic actions of these agents. Inhaled anesthetics such as isoflurane act to enhance responses to submaximal concentrations of GABA, but it is not clear whether their effect is mediated by an increase in the binding of the agonist or by changes in receptor gating behavior. To address this question, the authors studied the effects of isoflurane on a mutant GABA(A) receptor with a gating defect that decreases receptor sensitivity by lowering agonist efficacy. They then compared the effects of clinically relevant concentrations of isoflurane on the actions of GABA and piperidine-4-sulfonic acid (P4S), a partial agonist at the GABA(A) receptor.
The authors created a mutant of the GABA receptor alpha subunit (L277A) by site-directed mutagenesis. The mutant subunit was coexpressed with beta(2) and gamma(2S) subunits in HEK293 cells, and responses to GABA and P4S were recorded using the whole-cell patch clamp technique. EC values were determined for the full agonist GABA and the partial agonist P4S. The authors also determined the relative efficacy (epsilon) of P4S. These measurements were then repeated in the presence of isoflurane.
The concentration-response curve for GABA was shifted to the right (EC(50) = 278 microm) in the alpha(1)(L277A)beta(2)gamma(2S) mutant receptor, compared with the corresponding wild-type alpha(1)beta(2)gamma(2S) GABA(A) receptor (EC(50) = 16 microm). P4S is a partial agonist at both receptors, with a dramatically decreased relative efficacy at the mutant receptor (epsilon = 0.24). When the mutant receptor was studied in the presence of isoflurane, the concentration-response curves for both GABA and P4S were shifted to the left (EC(50) for GABA = 78 microm); the efficacy of P4S also increased significantly (epsilon = 0.40).
By studying a mutant GABA receptor with impaired gating, the authors were able to demonstrate clearly that isoflurane can increase the efficacy of a partial agonist, as well as increase agonist potency. These data suggest that the volatile anesthetic isoflurane exerts at least some of its effects on the GABA(A) receptor via alterations in gating rather than simply changing binding or unbinding of the agonist.
INHALED anesthetics prolong synaptic inhibition in the central nervous system at concentrations similar to those that induce hypnosis and unconsciousness in humans. 1,2A large body of evidence suggests that this effect is caused by modulation of the function of postsynaptic γ-aminobutyric acid type A (GABAA) receptors. For example, inhaled anesthetics enhance the response of GABAAreceptors to submaximal GABA in acutely isolated neurons 3,4and in heterologous expression systems, 5–7in which the GABA concentration–response curve is shifted to the left, with no increase in the maximal response. 3,4It is not clear whether the potentiating effects of volatile anesthetics at the GABAAreceptor are mediated by a decrease in agonist unbinding 8or by alterations in subsequent gating transitions. 9
The GABAAreceptor is made up of five subunits, each of which is proposed to contain four transmembrane segments (TM1–4) including a short extracellular loop between TM2 and TM3. The TM2–3 loop has been proposed to be involved in gating 10,11in other members of the gene family of ligand-gated ion channels. We have previously shown, by alanine-scanning mutagenesis, that mutations in the TM2–3 linker of the α2subunit can alter gating of the GABAAreceptor. 12In particular, mutation at position L277 in the α2subunit increases the EC50for GABA and reduces the maximal response of the partial agonist piperidine-4-sulfonic acid (P4S). Partial agonists have often been used to differentiate between changes in agonist binding and gating in mutant receptors. 13–16In addition, the use of partial agonists can discriminate between drugs that act primarily to alter agonist binding (and therefore have no effect on agonist efficacy) and those that exert their effects on gating transitions. 13,17,18We have combined the use of a partial agonist and a mutant GABAAreceptor with impaired gating to detect any potential effects of isoflurane on GABAAreceptor gating.
Materials and Methods
To create the mutant α1subunit, we introduced a single point mutation into the cDNA encoding the human GABAAreceptor α1subunit. 19Mutations were performed with the Quik Change site-directed mutagenesis kit (Stratagene, La Jolla, CA) with commercially produced primers of 24–30 bases in length (Operon Technologies, Alameda, CA). Successfully mutated clones were selected by the deletion of a unique site (Bsu I). Positive clones were confirmed by automated fluorescent DNA sequencing of the complete receptor subunit cDNA insert (Cornell DNA Sequencing Service, Ithaca, NY). All restriction enzymes were obtained from New England Biolabs (Beverley, MA).
Cell Culture and Transfection
Wild-type or mutant α1subunit cDNAs were coexpressed with the GABAAβ2and γ2Ssubunits via the plasmid vector pCIS2 in human embryonic kidney (HEK) 293 cells, as previously described. 20HEK 293 cells (American Type Tissue Culture Collection, Manassas, VA) were cultured on poly-d-lysine–treated coverslips (Sigma, St. Louis, MO) in Eagle minimum essential medium (Sigma) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT), l-glutamine (0.292 μg/ml; Life Technologies Inc., Grand Island, NY), penicillin G sodium (100 units/ml; Life Technologies Inc.), and streptomycin sulfate (100 μg/ml, Life Technologies Inc.). Cells were transfected using the calcium phosphate precipitation technique 21to achieve transient expression of human α1β2γ2Sor the corresponding mutant GABAAreceptor. Each coverslip of cells was transfected with approximately 6 μg of total DNA. The transfected cells were cultured for 24 h in an atmosphere containing 3% CO2before being removed and replaced with fresh culture medium in an atmosphere of 5% CO2.
Coverslips with the transfected cells were transferred after 48–72 h to a bath that was continuously perfused with extracellular saline. The extracellular saline contained 145 mm NaCl, 3 mm KCl, 1.5 mm CaCl2, 1 mm MgCl2, 5.5 mm d-glucose, and 10 mm HEPES, pH 7.4, at an osmolarity of 320–330 mOsm. Recordings were performed at room temperature using the whole-cell patch clamp technique as described previously. 22The patch pipette solution contained 147 mm N -methyl-d-glucamine hydrochloride, 5 mm CsCl, 5 mm K2ATP, 5 mm HEPES, 1 mm MgCl2, 0.1 mm CaCl2, and 1.1 mm EGTA, pH 7.2, at an osmolarity of 315 mOsm. The chloride equilibrium potential was therefore approximately 0 mV. Pipette-to-bath resistance was typically 5–10 MΩ. Cells were voltage clamped at −60 mV. All drugs were dissolved in extracellular medium and rapidly applied to the cell by local perfusion with laminar flow using a multichannel infusion pump (Stoelting, Wood Dale, IL). 19The loss of isoflurane using this perfusion device has been measured using gas chromatography and represents only 5–10% of the total applied drug concentration (Matthew D. Krasowski, M.D., University of Chicago, Chicago, IL, unpublished observations, 1998). A motor-driven switching system (BioLogic Rapid Solution Changer RSC-100; Molecular Kinetics, Pullman, WA) exchanged solutions around the cell within approximately 20 ms. The solution changer was driven by protocols in the acquisition program pCLAMP5 (Axon Instruments, Foster City, CA). Throughout the experiment, each cell was periodically challenged with a submaximal concentration of agonist to ensure that no cumulative desensitization or rundown of the GABAAreceptor currents had occurred. Responses were digitized (TL1–125 interface; Axon Instruments) using pCLAMP5 (Axon Instruments).
Concentration–effect data were fitted (Kaleidograph, Reading, PA) using the following equation (Hill equation)
where I/Imax(GABA)is the fraction of the maximally obtained GABA response, EC50is the concentration of agonist producing a half-maximal response, and nHis the Hill coefficient. Agonist responses in each cell were normalized to the maximal current that could be elicited by GABA. Relative efficacy (ε) was than determined by ε= Imax(P4S)/Imax(GABA).
Pooled data are represented as mean ± SEM. Statistical significance was determined at the P < 0.05 level by a two-tailed Student t test assuming unequal variance.
GABA and P4S (Sigma) were either dissolved directly into extracellular solution or stored overnight at 4°C in sealed Nalgene® tubes (Nalge/Nunc, Rochester, NY). All reagents were purchased from Sigma except for isoflurane, which has obtained from Abbott Laboratories (North Chicago, IL).
Pharmacology of the α1(L277A)β2γ2SMutant Receptor
The mutant GABAAreceptor α1(L277A)β2γ2Swas compared with the wild-type α1β2γ2SGABAAreceptor after transient expression in HEK 293 cells. In both receptors, brief applications of agonist produced saturable concentration-dependent inward currents (figs. 1A and 1B). The maximal amplitudes of GABA-induced currents and the Hill coefficient for GABA were similar in wild-type and mutant receptors (table 1). In the mutant receptor, the concentration–response curve for GABA was shifted far to the right (fig. 1C), with the EC50for GABA increased 17-fold compared with the wild-type, indicating that the mutant receptor is significantly less sensitive to GABA than the wild-type receptor.
P4S produces responses in wild-type α1β2γ2SGABAAreceptors that are approximately 74% of the maximal amplitude of GABA responses (fig. 2A). In contrast, α1(L277A)β2γ2Sreceptors exhibited maximal responses that were, on average, only 24% of the amplitude of the maximum current elicited by GABA (fig. 2B). In addition, the mutant receptor showed an increased EC50for P4S when compared with the wild-type receptor (fig. 2C). The data for EC50, efficacy (ε), and Hill coefficient (nH) are summarized in table 2.
Effects of Isoflurane on Wild-type and Mutant γ-Aminobutyric Acid Type A Receptors
Isoflurane (1 minimum alveolar concentration [MAC], 0.5 mm) increased the currents elicited by submaximal (EC20concentration) GABA at the wild-type receptor by 114% (fig. 3A). This increase in sensitivity to GABA is associated with a leftward shift in the GABA concentration–response curve (fig. 3B) and a significant decrease of the EC50from 17 to 5.9 μm (table 3;P < 0.01). When measuring the maximal current to GABA on individual cells before and during isoflurane application, there was a slight increase in maximal current in the presence of isoflurane, but this was not significant (data not shown).
In the mutant α1(L277A)β2γ2Sreceptor, isoflurane (1 MAC, 0.5 mm) also increased the potency of GABA. At the mutant receptor (fig. 3C), isoflurane potentiated the response to submaximal (EC20) GABA by 65%. Again, there was a significant leftward shift of the concentration–response curve for GABA at the mutant receptor in the presence of 0.5 mm isoflurane (P < 0.01), with a decrease in EC50from 132 to 78 μm (fig. 3D).
P4S is a partial agonist at the α1(L277A)β2γ2Smutant receptor. Isoflurane at 0.5 mm (equivalent to 1 MAC) enhances the response to a maximal concentration of P4S in a reversible manner. Figure 4Ashows data from a typical experiment from an individual cell expressing the mutant α1(L277A)β2γ2Sreceptor. The current elicited by a maximal concentration of P4S was increased in the presence of isoflurane. The concentration–response data also revealed a significant increase in relative efficacy of P4S (ε) from 0.27 to 0.4 in the presence of 0.5 mm isoflurane (P < 0.001;fig. 4B). In the presence of isoflurane there is also a leftward shift of the concentration–response curve for P4S in the mutant receptor (table 4). Increasing the concentration of isoflurane from 0.5 to 1.0 mm (2 MAC) produced no additional increase in efficacy (data not shown).
The results of this study strongly suggest that the volatile anesthetic isoflurane enhances GABAAreceptor gating. We used a partial agonist (P4S) together with a gating defective mutant of the GABAAreceptor to demonstrate an increase in potency and efficacy of the agonist in the presence of isoflurane.
Partial agonists have been used in the past to determine the effects of modulators on a particular receptor system. For example, the efficacy of 2-methyl-5-hydroxytryptophan at the 5-HT3receptor 23and taurine at the glycine receptor, 24can be enhanced by allosteric modulators. P4S has been shown to be a partial agonist at several GABAAreceptor subtypes, 12,16,25and its activity has been characterized in detail for the α1β2γ2Ssubunit combination, using single-channel recording techniques. 26
Our data show that P4S is a partial agonist with relatively high efficacy in the wild-type α1β2γ2SGABAAreceptor. In preliminary experiments, we found that isoflurane could increase the relative efficacy of P4S at the wild-type receptor (data not shown), but that these changes, although significant, were difficult to measure.
By introducing a mutation at L277 to Ala in the α1subunit, we were able to decrease potency of GABA and P4S and to decrease the relative efficacy of P4S from 74% to 24%. The rightward shift of the concentration–response curves for both agonists together with a simultaneous reduction in P4S efficacy suggests an impairment of receptor isomerization in the L277A mutant. 12,13The role of the TM2–3 extracellular loop may be similar in the GABAA, nicotinic acetylcholine, and glycine receptors. 24,27Mutations in the TM2–3 loop of the glycine receptor have been shown to increase closing rate constants (α) 28or reduce the single-channel conductance (γ). 11Mutations in this region of the glycine receptor do not alter the binding of the competitive glycine receptor agonist (3H)-strychnine, but do change the efficacy of the agonists β-alanine and taurine. 10,24Point mutations in the TM2–3 loop in the muscle type nicotinic acetylcholine receptor alter the efficacy of the partial agonist choline by increasing the opening rate of the receptor (β). 29Mutations in the TM2–3 linker of the GABAAreceptor have been shown to alter the effective efficacy of P4S, consistent with the primary effects of such mutations on receptor gating. 12
In our experiments, isoflurane increased the potency of GABA at the mutant receptor (α1(L277A)β2γ2S), but we did not observe a significant increase in maximal currents elicited by GABA in the presence of isoflurane at the mutant receptor. This is, in fact, not unexpected. Colquhoun 17has discussed the fact that, for an agonist with an already very high efficacy, further increases in efficacy cause only a parallel leftward shift of the dose–response curve, with no change in maximal current. A detailed model of GABAAreceptor activation 30defines the GABAAreceptor as a receptor with highly efficient gating when activated by GABA. The ratio of the isomerization rate constants (β/α) has been reported to be between 17.6 in hippocampal neurons 30and 10.7 in transfected HEK 293 cells 26when GABA is used as the agonist in outside-out patch recordings. We therefore suggest that GABA remains a highly efficient agonist, even at the α1(L277A)β2γ2Smutant receptor.
In contrast, isoflurane clearly increased the potency and relative efficacy of the partial agonist P4S at the mutant receptor. Changes in maximal response are easily observed with agonists with very low efficacy, as explained by Colquhoun. 17P4S has been shown to have decreased efficacy at the α1β2γ2Ssubunit combination. 26Since we assume that all receptor binding sites are occupied at 1 mm P4S in the L277A mutant, an increase in maximal response in this receptor can occur only if channel gating is facilitated by the anesthetic. 17In analogous studies, Downie et al. 31were able to show that isoflurane increased the maximum response of the glycine receptor to its low efficacy partial agonist taurine, and concluded that isoflurane enhances gating of the receptor.
In conclusion, isoflurane increases both the potency and efficacy of a partial agonist at the GABAAreceptor. This suggests that isoflurane exerts some or all of its effects on the GABAAreceptor by changing gating rather than via effects on agonist binding.