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Research ArticleArticle

Propofol and Other Intravenous Anesthetics Have Sites of Action on the γ-Aminobutyric Acid Type A Receptor Distinct from That for Isoflurane

Matthew D. Krasowski, Vladimir V. Koltchine, Caroline E. Rick, Qing Ye, Suzanne E. Finn and Neil L. Harrison
Molecular Pharmacology March 1998, 53 (3) 530-538; DOI: https://doi.org/10.1124/mol.53.3.530

Abstract

Both volatile and intravenous general anesthetics allosterically enhance γ-aminobutyric acid (GABA)-evoked chloride currents at the GABA type A (GABAA) receptor. Recent work has revealed that two specific amino acid residues within transmembrane domain (TM)2 and TM3 are necessary for positive modulation of GABAA and glycine receptors by the volatile anesthetic enflurane. We now report that mutation of these residues within either GABAA α2 (S270 or A291) or β1 (S265 or M286) subunits resulted in receptors that retain normal or near-normal gating by GABA but are insensitive to clinically relevant concentrations of another inhaled anesthetic, isoflurane. To determine whether receptor modulation by intravenous general anesthetics also was affected by these point mutations, we examined the effects of propofol, etomidate, the barbiturate methohexital, and the steroid alphaxalone on wild-type and mutant GABAA receptors expressed in human embryonic kidney 293 cells. In most cases, these mutations had little or no effect on the actions of these intravenous anesthetics. However, a point mutation in the β1 subunit (M286W) abolished potentiation of GABA by propofol but did not alter direct activation of the receptor by high concentrations of propofol. These data indicate that the receptor structural requirements for positive modulation by volatile and intravenous general anesthetics may be quite distinct.

The GABAA receptor is modulated positively by a wide variety of structurally diverse general anesthetics (Harris et al., 1995; Whiting et al., 1995). In particular, halogenated ethers such as enflurane and isoflurane (Nakahiro et al., 1989; Wakamori et al., 1991; Jones et al., 1992) along with intravenous anesthetic agents such as the barbiturates (Barker and Ransom, 1978), propofol (Hales and Lambert, 1991), etomidate (Uchida et al., 1995), and steroid anesthetics (Peters et al., 1988) all enhance the function of the GABAA receptor at clinically relevant concentrations. In addition, intravenous and volatile anesthetics can activate the GABAA receptor directly in the absence of GABA (Barker and Ransom, 1978; Robertson, 1989; Hales and Lambert, 1991; Yang et al., 1992).

The GABAA receptor is a heteromeric complex formed by different glycoprotein subunits (α1–6, β1–4, γ1–4, δ, and ε) that coassemble to form a chloride channel (Whitinget al., 1995). Most GABAA receptorsin vivo consist of pentameric complexes of α, β, and γ subunits with a stoichiometry of ααββγ (Chang et al., 1996), although receptors lacking the γ subunit can be expressed in vitro and are fully sensitive to general anesthetics (Pritchett et al., 1989; Jones et al., 1995).

GABAA receptors are members of a ligand-gated ion channel superfamily that also includes the glycine, serotonin3, GABA ρ (GABAC), and nicotinic acetylcholine receptors (Ortells and Lunt, 1995). GABAA receptors share significant amino acid sequence homology with these receptors (Ortells and Lunt, 1995). Glycine receptor function is modulated positively by clinical concentrations of volatile anesthetics (Harrison et al., 1993; Downie et al., 1996) but is affected only weakly by barbiturates (Koltchine et al., 1996; Masciaet al., 1996) and etomidate (Mascia et al., 1996). In addition, some general anesthetics have potent actions on neuronal (but not muscle) nicotinic acetylcholine receptors (Floodet al., 1997; Violet et al., 1997) or serotonin3 receptors (Jenkins et al., 1996). In contrast to other members of the ligand-gated channel superfamily, GABA ρ receptors are insensitive to nearly all anesthetic compounds, including propofol (Mihic and Harris, 1996), barbiturates (Shimada et al., 1992), volatile anesthetics (Harrison et al., 1993; Mihic and Harris, 1996), and steroid anesthetics (Mihic and Harris, 1996).

The dissimilar pharmacology of ρ receptors helped identify residues crucial for positive modulation by general anesthetics. Recently, Mihicet al. (1997) constructed glycine α1/GABA ρ1 receptor chimeras and characterized a 45-amino acid residue domain within the glycine α1 receptor that was necessary for positive modulation by enflurane. Mutagenesis within this domain led to the identification of specific amino acid residues in TM2 and TM3 that seem to be necessary for modulation or direct activation of GABAA and glycine receptors by enflurane.

The current study demonstrates that mutation of these specific residues in TM2 and TM3 also abolishes the enhancement of submaximal GABA-activated currents by isoflurane. Additional experiments were performed to test whether these specific mutations in TM2, TM3, or both also affected modulation and direct activation by intravenous general anesthetics.

Materials and Methods

Site-directed mutagenesis.

The S270I (i.e., serine at position 270 mutated to isoleucine), S270H, and A291W mutations of the human GABAA α2 subunit (Hadingham et al., 1993) and the M286W mutation of the GABAA β1 subunit (Hadingham et al., 1993) were introduced by the unique site elimination method (Deng and Nickoloff, 1992) with use of the USE kit (Pharmacia Biotech, Piscataway, NJ). The method uses a two-primer system in which one oligonucleotide primer encodes the desired mutation and the other alters a unique SspI restriction site on the pCIS2 plasmid to an MluI site. The mutagenic reaction mixture was digested with SspI restriction endonuclease to eliminate the parental template. Positive clones of transformed Escherichia coliDH5α (Pharmacia Biotech) then were screened for the appearance of theMluI site, and mutations were confirmed further by double-stranded sequencing (Sequenase 2.0; United States Biochemical, Cleveland, OH). Both MluI and SspI restriction enzymes were from New England Biolabs (Beverly, MA). The sequences and locations of the primers (Operon Technologies, Alameda, CA) used are α2(S270I): 5′-GACAACTCTAATCATCAGTGCTCGGAATTC-3′, corresponding to bases 879–908 of the α2 cDNA sequence; α2(S270H), 5′-GACAACTCTACACATCAGTGCTCGGAATTC-3′, corresponding to bases 879–908 of the α2 cDNA sequence; α2(A291W), 5′-CATGGACTGGTTTATTTGGGTTTGTTATGCATTTG-3′, corresponding to bases 936–970 of the α2 cDNA sequence; β1(M286); and 5′-GATTGATATTTATCTGTGGGGTTGCTTTGTG-3′, corresponding to bases 915–945 of the β1 cDNA sequence.

The S265I mutation in the β1 subunit was introduced with use of the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). The method uses the fact that DNA isolated from most strains ofE. coli is dam methylated and therefore susceptible to DpnI endonuclease digestion (target sequence, 5′-Gm6ATC-3′). Two oligonucleotide primers, containing the same desired mutations and complementary to each other, were extended during temperature cycling by means ofPfu DNA polymerase (Stratagene). The product was digested with DpnI (Stratagene) to eliminate the parental template and transformed into the XL-1 Blue strain of E.coli (Stratagene). Positive clones were confirmed again by double-stranded sequencing.

Cell culture and transfection.

Wild-type or mutant receptor cDNAs were expressed via the pCIS2 vector, which contains the strong promoter from cytomegalovirus and a polyadenylation sequence from Simian virus 40. HEK 293 cells (American Type Culture Collection, Rockville, MD) were cultured in Eagle’s minimal essential medium (Sigma Chemical, St. Louis, MO) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), l-glutamine (0.292 μg/ml; GIBCO BRL, Grand Island, NY), penicillin G sodium (100 units/ml; GIBCO BRL), and streptomycin sulfate (100 μg/ml; GIBCO BRL). For electrophysiological experiments, cells were plated onto glass coverslips coated with poly-d-lysine (Sigma). Each coverslip of cells was transfected individually according to the calcium phosphate precipitation technique (Pritchett et al., 1989; Harrison et al., 1993). There are reports of endogenous GABAA receptor subunit expression in HEK 293 cells (Ueno et al., 1996). Our own experience with HEK 293 cells to date does not concur with this finding. In fact, similar to other reports (Davies et al., 1997), we do not see significant GABA-induced currents in untransfected or sham-transfected HEK 293 cells or by transfection with either GABAA α2 or β1 subunit cDNAs alone (Koltchineet al., 1996).

Electrophysiology.

Electrophysiological recordings were performed at room temperature using the whole-cell patch-clamp technique as described previously (Harrison et al., 1993;Koltchine et al., 1996). The coverslips were transferred 24–72 hr after removal of the cDNA to a 70-ml chamber that was continuously perfused (2–3 ml/min) with extracellular medium containing 145 mm NaCl, 3 mm KCl, 1.5 mm CaCl2, 1 mmMgCl2, 5.5 mm d-glucose, and 10 mm HEPES, pH 7.4, osmolarity 320–330 mOsm. The intracellular solution contained 145 mm N-methyl-d-glucamine hydrochloride, 5 mm K2ATP, 5 mm HEPES/KOH, 2 mm MgCl2, 0.1 mmCaCl2, and 1.1 mm EGTA, pH 7.2, osmolarity 315 mOsm. Pipette-to-bath resistance was 4–6 MΩ. Cells were voltage-clamped at −60 mV. Because the intracellular and extracellular solutions contained equal concentrations of chloride, the chloride reversal potential was ≈0 mV.

GABA and anesthetics were rapidly (<50-msec exchange time) applied to the cell by local perfusion (Koltchine et al., 1996) using a motor-driven solution-exchange device (Bio Logic Rapid Solution Changer RSC-100; Molecular Kinetics, Pullman, WA). Laminar flow was maintained by applying all solutions at identical flow rates via a multichannel infusion pump (Stoelting, Wood Dale, IL). The solution changer was driven by protocols in the acquisition program pCLAMP5 (Axon Instruments, Foster City, CA). Responses were low-pass-filtered at 5 kHz, digitized (TL-1–125 interface; Axon Instruments) using pCLAMP5, and stored for off-line analysis.

Data analysis.

Modulator-induced potentiation of an agonist-induced current was defined as the percentage increase of the peak control agonist response (0% indicates no difference from control response). Concentration-response data were fitted (KaleidaGraph, Reading, PA) with the logistic equation: I/Imax = 100 * [drug]n/([drug]n + (EC50 )n), where I/Imax is the percentage of the maximum obtainable agonist response, EC50 is the concentration producing a half-maximal response, and n is the Hill coefficient. The current elicited by direct activation of the receptor by an anesthetic compound was expressed relative to the maximal current that could be elicited by GABA. Any direct activation produced by a modulator during pre-equilibration was subtracted from the total current elicited by coapplication of GABA and modulator. The subtraction of direct anesthetic activation is appreciable only at high anesthetic concentrations (e.g., ≥10 μmpropofol or etomidate). In general, for the screening of potentiation, concentrations of anesthetics were chosen carefully (see Results) so little or no direct activation contributed to the observed current. Pooled data are presented throughout as mean ± standard error. Statistical significance was determined by Student’s two-tailed, unpaired t test.

Throughout this study, potentiation by the various general anesthetic agents was always assessed with test concentrations of GABA that correspond to EC20 value on the concentration-response curve for the particular receptor under study. In this fashion, the percentage potentiation produced by coapplication of a given modulator can be compared across different receptors and will not be influenced by potential shifts in GABA concentration-response curves among receptors. Across all the potentiation experiments for wild-type and mutant receptors reported here, the actual percentage of maximal GABA response for the test concentrations used were: α2β1 wild-type (19.7 ± 2.5%, 30 experiments), α2(S270I)β1 (22.3 ± 2.5%, 33 experiments), α2(S270H)β1 (22.2 ± 2.0%, 35 experiments), α2(A291W)β1 (20.5 ± 2.5%, 25 experiments), α2β1(S265I) (18.6 ± 2.6%, 53 experiments), and α2β1(M286W) (19.4 ± 2.6%, 36 experiments).

Drugs.

Stock solutions of GABA (Sigma) and anesthetic compounds were diluted into extracellular solution daily before use. The other drugs used in this study were propofol (2,6-diisopropylphenol; Aldrich, Milwaukee, WI), methohexital sodium (Brevital sodium; Eli Lilly and Co., Indianapolis, IN), alphaxalone, picrotoxin (both from Research Biochemicals, Natick, MA), and isoflurane (Forane; Ohmeda Caribe, Guayama, PR). Propofol, etomidate, picrotoxin, and alphaxalone were first prepared as stock solutions in dimethylsulfoxide (Sigma) before being dissolved in the extracellular medium. The maximum final concentration of dimethylsulfoxide was 0.05% (v/v), which was determined during carrier control experiments to have no significant effect on GABA-induced currents in the receptor constructs analyzed in this study. Preparation and measurement of isoflurane solutions have been described previously (Jones et al., 1992). The MAC equivalent for isoflurane at 25° used for this study was 0.5 mm (Jones et al., 1992). Research-grade etomidate was a generous gift from Prof. Alfred Doenicke (Institute of Anesthesiology, Ludwig Maximilians University of München, Germany).

Results

Expression and characteristics of wild-type and mutant receptors.

Six wild-type and mutant GABAAreceptors were expressed by transfection of HEK 293 cells: α2β1 wild-type, α2(S270I)β1, α2(S270H)β1, α2(A291W)β1, α2β1(S265I), or α2β1(M286W). After transient expression in HEK 293 cells, all of the wild-type and mutant receptors tested in this study produced inward currents in response to application of GABA via the rapid solution changer. The EC50 and Hill slope values estimated from GABA concentration-response curves for receptors that contain either mutant α2 or β1 subunits demonstrate that these receptors retain GABA concentration-response relationships that are similar to those of wild-type α2β1 receptors: α2β1 wild-type (EC50 = 8.7 ± 0.4 μm, nH = 1.9 ± 0.2, nine experiments), α2(S270I)β1 (EC50 = 14.6 ± 0.1 μm,nH = 2.4 ± 0.1, six experiments), α2(S270H)β1 (EC50 = 3.5 ± 0.2 μm, nH = 1.5 ± 0.1, five experiments), α2(A291W)β1 (EC50 = 2.4 ± 0.1 μm, nH = 1.7 ± 0.2, five experiments), α2β1(S265I) (EC50 = 37.5 ± 8.5 μm, nH = 1.2 ± 0.2, seven experiments), and α2β1(M286W) (EC50 = 8.7 ± 0.4 μm, nH = 0.8 ± 0.2, seven experiments).

The EC50 values for the mutant receptors do not differ by >4.3-fold from wild-type. The Hill slopes for the GABA concentration-response relationships for the α2β1(S265I) and α2β1(M286W) mutant receptors are significantly lower compared with wild-type α2β1 receptors (p < 0.001 and < 0.05, respectively). In addition, the maximal current amplitude of all mutant receptors in response to GABA did not differ by more than 1.6-fold from wild-type: α2β1 wild-type (699 ± 75 pA, 37 experiments), α2(S270I)β1 (437 ± 33 pA, 46 experiments), α2(S270H)β1 (606 ± 88 pA, 45 experiments), α2(A291W)β1 (1032 ± 157 pA, 34 experiments), α2β1(S265I) (694 ± 97 pA, 58 experiments), and α2β1(M286W) (557 ± 90 pA, 59 experiments).

An interesting property of mutations at the A291 position in TM3 (GABAA α2 numbering) is the propensity to form tonically open receptors. In particular, GABAAα2(A291W)β1(M286W) and glycine α1(A288W) mutant receptors seem to be tonically open in the absence of agonist. The application of picrotoxin results in “outward” currents, consistent with closure of open channels (Mihic et al., 1997). For the GABAA α2(A291W)β1(M286W) mutant receptor, picrotoxin (1–100 μm) produces outward currents whose maximal amplitude was 38.5 ± 6.5% (six experiments) of the amplitude of the maximal inward current produced by GABA. Neither the α2(A291W)β1 nor the α2β1(M286W) mutant receptors seem to be tonically active because application of picrotoxin at concentrations up to 100 μm resulted in no deflection of the base-line current (four experiments for each). Consistent with the approach taken in earlier work (Mihic et al., 1997), we did not attempt anesthetic modulation or direct activation experiments on tonically open channels such as the GABAAα2(A291W)β1(M286W) mutant receptor.

Mutations in TM2 and TM3 of the GABAA α2 and β1 subunits abolish positive modulation by isoflurane.

At the clinically relevant concentrations of 0.25 and 0.5 mm (0.5 and 1.0 MAC equivalents, respectively), isoflurane strongly enhanced responses to an EC20 concentration of GABA in wild-type α2β1 receptors (Fig. 1A). In contrast, coapplication of isoflurane up to 1.0 mm (2 MAC) produced no enhancement of submaximal GABA currents at mutant α2(S270H)β1 or α2(A291W)β1 receptors (Fig. 1, B and C). In fact, all five mutant receptors studied in this report were insensitive to isoflurane concentrations up to 1.0 mm (Table1). To further examine the mechanism by which these mutations altered potentiation by isoflurane, we examined whether the addition of a γ2s subunit to one of the mutant receptors would lead to any potentiation by isoflurane. Submaximal GABA currents at the α2(A291W)β1γ2s mutant receptor, however, also were insensitive to potentiation by isoflurane: potentiation of EC20 GABA currents by 0.5 and 1.0 mmisoflurane was −3.8 ± 2.7% and −8.3 ± 4.7%, respectively (five experiments for both concentrations; negative values indicate slight inhibition).

Figure 1
Figure 1

Mutations within TM2 and TM3 abolish positive modulation by isoflurane. A, Submaximal GABA currents in wild-type GABAA α2β1 receptors are strongly enhanced by coapplication of clinically relevant concentrations of isoflurane (0.25 and 0.5 mm). B and C, In contrast, submaximal GABA currents in α2(S270H)β1 or α2(A291W)β1 mutant receptors are not enhanced by coapplication of isoflurane concentrations up to 1 mm. Individual recordings are from HEK 293 cells transfected with cDNAs encoding the indicated subunit combination.

View this table:
Table 1

Potentiation of wild-type and mutant receptors by anesthetics

Propofol potentiation of GABA.

A propofol concentration of 1 μm was chosen to assay GABA potentiation because it is within the estimated clinically relevant concentration range (Franks and Lieb, 1994) and produces no direct activation in most of the receptors tested (see below). Mutations in the α2 subunit had little or no effect on GABA potentiation by 1 μm propofol (Fig.2, Table 1). A point mutation in TM3 of the β1 subunit (M286W), however, eliminated GABA potentiation by 1 μm propofol (Table 1). In fact, submaximal GABA currents at the α2β1(M286W) mutant receptor were not enhanced by propofol at concentrations up to 10 μm (Fig.3). The addition of a γ2s subunit did not alter the effect of this β1 mutation because submaximal currents at the α2β1(M286W)γ2s mutant receptor also were insensitive to potentiation by propofol at concentrations up to 10 μm (Fig. 3B).

Figure 2
Figure 2

Mutations in the GABAA α2 subunit that abolish potentiation by isoflurane do not alter potentiation by propofol (PRO), etomidate (ETO), and methohexital (MTX). Submaximal GABA currents in wild-type GABAA α2β1 receptors are strongly enhanced by anesthetic concentrations of propofol (1 μm), etomidate (10 μm), and methohexital (5 μm). Similarly, submaximal GABA currents at α2(S270I)β1 or α2(S270H)β1 mutant receptors are enhanced by propofol, etomidate, and methohexital. Individual recordings are from HEK 293 cells transfected with cDNAs encoding the indicated subunit combinations.

Figure 3
Figure 3

Potentiation by both propofol and isoflurane is abolished in the α2β1(M286W) mutant receptor. A, Submaximal GABA currents in the α2β1(M286W) receptor are not enhanced by coapplication of 1.0 mm isoflurane (ISO) or supra-anesthetic propofol (PRO) concentrations (5 and 10 μm). Recording is from a single HEK 293 cell transfected with cDNAs encoding the α2β1(M286W) receptor combination. B, Concentration-response relationships for potentiation of GABA by propofol. For wild-type (WT) GABAA α2β1 receptors, significant potentiation of an EC20 test concentration of GABA occurs at all concentrations of ≥0.2 μm (p < 0.05 for each concentration ≥0.2 μm; 5–12 experiments for each data point). From the curve fit, the EC50 value for potentiation of wild-type α2β1 receptors by propofol is 1.6 μmwith a Hill slope of 1.1. In contrast, propofol (≤10 μm) does not potentiate submaximal GABA currents at α2β1(M286W) (•, 6–9 experiments) or α2β1(M286W)γ2s (▪, 7 experiments) mutant receptors.

Etomidate potentiation of GABA.

Etomidate (1–20 μm) potentiated submaximal GABA currents at wild-type α2β1 receptors with an estimated EC50 value of 3.4 μm, Hill slope of 1.6, and predicted maximal potentiation (Emax) of 216% (six or seven experiments for all concentrations). Etomidate (10 μm) significantly potentiated submaximal GABA currents in all wild-type and mutant receptors tested (Table 1). The magnitude of potentiation by etomidate was markedly reduced in the α2(A291W)β1 mutant receptor, whereas all other mutant receptors retained normal potentiation by 10 μm etomidate (Table 1).

Methohexital potentiation of GABA.

Previous work from our laboratory has shown that the barbiturate methohexital induces GABA potentiation, direct activation, and blocking effects on wild-type GABAA α2β1 receptors (Koltchine et al., 1996). A concentration of 5 μm methohexital was used for the potentiation experiments because it produces substantial GABA potentiation without any direct activation of GABAA α2β1 receptors (Koltchine et al., 1996). Methohexital potentiated the action of GABA at all receptors, although two receptors harboring mutations in TM3, α2(A291W)β1 and α2β1(M286W), showed significantly lesser degrees of potentiation by 5 μm methohexital (Table 1).

Alphaxalone potentiation of GABA.

None of the mutant receptors studied showed altered potentiation of submaximal GABA currents by 1 μm alphaxalone relative to wild-type α2β1 receptors (Table 1).

Effects of mutations in TM2 and TM3 on direct receptor activation by intravenous and volatile anesthetics.

Direct activation by isoflurane was not studied in detail because isoflurane produces only minimal direct activation (5–10% of maximal GABA currents) in wild-type GABAA α2β1 receptors. The direct activation of wild-type α2β1 receptors by isoflurane (Fig. 1A) was not evident in mutant receptors (Fig. 1, B and C). In fact, concentrations of isoflurane up to 5 mm (10 MAC) failed to elicit any direct activation of the α2(A291W)β1 mutant receptor (data not shown).

Direct GABAA receptor activation by propofol.

Direct activation of wild-type GABAA α2β1 receptors by propofol also was described previously (Jones et al., 1995). Concentrations of propofol of >50 μmcan produce a profound block of the effect of GABA responses (Hales and Lambert, 1991), which is often followed by a “rebound” or “surge” current during washout. In our experiments, blocking and rebound effects were most evident at propofol concentrations of ≥100 μm, although small rebound currents immediately after washout of applied propofol were occasionally evident at 50 μm (Fig. 4). A propofol concentration of 50 μm thus was suitable to compare near-maximal direct activation by propofol in different receptors, with minimal interference from block.

Figure 4
Figure 4

Direct receptor activation by supra-anesthetic concentrations of propofol (PRO), etomidate (ETO), and methohexital (MTX) is similar to that for wild-type GABAA α2β1 receptors in most mutant receptors, except for α2β1(S265I), in which direct activation by propofol is reduced markedly. Wild-type GABAAα2β1 receptors are activated directly by propofol (50 μm), etomidate (50 μm), and methohexital (200 μm). Propofol, etomidate, and methohexital also directly activate α2(S270I)β1, α2β1(S265I), and α2β1(M286W) mutant receptors. Direct activation by propofol is reduced markedly in α2β1(S265I) but not α2(S270I)β1 receptors. Horizontal scale bar, 10 sec for direct activation by propofol, etomidate, and methohexital and 2 sec for the maximal GABA applications. The “rebound” currents at the tail end of drug applications are evident in some of the propofol and methohexital traces. Individual recordings are from HEK 293 cells transfected with cDNAs encoding the indicated subunit combinations.

Mutations in the α2 subunit had little effect on direct activation by 50 μm propofol (Fig. 4; Table2). In fact, during the course of the potentiation experiments, it was noted that the receptor mutant α2(A291W)β1 exhibited noticeable direct activation by 1 μm propofol, a situation not seen in the wild-type α2β1 receptors. Further examination of the direct activation by propofol revealed that the α2(A291W)β1 mutant receptor has >10-fold higher apparent affinity for propofol than the wild-type α2β1 receptor, as shown by the leftward shift in the concentration-response relationship for direct activation by propofol (Fig. 5A). The GABA concentration-response curve for the α2(A291W)β1 receptor is shifted to the left in a similar manner.

View this table:
Table 2

Intravenous anesthetic direct activation data for wild-type and mutant receptors

Figure 5
Figure 5

Concentration-response relationships for direct activation of GABAA α2β1 receptors by propofol and etomidate. A, For wild-type (WT) α2β1 receptors (○), propofol produces significant direct activation at all concentrations of ≥5 μm (p < 0.05 for each concentration ≥5 μm, 4–8 experiments). The direct activation of wild-type α2β1 receptors by propofol has an EC50 value of 10.5 μm, Hill slope of 2.6, and Emax value of 52.0% of the maximal GABA current. In contrast, the α2(A291W)β1 receptor is more sensitive than the wild-type α2β1 receptor to the direct actions of propofol (•). Propofol produces significant direct activation at all concentrations of ≥0.5 μm at the α2(A291W)β1 receptor (p < 0.01 for each concentration ≥0.5 μm, 4–13 experiments). The direct activation of α2(A291W)β1 receptors by propofol has an EC50 value of 1.0 μm, Hill slope of 1.2, and Emax value of 39.8%. The α2β1(M286W) mutant receptor, although insensitive to potentiation by propofol, is still directly gated by propofol (▪) at all concentrations of ≥2 μm (p< 0.05 for each concentration ≥ 2 μm, 6–15 experiments). The direct activation of α2β1(M286W) receptors by propofol has an EC50 value of 9.5 μm, Hill slope of 2.2, and Emax value of 39.5%. B, For wild-type (WT) α2β1 receptors (○), etomidate produces significant direct activation at all concentrations of ≥5 μm (p < 0.05 for each concentration ≥5 μm, 4–7 experiments). The direct activation of wild-type α2β1 receptors by etomidate has an EC50 value of 10.7 μm, Hill slope of 1.9, and Emax value of 47.5%. Similar to propofol, the α2(A291W)β1 receptor is more sensitive to the direct actions of etomidate (•). Etomidate produces significant direct activation at all concentrations of ≥0.5 μm at the α2(A291W)β1 receptor (p < 0.05 for each concentration ≥0.5 μm, 7–10 experiments). The direct activation of α2(A291W)β1 receptors by etomidate has an EC50 value of 1.9 μm, Hill slope of 1.2, and Emax value of 31.4%. The α2β1(M286W) mutant receptor is directly gated by etomidate (▪) at all concentrations of ≥10 μm(p < 0.01 for each concentration ≥10 μm, 7–19 experiments). The direct activation of α2β1(M286W) receptors by etomidate has an EC50 value of 15.1 μm, Hill slope of 2.5, and Emax value of 23.5%. Error bars, standard error.

Interestingly, although submaximal GABA currents at the α2β1(M286W) mutant receptor are not potentiated by propofol (see above), the direct activation by propofol of this mutant receptor was not different from its effect on wild-type receptors (Fig. 5A; Table 2). Propofol (50 μm) still activated the α2β1(S265I) mutant receptor directly but elicited significantly smaller maximal currents compared with wild-type α2β1 receptors (p < 0.001 compared with wild-type; Fig. 4, Table 2).

Direct GABAA receptor activation by etomidate.

It has been reported in experiments with Xenopus laevis oocytes that the direct action of etomidate is nearly absent or that etomidate has substantially reduced efficacy and/or apparent affinity in β1 compared with β2 or β3 subunit-containing receptors (Hill-Venning et al., 1997; Sanna et al., 1997). In our experiments using HEK 293 cells, etomidate produced significant direct effects in wild-type α2β1 receptors in the absence of GABA (Figs. 4 and 5B, Table 2). The direct activation by etomidate at wild-type α2β1 receptors had an EC50 value of 10.7 μm, a much higher apparent affinity than seen in experiments with β1-containing subunits in X. laevisoocytes (Hill-Venning et al., 1997; Sanna et al., 1997).

As with propofol, etomidate has an increased apparent affinity in the α2(A291W)β1 mutant receptor, as indicated by a leftward shift in the etomidate direct activation concentration-response curve (Fig. 5B). Direct activation by 50 μm etomidate of the α2β1(S265I) mutant receptor also was reduced slightly compared with the wild-type α2β1 receptor (Table 2). Although high concentrations of etomidate can produce channel block and rebound currents at GABAA receptors (Robertson, 1989), these were not seen during our experiments at etomidate concentrations up to 100 μm.

Direct GABAA receptor activation by methohexital.

At high concentrations, methohexital produces substantial direct activation of GABAA α2β1 receptors (Koltchineet al., 1996); 200 μm methohexital was chosen for the direct activation experiments because at this concentration, methohexital elicits a large direct effect with minimal blocking effects. Direct activation of all the mutant receptors by methohexital was similar to that for the wild-type α2β1 receptor (Fig. 4, Table2). Direct activation by alphaxalone was not studied because it is not prominent in GABAA α2β1 receptors (C. E. Rick, unpublished observations).

Discussion

Actions of general anesthetics on wild-type GABAAα2β1 receptors.

Submaximal GABA currents at wild-type human GABAA α2β1 receptors were potentiated by all the volatile and intravenous anesthetics studied. Thus, in agreement with other published studies, the presence of the γ subunit is not required for potentiation by propofol (Jones et al., 1995), methohexital (Koltchine et al., 1996), etomidate (Uchida et al., 1995; Sanna et al., 1997), alphaxalone (Horne et al., 1993), or isoflurane (Harrisonet al., 1993; Mihic et al., 1994).

A mutation in TM3 of the α2 subunit alters the concentration-response relationship for GABA and for direct receptor activation by propofol and etomidate.

Although the GABA concentration-effect relationships for the mutants analyzed in this study mostly were very similar to that in the α2β1 wild-type receptor, a few changes were evident. A receptor with a mutation in TM3 of the α2 subunit, α2(A291W)β1, has a higher apparent affinity for GABA than the wild-type α2β1 receptor, as indicated by a leftward shift in the GABA concentration-response curve. In addition, the concentration-response curves for direct activation by etomidate and propofol were shifted to the left of those for the wild-type α2β1 receptor by ≈6- and ≈10-fold, respectively. Thus, for the α2(A291W)β1 mutant receptor, the apparent affinities for activation by GABA, etomidate, and propofol are all increased relative to wild-type. Also, the receptors containing mutations in the β1, but not α2, subunit had significantly lower Hill slopes compared with the wild-type α2β1 receptor for their GABA concentration-response relationships. These changes suggest altered gating mechanisms in these mutant receptors.

The higher apparent affinity of GABA, etomidate, and propofol for the α2(A291W)β1 mutant receptor may be related to the observation that mutations at the 291 position in TM3 (GABAA α2 numbering) have the propensity for producing tonically open GABAA or glycine receptors. This, however, is observed only in the glycine α1(A288W) and GABAA α2(A291W)β1(M286W) mutant receptors (Mihic et al., 1997). In other words, tonically active receptors are produced only when all five subunits of the presumed pentameric receptor contain the mutation to tryptophan in TM3.

A mutation in TM3 of the β1 subunit abolishes potentiation of GABA but not direct receptor activation by propofol.

For the most part, the mutations analyzed in this study had little effect on GABA potentiation and/or direct activation by the intravenous anesthetics propofol, etomidate, methohexital, or alphaxalone. An exception is the α2β1(M286W) mutant receptor, at which propofol concentrations of ≤10 μm failed to enhance submaximal GABA currents. The addition of a γ2s subunit did not alter the deleterious effect of the β1(M286W) mutation because the α2β1(M286W)γ2s mutant receptor also was insensitive to propofol potentiation of GABA. In contrast to the lack of potentiation, propofol still directly activates this mutant receptor, with a concentration-response relationship that overlaps that for the wild-type α2β1 receptors.

Although the relationship between agonist potentiation and direct activation by intravenous anesthetics at the GABAA receptor is poorly understood, there are examples of subunit combinations in which one effect is present without the other. For example, receptors containing the GABAA α4 subunit show agonist potentiation but not direct receptor activation by propofol and pentobarbital (Waffordet al., 1996); a similar situation is seen withDrosophila GABA receptors (Belelli et al., 1996). In the current study, a receptor with a mutation in TM2 of the β1 subunit, α2β1(S265I), shows markedly reduced direct activation by propofol but retains normal GABA potentiation. These results suggest distinct requirements for potentiation and direct activation by intravenous general anesthetics. Also, in conjunction with the results of previous studies (Sanna et al., 1995a, 1995b), our results further implicate the β subunit as the major determinant influencing propofol actions at the GABAAreceptor.

Recent work has demonstrated that mutation of N289 within the GABAA β3 subunit (homologous with S265 in the β1 subunit) to methionine abolishes direct activation and GABA potentiation by etomidate (Belelli et al., 1997). Although none of the TM2 mutants analyzed in this study significantly reduced GABA potentiation by etomidate, direct activation of the α2β1(S265I) and α2β1(M286W) mutant receptors was reduced significantly compared with wild-type. It will be interesting to determine whether other residues within or near TM2 of the β subunit also alter the modulatory effects or direct activation by etomidate.

Residues within TM2 and TM3 of the GABAA receptor may form part of a volatile ether anesthetic binding site.

The results from this study demonstrate that specific residues within both TM2 and TM3 of GABAA α2 and β1 subunits are necessary for positive receptor modulation by the halogenated methyl ethyl ether anesthetic isoflurane. This extends the findings from a previous study that these residues also are critical for positive modulation by the related volatile anesthetic enflurane (Mihic et al., 1997). These same specific residues within TM2 and TM3 seem to be critical for modulation by n-alkanols (Mihic et al., 1997). In this study, mutations in either the α2 or β1 subunits in TM2 or TM3 abolished modulation by isoflurane. Similar to the situation seen with propofol potentiation of GABA at the α2β1(M286W)γ2s mutant receptor, the α2(A291W)β1γ2s mutant receptor was as insensitive to isoflurane potentiation of GABA as the α2(A291W)β1 mutant receptor. This is consistent with the hypothesis that the γ subunit is not a major determinant of volatile anesthetic modulation.

The mutations analyzed in the current study all result from the replacement of a smaller amino acid by a larger amino acid (e.g., serine to isoleucine or histidine, alanine or methionine to tryptophan). We hypothesize that the serine in TM2 (for α2 or β1 subunits) and alanine (α2 subunit) or methionine (β1 subunit) in TM3 forms part of a binding site for enflurane, isoflurane, andn-alkanols. If residues within TM2 and TM3 of the GABAA α and β subunits do indeed form part of a volatile anesthetic binding pocket, then substitution of larger amino acid residues at those positions (e.g., those found at the corresponding positions in the GABA ρ1 receptor) may sterically hinder volatile anesthetic binding and thereby ablate positive modulation of receptor function.

In summary, the results of this study indicate that the receptor structural requirements for volatile and intravenous general anesthetic modulation of the GABAA receptor may be quite distinct. Future work with chimeric and additional mutant receptor subunits will attempt to uncover whether TM3 in the β subunit contains a binding site for propofol or whether mutations such as β1(M286W) instead interfere with transduction step or steps necessary for agonist potentiation by propofol.

Acknowledgments

We are thankful to Dr. S. J. Mihic for helpful suggestions on this manuscript and for sharing unpublished data. We also thank Dr. D. S. McGehee for careful reading of the manuscript and A. Kung, L. Brady, and M. Ruan for technical assistance.

Footnotes

    • Received August 8, 1997.
    • Accepted November 11, 1997.

  • Send reprint requests to: Matthew D. Krasowski, Whitman Laboratory, University of Chicago, 915 East 57th Street, Room 202, Chicago, IL 60637. E-mail:kra3{at}harper.uchicago.edu

  • This work was supported by National Institutes of Health Grants GM45129, GM00623, and GM56850 (N.L.H.) and training grants from National Institute of Mental Health (M.D.K.) and National Institute on Drug Abuse (V.V.K.).

Abbreviations

GABAA
γ-aminobutyric acid type A
EGTA
ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HEK
human embryonic kidney
TM
transmembrane domain
MAC
minimum alveolar concentration

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Propofol and Other Intravenous Anesthetics Have Sites of Action on the γ-Aminobutyric Acid Type A Receptor Distinct from That for Isoflurane

Matthew D. Krasowski, Vladimir V. Koltchine, Caroline E. Rick, Qing Ye, Suzanne E. Finn and Neil L. Harrison
Molecular Pharmacology March 1, 1998, 53 (3) 530-538; DOI: https://doi.org/10.1124/mol.53.3.530
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