Mapping General Anesthetic Sites in Heteromeric Gamma-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes

Abstract

Intravenous general anesthetics including propofol, etomidate, alphaxalone, and barbiturates, produce important actions by enhancing gamma-aminobutyric acid type A (GABA-A) receptor activation. Here we review scientific studies that have located and mapped IV anesthetic sites using photoaffinity labeling and substituted cysteine modification-protection. These anesthetics bind in transmembrane pockets between subunits of typical synaptic GABA-A receptors, and drugs that display stereoselectivity also show remarkably selective interactions with distinct interfacial sites. These results suggest strategies for developing new drugs that selectively modulate distinct GABA-A receptor subtypes.

Introduction

GABA_ARs are members of the pentameric ligand-gated ion channel (pLGIC) superfamily, the main inhibitory neurotransmitter receptors in the central nervous system, and major targets of many, but not all, general anesthetics (1,2). The GABA_AR is assembled from five homologous subunits arranged pseudo-symmetrically around a central transmembrane chloride-conducting pore (Figure 1). Each subunit has an extracellular domain (ECD) with over 200 amino acids, a transmembrane domain with four membrane-spanning α-helices (M1 to M4), and a variable-size intracellular loop between the M3 and M4 helices (3). The physiological roles, pharmacological properties and distribution of GABA_ARs vary with the subunit composition. Most native GABA_ARs contain two α and two β subunits with the fifth subunit being another β, a γ, or a δ. Synaptic GABA_ARs mostly contain γ2-subunits, although these subunits are also found extrasynaptically. The δ-subunit is exclusively extrasynaptic (4,5). No high resolution crystal structures of heteromeric GABA_ARs are available. Homology models are based on crystal structures of related homomeric pLGICs from bacteria, nematodes, and human β3 GABA_ARs (6–8).

Figure 1. The locations of modulatory sites in a structural model of α1β3γ2L GABA_ARs.

The structure shown was obtained by homology modeling based on the GluCl chloride channel (3RHW.pdb) (7,26). The five subunits are arranged around a central ion–conducting pore and colored as follows: α1, yellow; β3, red, and γ2 green. The α-helices are represented as cylinders, β-sheets as flat planks and loops as strings. A. A side view of the α1β3γ2L GABA_AR homology model shows the extracellular domain (ECD) and the transmembrane domain (TMD). The intracellular domain is not modeled because no structural information is available. B. The extracellular domain viewed from the synapse, showing residues involved in agonist and BDZ binding. By convention, subunit order is counted in an anticlockwise direction (arrow). Important amino acids associated with various sites are distinguished by the color of their carbon atoms: agonist site, dark green (α1 Phe-65; β3 Tyr-157 & −205, Phe-200); benzodiazepine site (BDZ), orange (α1 His-102 & −210); azietomidate site, cyan (β3 Met-286 & Val-290, α1 Met-236); main R–mTFD-MPAB site, goldenrod (γ2 Ser-301, β3 Met-227). Red and blue atoms are oxygen and nitrogen respectively.

GABA_AR gating between resting (closed) and ion-conducting (open) states involves a widespread conformation change coupling GABA binding at two ECD agonist sites between subunits (both β⁺ – α⁻ interfaces) and the transmembrane ion pore about 50 Å away (Figure 1). Volatile anesthetics, propofols, etomidates, barbiturates, steroids and alcohols all similarly enhance GABA_AR–mediated currents, suggesting that GABA_ARs are major contributors to the anesthetic state (9–11). Convincing in vivo evidence that some high affinity general anesthetics act through GABA_ARs comes from studies of knock-in mice bearing a single amino acid mutation at position 265 in the GABA_AR β3 subunit (β3N265M in the M2 helix) (12). This substitution reduces GABA_AR sensitivity to propofol and etomidate in vitro (13) and β3N265M mice exhibit greatly reduced sensitivity to the anesthetic effects of etomidate, propofol, and pentobarbital (12,14), but not volatile or steroid anesthetics.

Anesthetics potentiate GABA_AR gating in the presence of GABA by binding with much higher affinity to the open state than to the resting state, increasing the fraction of open channels and the duration of channel openings (15). In addition, high concentrations of anesthetics directly activate (agonize) GABA_ARs. Evidence suggests that both of these anesthetic actions are mediated by the same mechanism and the same allosteric sites (16). In essence, resting-state receptors have very low open probability and by binding with much higher affinity to the open state than to the resting state, high anesthetic concentrations induce a modest fraction of receptors to activate (allosteric agonism). When receptors are already partially activated by an orthosteric agonist such as GABA, much lower concentrations of anesthetics shift many more receptors toward opening (co-agonism). Quantitative allosteric co-agonist models can account for both modulation and agonism of synaptic GABA_ARs by etomidate and propofol (16,17). For anesthetics to exhibit different affinities for distinct functional receptor states, their binding sites must change shape during the state transition (18). This review summarizes progress toward our long-term goal to understand both where anesthetics interact with GABA_ARs and the conformational changes underlying their state-selective binding.

Overall Strategy for Mapping Functional General Anesthetic Sites

The overall strategy of our research program is 1) to identify GABA_AR binding sites in an unbiased (hypothesis-free) manner using photolabeling with analogs of high-affinity anesthetics, and 2) to investigate the functional roles of specific amino acids at or near photolabeled sites using mutational, biochemical, and functional methods. These data are interpreted using both GABA_AR structural homology models and the functional principles of allosterism briefly described above.

Photolabeling Strategy

Covalent photo-modification of target proteins overcomes weak affinity and transient binding site occupation by general anesthetics (19).

Our goal of identifying anesthetic binding sites in GABA_ARs has required a number of innovative strategic developments for each of the following steps:

1. Design, synthesize and pharmacologically characterize photo-reactive analogs of potent general anesthetics known to modulate GABA_ARs. Using multiple photolabel derivatives of the same parent drug reduces the likelihood of misinterpreting results.

2. Develop cell lines that express affinity-tagged GABA_ARs of physiologically significant subunit compositions, in quantities suitable for biochemical characterization, and Edman amino acid sequencing.

3. Develop methods to affinity-purify and functionally reconstitute expressed GABA_ARs while maintaining allosteric linkages to GABA and anesthetic sites.

4. Radiolabel (tritiate) the best photolabels, photolyze them while bound to GABA_ARs, purify radiolabeled peptides, and apply Edman sequencing to identify photo-adducted amino acids.

5. Assess the functional significance of photolabeled sites, using both photochemical and other techniques. Based on allosteric principles, GABA is expected to enhance anesthetic photo-incorporation. Parent drugs and those with overlapping sites are expected to competitively reduce rates of photolabel incorporation. Non-photochemical structure-function techniques include testing whether mutations at the photolabeled site alter sensitivity to parent drug, and SCAMP (see below).

Edman versus mass spectroscopy for photo-adduct location

Edman degradation is a biochemical method that sequentially cleaves one N-terminal amino acid at a time from a polypeptide. The free modified amino acid is then isolated and analyzed for the presence of radioactive photo-adducts or identification of its sidechain. We and others (related review by Woll et al. (20) in this issue of the journal) have also used mass spectroscopy (MS) to identify photo-adducted peptides and amino acids in molecular targets where anesthetics bind. MS precisely determines the mass:charge ratio of molecules moving through an electric field, and when coupled with methods for further fragmentation of peptides isolated in an ion trap (MS/MS), the amino acid sequence and the locations of photo-adducts can be determined.

MS requires far less protein than Edman degradation and target protein purification is also unnecessary. MS requires no radioactivity, although isotopic tracing (e.g. with deuterium) can improve adduct identification (21). For small soluble target proteins such as myokinase (MW ≈ 21 kDa) labeled at high efficiency, the precision of MS enables both identification of photo-incorporation sites, and the stoichiometry of sites (22). On the other hand, because MS depends on ionization of protein fragments, it is less successful in hydrophobic protein regions such as the transmembrane helices where anesthetics bind to GABA_ARs. Anesthetic adduction typically increases the hydrophobicity of peptides, exacerbating this problem. Another weakness of MS is that quantifying and comparing photo-adduction efficiency is very difficult (23). Methods used to fragment peptides during MS analysis can also degrade photo-adducts, complicating adduct identification.

In contrast to MS, a major advantage of combining tritiated photolabels with Edman sequencing is the ability to track and quantify radioactivity and normalize it to protein at every analytical step. Thus, photo-adduction levels (e.g. in cpm/pmol of protein) can be quantitatively compared in different receptor conformations (e.g. resting vs. GABA-bound). Competition with anesthetics can also be assessed under conditions reflecting initial photolabel incorporation rate. These advantages have led us to favor Edman sequencing for much of our photolabel research. Disadvantages of Edman sequencing include the additional synthetic chemistry required to produce stable high-level radiolabeling. Edman sequencing also needs large quantities of purified homogeneous GABA_ARs, achieved only recently with our stable inducible cell lines (24). Receptor yields from these cell lines are sufficient for identifying photo-adducted GABA_AR amino acids in the resting and desensitized states (25,26), and for spectroscopic studies (27). Freeze-clamp photolabeling of GABA_ARs in the transient open state will require still more protein (28)

Choosing parent anesthetics

High affinity for targets is a key feature of successful anesthetic photolabels. Generally, drugs acting at concentrations above 50 µM show little target or site specificity, and their potency is predicted by hydrophobicity alone (29). For example, anesthetic potencies of methanol through 1-octanol are predicted by their octanol/water partition coefficients (30) (Figure 2). Clinical volatile anesthetics all act at free plasma concentrations above 100 µM, with potencies paralleling hydrophobicities, and can interact at many binding sites large enough to accommodate them (31,32). In contrast, for anesthetics with EC50s below 50 µM the correlation with hydrophobicity breaks down and significant stereoselectivity emerges (yellow zone, Figure 2). For further illustration, the volatile anesthetic bromoform (IC₅₀ = 125 µM) occupies eleven sites in a crystal structure of a bacterial pLGIC, (33) (Figure 3A), whereas ketamine (IC₅₀ = 58 µM) interacts stereoselectively with a single class of intrasubunit sites in another pLGIC (34) (Figure 3B). On this basis, we have focused on developing photolabels that act below 10 µM and, wherever possible, exhibit stereoselectivity, including derivatives of etomidate, propofol, barbiturates, and alphaxalone.

Figure 2. The importance of potency in governing selective binding to general anesthetic sites.

The correlation between EC50 for loss of righting reflexes (LoRR) in tadpoles and octanol/water partition coefficient holds well for anesthetics with EC50s above ~50 µM (Pearson correlation coefficient = −0.987; P < 0.0001). More potent agents display weak dependence on partition coefficient (Pearson correlation coefficient = −0.209; P = 0.49; yellow box). Some of the most successful photolabels lie in this group and are more potent than predicted by hydrophobicity (points with light blue fill). Data sources are given in (46).

Figure 3. Selectivity of general anesthetic binding to crystallized homomeric cation–conducting pLGICs is dependent on potency.

A. Bromoform (brown), a low potency drug, occupies eleven sites in three different classes on ELIC (33): (1) 5 homologous intrasubunit sites in the extracellular domain (ECD); (2) 5 homologous sites in the lipid-protein interface of the transmembrane domain, and (3) a single site in the channel lumen at the interface of all five subunits. B. Ketamine (carbons grey, nitrogen blue, chlorine green) occupies a single class of 5 intersubunit sites on GLIC (34), which potently and stereoselectively inhibits. Channel subunits are shown as ribbons and are colored arbitrarily. The anesthetics are shown space filled and colored conventionally.

Pharmacological characterization of anesthetic photolabels

Table 1 shows the structures of photolabels based on etomidate, mephobarbital, and propofol that are described below.

Table 1.

Structures of Potent Anesthetics and Photolabel Derivatives

Name	R1	R2	Name	R3	Name	R4	R5	R5
R-Etomidate	H		R-Mephobarbital	H	Propofol		H	H
R-Azi- etomidate	H				AziPm	H		H
R-TDBzl- etomidate	H		R–mTFD- Mephobarbital (R–mTFD- MPAB)		o-Propofol diazirine		H	H
R-pTFD- etomidate					p-Azipentyl-Pro		H

^*Chiral center

The first successful anesthetic photolabel in GABA_ARs, R-azietomidate, acts just like R-etomidate both in vivo and in vitro. Its potency for loss of righting reflexes (LoRR) in tadpoles (EC50 ≈ 2 µM) is identical to R-etomidate’s, and both S-enantiomers are about 10-fold less potent (Figure 2) (35). In GABA_AR β3N265M mice, sleep times after intraperitoneal R-etomidate and R-azietomidate were equally attenuated relative to wild-type (36). Both drugs enhanced currents induced by low GABA concentrations in α1β2γ2L GABA_ARs with similar potency, efficacy and enantioselectivity (35).

A second–generation etomidate photolabel, R-TDBzl-etomidate, is an aromatic diazirine, reactive at a wider range of amino acid side chains than aliphatic diazirines. It is more potent than R-etomidate both in tadpoles (LoRR EC50 ≈ 700 nM) and in enhancing GABA_AR currents (37).

R–mTFD-MPAB is a derivative of mephobarbital, but 25-fold more potent, with a tadpole LoRR EC50 of 3.7 µM, and its S-enantiomer is 10-fold less potent (38,39). R–mTFD-MPAB enhances GABA_AR currents with an EC50 of 2.1 µM. It induces anesthesia in wild type and β3N265M mice with equal potency (personal communication from Robert Pearce, MD-PhD,Univ. Wisconsin at Madison), suggesting action via sites distinct from the etomidate sites.

Propofols are relatively small general anesthetics that nonetheless exhibit high potency and strong structure activity relationships (40). Three propofol photolabels have been described. AziPm (m-diaziryl-propofol) has potency similar to propofol in tadpoles (EC50 ≈ 3 µM), but modulates α1β2γ2L GABA_AR with less efficacy than propofol (41). Ortho-propofol diazirine (o-PD) causes LoRR in rats with EC50 ≈ 14.7 mg/kg (vs. 4.7 mg/kg for propofol). It enhances α1β2γ2S receptor currents with efficacy comparable to propofol (21). o-PD is chemically unstable, making it unsuitable for approaches based on tritiation and Edman sequencing. It has been used for photolabeling with MS analysis (21). 4-Azipentyl-propofol contains an aliphatic diazirine at the para position. It produces tadpole LoRR with EC50 ≈ 3.2 µM and enhances α1β2γ2L receptor currents with efficacy similar to propofol (42).

Based on pharmacological interactions, sites where alphaxalone and related neurosteroids modulate GABA_ARs are likely different from other anesthetics (43). Photolabeling of GABA_ARs has succeeded with one steroid, 6-azi-pregnanolone (6-AziP). This compound enhances α1β2γ2L GABA_AR currents with EC50 ≈ 3 µM (44).

Anesthetic Photolabeling Results in GABA_ARs

Potent anesthetic photolabels identify anesthetic sites in transmembrane subunit interfaces.

Etomidate binds at β⁺ – α⁻ interfaces

Both [³H]azi-etomidate and [³H]TDBzl-etomidate photolabel α1β3 GABA_ARs at β3M286 in M3 and α1M236 in M1 (25,45). [³H]TDBzl-etomidate also labels β3V290, one helical turn intracellular from M286 in M3. Photoincorporation by these etomidate photolabels is inhibited by etomidate and propofol, but not mTFD-MPAB or alphaxalone (26). These residues all abut the inter-subunit spaces between β3-M3 helices (the “β3⁺” face) and α1-M1 helices (the “α1⁻” face; Figures 1 and 4). A photolabel moiety was added at the other end of etomidate, creating pTFD-etomidate (Table 1), but this compound did not modulate GABA_ARs (46), suggesting that the phenyl ring occupies a sterically restricted environment. In silico docking suggests the phenyl ring projects towards the M2 helices, coming close to β3N265, where mutations produce dramatic effects on etomidate sensitivity (SCAMP studies of this site are discussed below) (25,26,47).

Figure 4. Selectivity of general anesthetics for intersubunit sites in α1β3γ2L GABA_ARs.

The homology model and coloring are as in Figure 1, and the view of the transmembrane domain () is from the synaptic side with the extracellular domain removed. Subunit interfaces are labeled (note that “+” corresponds to M3 helices and “−” corresponds to M1 helices). Photolabels that selectively bind in each interface are identified in colored boxes, and white boxes identify the general anesthetics that compete with photolabels for occupancy of that interface.

R–mTFD-MPAB binds at γ⁺ – β⁻ and α⁺ – β⁻ interfaces

R–mTFD-MPAB does not photolabel the etomidate sites. Instead it photoincorporates at α1A291 and α1Y294 (both in M3), β3M227 (M1), and γ2S301 (M3) (26). These residues are homologs of those labeled by etomidate derivatives, but located in both γ2⁺ – β3⁻ and α1⁺ – β3⁻ interfaces of α1β3γ2L GABA_ARs (Figure 4). R-mTFD-MPAB photoincorporation is inhibited by mTFD-MPAB and propofol, but not by etomidate or alphaxalone. R–mTFD-MPAB modulates [³H]muscimol binding in α1β3γ2L and α1β3 GABA_ARs with EC50s of 2 and 30 µM respectively, suggesting that it has higher affinity for the γ2⁺ – β3⁻ than for the interface α1⁺ – β3⁻ interface (27).

Propofol binds at β⁺ – α⁻, γ⁺ – β⁻, and α⁺ – β⁻ interfaces

Propofol binding sites have been explored with competition studies, described above, and two photolabel analogs. After o-PD photolabeling of β3 (homomeric) and α1β3 GABA_ARs, mass spectrometry detected a single adducted residue: β3H267 in M2 (21). However, propofol displacement of o-PD was not demonstrated. The location of β3H267 is at the top of M2 on the β⁻ face (8). In contrast, propofol–displaceable photoincorporation of [³H]aziPm occurred at both in β3⁺ – α1⁻ interfaces (β3M286, α1M236 and α1I239) and in α1⁺ – β3⁻ interfaces (β3M227) in α1β3 GABA_ARs (47). These results suggest that AziPm binds to all four interfaces labeled by azietomidate and mTFD-MPAB. This inference is also supported by propofol displacement experiments (26) and quantitative analyses of α1β2γ2L GABA_AR activity showing that etomidate acts via two equivalent sites, while propofol acts via more than two allosteric sites (16,17). Further research is needed to establish the relative affinities and efficacies of these sites, and their dependence on subunit composition.

6-AziP binds at β3⁺

Homomeric β3 GABA_ARs were photolabeled with 6-AziP (48). MS analysis identified β3F302 as the only photolabeled residue. This residue is located on β3-M3, three to four helical turns intracellular from β3M286 and β3V290, the residues photolabeled by TDBzl-etomidate. Displacement by other steroids such as alphaxalone was not demonstrated.

Do anesthetics bind at α⁺ – γ⁻ interfaces?

No photolabeling has been detected in γ2-M1 using any of the anesthetic derivatives described above. This suggests, albeit inconclusively, that residues photolabeled on α1-M3 are from sites in the α⁺ – β⁻ interfaces.

Anesthetic binding at β3⁺ – β3⁻ interfaces

Azietomidate photolabels β3M227 in α1β3 GABA_ARs, but no incorporation is found in α1-M3 (25). Thus, this etomidate-displaceable labeling is in the β3⁺ – β3⁻ interface, consistent with etomidate activation of β3 homomeric receptors (49). Interestingly, etomidate does not displace R-mTFD-MPAB photolabeling at β3M227 in α1β3 receptors, indicating that mTFD-MPAB binds very weakly to β3⁺ – β3⁻ interfaces (26,47).

Substituted Cysteine Modification-Protection (SCAMP) Strategy

Anesthetic photolabeling has located a number of amino acid contacts in GABA_AR inter-subunit pockets, but almost certainly has missed others. Thus, complementary techniques are needed to further probe anesthetic-receptor contacts near photolabel sites. One successful approach to extending the map of anesthetic contacts is SCAMP, illustrated in Figure 5. Briefly, a side-chain hypothesized to be in or near an anesthetic binding site is mutated to cysteine, providing a free sulfhydryl. These cysteine substituted receptors are then exposed to sulfhydryl-reactive chemical probes, and after washout of these reagents, electrophysiology is used to detect whether covalent bond formation produced an irreversible functional change. This is equivalent to real-time mutagenesis at the side-chain of interest. If a functional modification signal is observed, then the rates of modification in the absence and presence of anesthetic are compared. Drug occupancy will reduce the rate of covalent bond formation by steric competition if the substituted cysteine is in the binding pocket. This interpretation is strengthened if other classes of anesthetic fail to block modification. However, if all anesthetics inhibit modification, this could indicate a noncompetitive mechanism such as allosteric action.

Figure 5. Substituted Cysteine Modification and Protection (SCAMP).

Top: Transmembrane cross-sections of both open and closed receptors are diagrammed. Anesthetic site affinity or accessibility is enhanced by activating GABA_A receptors with GABA. Bottom: A close-up view of one interfacial pocket is shown with engineered sulfhydryls, one of which is in the anesthetic site. Modification of both sulfhydryls by p-chloromercuribenzene sulfonate (pCMBS), and protection of one sulfhydryl by bound anesthetic are also depicted. Experiments are performed with one cysteine substitution at a time.

Several experimental criteria must be met when applying SCAMP to sites such as those for anesthetics that are coupled to channel gating (agonist sites). First, the anesthetic drug must still modulate or activate mutant receptors, signifying that normal drug-site interactions are retained. Second, the mix of receptor states in both control modification and anesthetic protection studies must be similar. Because anesthetics tend to activate receptors, this condition can often be met by including GABA in both control modification and anesthetic protection experiments. Third, protection is best studied using anesthetic concentrations that occupy a large fraction of sites, to more effectively block covalent modification. GABA also enhances anesthetic affinity and increases the fraction of drug-occupied binding sites. However, cysteine substitution may reduce GABA efficacy, requiring modified control conditions. Finally, GABA often increases the rate of modification at cysteine substituted positions in transmembrane helices, reducing the concentrations of probe chemicals needed in experiments.

Anesthetic SCAMP Results in GABA_ARs

The βM286 residue

SCAMP was used to investigate anesthetic sites in GABA_ARs several years before anesthetic photolabeling. Bali and Akabas (50) introduced cysteine at two β2 positions, β2N265 and β2M286, where other mutations were known to influence propofol sensitivity in α1β2γ2 receptors (Figure 6 shows these residues in β3, which is highly homologous to β2 in this region). Both residues are predicted to contribute to the “β⁺” transmembrane interface, although β2N265 may also be accessible from the “β⁻” interfacial pocket (51). To achieve comparable mixes of receptor states during control and protection experiments, Bali and Akabas used a strategy different from ours, likely resulting in under 50% anesthetic site occupancy. They found that propofol protected β2M286C from modification, but did not protect β2N265C. We later investigated the β2M286C mutation in a detailed study of etomidate protection, which revealed uniquely useful features of this mutation (52). The β2M286C mutation weakened etomidate modulation of GABA-elicited responses, and eliminated direct activation by high etomidate concentrations. Covalent bond formation between β2M286C and p-chloromercuribenzenesulfonate (pCMBS), a small water-soluble sulfhydryl-specific reagent, produced clear functional changes that were blocked in the presence of etomidate. The functional features of the βM286C mutation also enabled studies of etomidate-dependent protection in both the absence of GABA (almost entirely resting state receptors) and in the presence of GABA (activated and desensitized receptors). Protection studies revealed that etomidate concentrations occupying half of the receptor sites in the absence of GABA were about 10-fold higher than those in the presence of GABA, consistent with a quantitative functional model of etomidate co-agonism in α1β2M286Cγ2L GABA_ARs (16,52).

Figure 6. Combining photolabel and SCAMP results to map the β+ – α⁻ interfacial anesthetic sites.

The homology model is based on the GluCl chloride channel (3RHW.pdb) (7,26). The five subunits are colored as follows: α1, yellow; β3, red, and γ2 green. The transmembrane helical backbones are depicted as ribbons. Residues in this site identified with either photolabeling or SCAMP are shown as space-filling atomic models and labeled. Contact residues are coded by coloring their carbons. Residues photolabeled by etomidate and propofol derivatives show cyan and pink carbons, respectively. Grey carbons indicate binding site residues identified by SCAMP with either etomidate or propofol. Red and blue atoms are oxygen and nitrogen respectively. Also shown in ball and stick mode are three residues in the γ⁺ – β⁻ inter-subunit region that are discussed in the text: γ2S301, β3M227, and β3H267.

The α-M1 helix

Photolabeling at αM236 by azi-etomidate (45) provided the first hint that the α-M1 transmembrane helix abuts bound etomidate in β⁺–α⁻ interfacial pockets. To further define the role of α-M1 in etomidate binding and modulation, we applied SCAMP to a series of 14 α1-M1 residues, from α1Q229 to α1Q242 spanning over three helical turns, in α1β2γ2L receptors (53). We found interesting functional phenotypes associated with these cysteine substitutions. For example, in α1M236Cβ2γ2L receptors GABA was a partial agonist, activating only about 25% of receptors, while etomidate alone activated more than 95% of receptors. Remarkably, these findings remained consistent with our allosteric co-agonist model. Most of the other α1-M1 cysteines we studied also displayed reduced receptor sensitivity to GABA, while retaining etomidate sensitivity. Thus, the entire α1-M1 region we studied was linked to channel gating.

For SCAMP studies of α1M236Cβ2γ2L, we established similar distributions of receptor states in both control modification and etomidate protection experiments by including alphaxalone (which does not bind at etomidate sites) with GABA in controls. This combination fully activated the mutant receptors, matching the effects of etomidate plus GABA. Etomidate protected α1M236C from covalent modification by pCMBS, confirming steric proximity. Etomidate protection was also observed at both α1L232C and α1T237C (Figure 6), neither of which was photolabeled by etomidate derivatives. Thus, etomidate apparently contacts one face of the outer α1-M1 helix over ~1.5 helical turns. Moreover, etomidate did not protect any cysteine substituted sidechains predicted to face the intra-subunit pocket formed by the four α1 transmembrane helices. This further supports the idea that sites where anesthetics act as allosteric agonists are located between adjacent subunits. Subsequent studies also demonstrated propofol protection at α1M236C (see below).

The βN265 residue

Despite the remarkable effects of mutations at βN265, Bali and Akabas (50) failed to demonstrate propofol protection and no anesthetic photolabels have adducted this locus. We examined etomidate interactions at βN265 using SCAMP (54), because βN265 mutations appear to affect etomidate sensitivity more than propofol. The β2N265C mutation eliminated modulation or activation by etomidate at concentrations (300 µM) more than100-fold higher than those affecting wild-type GABA_ARs. Similarly high concentrations of etomidate did not protect β2N265C from pCMBS modification. Because α1β2N265Cγ2L violates one requirement for interpretation of SCAMP protection data (sensitivity to the anesthetic), these negative results fail to reveal how the mutation alters etomidate-receptor interactions: it could obliterate etomidate binding; etomidate could bind without contacting β2N265, or the mutation could eliminate drug efficacy, by decoupling the site from channel gating.

To test for binding effects of βN265 mutations, we exploited α1M236C, already established as protected by etomidate (53). In a wild-type background, α1M236C was readily protected by etomidate (as low as 3 µM) and propofol, but not by alphaxalone. When the β2N265M mutation was added, etomidate concentrations below 300 µM did not protect α1M236C, indicating low anesthetic site occupancy in both the absence and presence of GABA. Similar results were found with propofol. These findings, although indirect, indicate that the β2N265 mutations strongly affect etomidate and propofol binding in the β⁺–α⁻ interfacial pockets, supporting the hypothesis that β2N265 is a key contact point.

The βH267 residue

As noted above, photolabeling with o-PD identified β3H267 as a possible contact for propofol (21). We explored both the pharmacological effects of the β3H267C mutation, and the ability of four potent anesthetics to protect this sidechain from chemical modification (55). The α1β3H267Cγ2L receptors retain most of the gating features of wild-type: low spontaneous activation, normal GABA EC₅₀, and high GABA efficacy. However, the β3H267C mutation selectively sensitizes receptors to direct activation by propofol and the barbiturate photolabel R-mTFD-MPAB. SCAMP experiments reveal that mTFD-MPAB protects β3H267C from pCMBS modification, while etomidate, alphaxalone, and propofol do not. Thus, while photolabeling shows that propofol and mTFD-MPAB sites overlap, β3H267 abuts the mTFD-MPAB binding site, but is far enough from bound propofol to allow pCMBS unimpeded access. As expected, β3H267 does not interact with either etomidate or alphaxalone, two anesthetics with sites that do not overlap with those of mTFD-MPAB.

This result also has implications for structural GABA_AR homology models based on crystallized homomeric pLGICs. Homology models based on both β3 homomeric GABA_ARs (8) and on GLIC (6) have sidechains bridging the “β^‒“ transmembrane interfaces, separating them into two non-contiguous pockets: one near the ion channel adjacent to βH267, and another near the lipid-protein interface that includes residues photolabeled by both mTFD-MPAB and azi-Pm (βM227, αA291, αY294, and γS301). SCAMP results indicate that mTFD-MPAB binds in a single contiguous pocket that abuts all of these residues, favoring homology models based on ivermectin-bound GluCl channels (7).

Summary and Conclusions

Anesthetic molecular mechanisms in GABA_ARs

The conclusion that etomidate, barbiturate and propofol binding sites on GABA_ARs are located in homologous pockets between adjacent subunits in the transmembrane domain is supported by both photolabeling and SCAMP studies. Mutations at photolabeled inter-subunit sites alter both receptor function and anesthetic sensitivity, supporting the pharmacological relevance of these sites (56–58). Molecular modeling studies also suggest that anesthetic binding to inter-subunit pockets in GABA_ARs correlates with drug potency (59). The identification of inter-subunit anesthetic sites is an important shift from previous hypotheses proposing anesthetic sites within the transmembrane four helix bundles of GABA_AR subunits (i.e. intra-subunit sites) (60). Anesthetic sites on GABA_ARs are also distinguished from those on cationic channels of the pLGIC superfamily. In cationic channels, crystallography reveals general anesthetics binding in intra-subunit pockets within the four helix bundles of GLIC (61). In muscle type acetylcholine receptors, inhibitory sites are in the channel lumen, and four helix bundles (62–64).

Five homologous inter-subunit transmembrane sites are potentially formed by each GABA_AR pentamer. In physiologically relevant α1β3γ2 heteropentamers, four classes of distinct interfacial sites are predicted (Figure 4), and anesthetic photolabeling has revealed remarkable selectivity for particular inter-subunit sites. In α1β3γ2 GABA_ARs, etomidate sites (β⁺ – α⁻ interfaces) are favored over R-mTFD-MPAB sites by R- and S-etomidate (>100-fold) and propofol (1.5-fold), whereas the R–mTFD-MPAB sites (γ⁺ – β⁻ and to a lesser extent α⁺ – β ⁻ interfaces) are favored by R-mTFD-MPAB (~60-fold), phenobarbital (13-fold), pentobarbital (8-fold) and thiopental (1.6-fold). However, the mTFD-MPAB sites cannot be regarded as universal barbiturate sites because brallobarbital favors the etomidate site (26). Preliminary SCAMP studies are consistent with these conclusions. The remaining α1⁺ – γ2⁻ interface has not been unequivocally photolabeled, and SCAMP will be useful in testing whether any anesthetics bind near γ2-M1.

Subunit selective drug development

The subunit selectivity of various potent general anesthetics raises the possibility that new clinical drugs can be developed that act selectively on specific GABA_AR subtypes. For example, etomidate acts selectively on receptors with β2 or β3 subunits, but not β1 (65). However, nearly all GABA_ARs contain β⁺ – α⁻ interfaces, so etomidate derivatives act on a wide range of GABA_A networks. In contrast, drugs like mTFD-MPAB may select for synaptic GABA_ARs, all of which contain γ2 subunits, rather than δ-subunit containing extrasynaptic receptors.

We do not yet know how the many physiological components underlying the general anesthetic state would respond to agents of these two classes, but the principle has already been illustrated for benzodiazepines and similar drugs that bind selectively at distinct ECD interfaces (66,67). These act in the αx⁺ – γ2 ⁻ and αx⁺ – βy⁻ interfaces in the ECD (Figure 1). Studies in transgenic animals show that classical benzodiazepines act at α1⁺ – γ2 ⁻ interfaces to produce sedative and anticonvulsant effects, while α2⁺ – γ2 ⁻ interfaces mediate anxiolysis, and α5⁺ – γ2 ⁻ interfaces mediate learning and memory effects (68). Considering that the GABA_A receptor family includes a large set of different subunit isoforms (α1–6; β1–3; γ1–3; δ; ε; π; ρ1–3; θ), that are distributed differentially throughout the central nervous system (4), it is clear that much remains to be discovered.

Structure activity relationships acquired during photolabel development have incidentally also proven useful in developing novel clinical anesthetics. Specifically, the development of different etomidate photolabels helped identify parts of the molecule that could be modified without loss of potency and efficacy (35,37,46). This information contributed to the design of short-acting etomidate derivatives for clinical use (69,70).

Future directions

Photolabeling and SCAMP are being applied to a number of important questions related to mechanisms of general anesthesia. One challenging goal is locating sites where neurosteroid anesthetics (e.g. alphaxalone) bind to heteromeric GABA_ARs. Another goal is to better understand state-dependent conformational changes and anesthetic binding, particularly in the transient open state. Sites where low affinity anesthetics act in heteromeric GABA_ARs are largely unmapped, but there is evidence that some bind in the inter-subunit pockets where potent IV drugs act (51,71). These approaches are also being applied to map inhibitory (convulsant) sites on GABA_ARs (72).

Focus and Scope.

This review focuses on progress in our research aimed at mapping binding sites for IV general anesthetics in heteromeric γ-aminobutyric acid type A receptors (GABA_ARs) using two complementary techniques: photolabeling and substituted cysteine modification-protection (SCAMP). We include sections on both research strategy and results for each approach. Results are discussed in the context of both models of GABA_AR molecular structure and models of anesthetic-induced receptor modulation.