High affinity for targets is a key feature of successful anesthetic photolabels. In general, drugs acting at concentrations >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 coefficients30 (Figure 2). Clinical volatile anesthetics all act at free plasma concentrations >100 µM, with potencies paralleling hydrophobicities and can interact at many binding sites large enough to accommodate them.32,33 In contrast, for anesthetics with 50% effective concentrations (EC50s) <50 µM, the correlation with hydrophobicity breaks down and significant stereoselectivity emerges (yellow zone; Figure 2). For further illustration, the volatile anesthetic bromoform (half-maximal inhibitory concentration = 125 µM) occupies 11 sites in a crystal structure of a bacterial pLGIC34 (Figure 3A), whereas ketamine (half-maximal inhibitory concentration = 58 µM) interacts stereoselectively with a single class of intrasubunit sites in another pLGIC35 (Figure 3B). On this basis, we have focused on developing photolabels that act <10 µM and, wherever possible, exhibit stereoselectivity, including derivatives of etomidate, propofol, barbiturates, and alphaxalone.
PHARMACOLOGIC CHARACTERIZATION OF ANESTHETIC PHOTOLABELS
Table 1 shows the structures of photolabels based on etomidate, mephobarbital, and propofol that are described below.
The first successful anesthetic photolabel in GABAARs, R-azietomidate, acts just like R-etomidate both in vivo and in vitro. Its potency for loss of righting reflexes (LoRR) in tadpoles (EC50 approximately 2 μM) is identical to R-etomidate’s, and both S-enantiomers are approximately 10-fold less potent (Figure 2).36 In GABAAR β3N265M mice, sleep times after intraperitoneal R-etomidate and R-azietomidate were equally attenuated relative to wild type.37 Both drugs enhanced currents induced by low GABA concentrations in α1β2γ2L GABAARs with similar potency, efficacy, and enantioselectivity.36
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 approximately 700 nM) and in enhancing GABAAR currents.38
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.39,40 R-mTFD-MPAB enhances GABAAR 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, University of Wisconsin at Madison, Madison, WI), 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.41 Three propofol photolabels have been described. AziPm (m-diaziryl-propofol) has potency similar to propofol in tadpoles (EC50 approximately 3 μM), but it modulates α1β2γ2L GABAAR with less efficacy than propofol.42 ortho-Propofol diazirine (o-PD) causes LoRR in rats with EC50 approximately 14.7 mg/kg (vs 4.7 mg/kg for propofol). It enhances α1β2γ2S receptor currents with efficacy comparable to propofol.22 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.22 4-Azipentyl-propofol contains an aliphatic diazirine at the para position. It produces tadpole LoRR with EC50 approximately 3.2 μM and enhances α1β2γ2L receptor currents with efficacy similar to propofol.43
On the basis of pharmacologic interactions, sites where alphaxalone and related neurosteroids modulate GABAARs are likely different from other anesthetics.44 Photolabeling of GABAARs has succeeded with 1 steroid, 6-azi-pregnanolone (6-AziP). This compound enhances α1β2γ2L GABAAR currents with EC50 approximately 3 μM.45
ANESTHETIC PHOTOLABELING RESULTS IN GABAARS
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 GABAARs at β3M286 in M3 and α1M236 in M1.26,46 [3H]TDBzl-etomidate also labels β3V290, 1 helical turn intracellular from M286 in M3. Photoincorporation by these etomidate photolabels is inhibited by etomidate and propofol, but not mTFD-MPAB or alphaxalone.4 These residues all abut the intersubunit 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 GABAARs,31 suggesting that the phenyl ring occupies a sterically restricted environment. In silico docking suggests the phenyl ring projects toward the M2 helices, coming close to β3N265, where mutations produce dramatic effects on etomidate sensitivity (SCAMP studies of this site are discussed below).4,26,47
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).4 These residues are homologs of those labeled by etomidate derivatives, but located in both γ2+-β3− and α1+-β3− interfaces of α1β3γ2L GABAARs (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 GABAARs 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 2 photolabel analogs. After o-PD photolabeling of β3 (homomeric) and α1β3 GABAARs, mass spectrometry detected a single adducted residue: β3H267 in M2.22 However, propofol displacement of o-PD was not demonstrated. The location of β3H267 is at the top of M2 on the β− face.9 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 GABAARs.47 These results suggest that AziPm binds to all 4 interfaces labeled by azietomidate and mTFD-MPAB. This inference is also supported by propofol displacement experiments4 and quantitative analyses of α1β2γ2L GABAAR activity showing that etomidate acts via 2 equivalent sites, whereas propofol acts via >2 allosteric sites.17,18 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 GABAARs were photolabeled with 6-AziP.48 MS analysis identified β3F302 as the only photolabeled residue. This residue is located on β3-M3, 3 to 4 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 GABAARs, but no incorporation is found in α1-M3.26 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.4,47
Anesthetic photolabeling has located a number of amino acid contacts in GABAAR intersubunit 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. In brief, 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.
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 GABAARs
The βM286 Residue
SCAMP was used to investigate the anesthetic sites in GABAARs several years before anesthetic photolabeling. Bali and Akabas50 introduced cysteine at 2 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 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 approximately 10-fold higher than those in the presence of GABA, consistent with a quantitative functional model of etomidate coagonism in α1β2M286Cγ2L GABAARs.17,52
The α-M1 Helix
Photolabeling at αM236 by azi-etomidate46 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 3 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 approximately 25% of receptors, whereas etomidate alone activated >95% of receptors. Remarkably, these findings remained consistent with our allosteric coagonist 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 approximately 1.5 helical turns. Moreover, etomidate did not protect any cysteine-substituted side chains predicted to face the intrasubunit pocket formed by the 4 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 Akabas50 failed to demonstrate propofol protection, and no anesthetic photolabels have adducted this locus. We examined the etomidate interactions at βN265 using SCAMP54 because βN265 mutations appear to affect etomidate sensitivity more than propofol. The β2N265C mutation eliminated modulation or activation by etomidate at concentrations (300 μM) >100-fold higher than those affecting wild-type GABAARs. 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 <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.22 We explored both the pharmacologic effects of the β3H267C mutation and the ability of 4 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, whereas 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, 2 anesthetics with sites that do not overlap with those of mTFD-MPAB.
This result also has implications for structural GABAAR homology models based on crystallized homomeric pLGICs. Homology models based on both β3 homomeric GABAARs9 and on GLIC8 have sidechains bridging the “β−” transmembrane interfaces, separating them into 2 noncontiguous 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 aziPm (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 the Glutamate-gated Chloride Channel (GluCl) from Caenorhabditis elegans.3
Anesthetic Molecular Mechanisms in GABAARs
The conclusion that etomidate-, barbiturate-, and propofol-binding sites on GABAARs are located in homologous pockets between adjacent subunits in the transmembrane domain is supported by both photolabeling and SCAMP studies. Mutations at photolabeled intersubunit sites alter both receptor function and anesthetic sensitivity, supporting the pharmacologic relevance of these sites.56–58 Molecular modeling studies also suggest that anesthetic binding to intersubunit pockets in GABAARs correlates with drug potency.59 The identification of intersubunit anesthetic sites is an important shift from previous hypotheses proposing anesthetic sites within the transmembrane 4 helix bundles of GABAAR subunits (ie, intrasubunit sites).60 Anesthetic sites on GABAARs are also distinguished from those on cationic channels of the pLGIC superfamily. In cationic channels, crystallography reveals general anesthetics binding in intrasubunit pockets within the 4 helix bundles of GLIC.61 In muscle-type acetylcholine receptors, inhibitory sites are in the channel lumen, and 4 helix bundles.62–64
Five homologous intersubunit transmembrane sites are potentially formed by each GABAAR pentamer. In physiologically relevant α1β3γ2 heteropentamers, 4 classes of distinct interfacial sites are predicted (Figure 4), and anesthetic photolabeling has revealed remarkable selectivity for particular intersubunit sites. In α1β3γ2 GABAARs, 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 (approximately 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.4 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 GABAAR subtypes. For example, etomidate acts selectively on receptors with β2 or β3 subunits, but not β1.65 However, nearly all GABAARs contain β+-α− interfaces, so etomidate derivatives act on a wide range of GABAA networks. In contrast, drugs such as mTFD-MPAB may select for synaptic GABAARs, all of which contain γ2 subunits, rather than δ subunit containing extrasynaptic receptors.
We do not yet know how the many physiologic components underlying the general anesthetic state would respond to agents of these 2 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, whereas α2+-γ2− interfaces mediate anxiolysis, and α5+-γ2− interfaces mediate learning and memory effects.68 Considering that the GABAA 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,6 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.31,36,38 This information contributed to the design of short-acting etomidate derivatives for clinical use.69,70
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 (eg, alphaxalone) bind to heteromeric GABAARs. 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 GABAARs are largely unmapped, but there is evidence that some bind in the intersubunit pockets where potent IV drugs act.51,71 These approaches are also being applied to map inhibitory (convulsant) sites on GABAARs.72
Name: Stuart A. Forman, MD, PhD.
Contribution: This author helped in the study conception, wrote the manuscript, create the figures, and edited the manuscript.
Conflicts of Interest: Stuart A. Forman is a member of the ABP700 Scientific Advisory Board for the Medicines Company.
Name: Keith W. Miller, DPhil.
Contribution: This author helped in the study conception, wrote the manuscript, create the figures, and edit the manuscript.
Conflicts of Interest: The author has no conflicts of interest to declare.
This manuscript was handled by: Gregory J. Crosby, MD.
We acknowledge the important contributions of colleagues in our research program: Shaukat Husain, DPhil (Department of Anesthesia Critical Care and Pain Medicine, Massachusetts General Hospital), Karol Bruzik, PhD (Medicinal Chemistry and Pharmacognosy, University Illinois Chicago), Jonathan Cohen, PhD (Neurobiology, Harvard Medical School), David Chiara, MD, PhD (Neurobiology, Harvard Medical School), and Douglas Raines, MD (Department of Anesthesia Critical Care and Pain Medicine, Massachusetts General Hospital). Molecular graphics images were produced using the University of California San Francisco Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco.73
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