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Locations of binding sites for GABA, benzodiazepines, R-mTFD-MPAB, and etomidate in an (1) 2 (3) 2 GABA A R. GABA-binding sites are in the ECD at the interface between the and subunit referred to as the -subunit interface, and with that nomenclature continued in a counterclockwise direction, the benzodiazepine site is at the -subunit interface. Depicted in the TMD are the locations of the four transmembrane helices (M1-M4) in each subunit, the etomidate-binding sites at thesubunit interfaces that contain the GABA-binding sites in the ECD, and the R-mTFD-MPAB sites at the -and -subunit interfaces.
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![FIGURE 4. Pharmacological specificity of S-[ 3 H]mTFD-MPPB and R-[ 3...](publication/280582153/figure/fig4/AS:928035058487297@1598272208419/Pharmacological-specificity-of-S-3-HmTFD-MPPB-and-R-3-HmTFD-MPAB-photoincorporation_Q320.jpg)
In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit ane...
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... transmembrane domain (TMD) of 132 GABA A Rs based upon the locations of residues photolabeled by analogs of etomidate, mephobarbital, and propofol in homology models based on published structures of several homologous members of the Cys-loop superfamily of pentameric ligand-gated ion channels (17)(18)(19), including one of a human 3 GABA A R ( Fig. 1) (20). Photoreactive etomidate analogs identify a high affinity binding site for etomidate at the -subunit interfaces, based upon the photolabeling of amino acids in the subunit M3 and subunit M1 transmembrane helices (21,22). A mephobarbital analog, R-[ 3 H]mTFD-MPAB, photolabeled amino acids in the M1, M3, and M3 transmembrane ...
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... 3 H]mTFD-MPAB photolabeling. Similar to R-mTFD-MPAB, S-mTFD-MPAB acts as an anesthetic, but with 10-fold lower potency, and it acts as a low efficacy potentiator of GABA responses (25). In contrast, for mTFD-MPPB, differing from mTFD-MPAB by only two more hydrogen atoms ( [1-methyl-5-propyl-5-(m-trifluorometh- yl-diazirynylphenyl]barbituric acid, Fig. 1), the R-enantiomer acts as an anesthetic in vivo and potentiated GABA responses for expressed 132 GABA A R, whereas the S-enantiomer acts as a convulsant and inhibits GABA responses (26). Thus, the enantiomers of mTFD-MPPB mirror the actions of S-and R-MPPB in vivo and in ...
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... dependence of nonradioactive S-mTFD-MPPB inhibition of S-[ 3 H]mTFD-MPPB photoincorporation in the 56-kDa gel band from 132 GABA A Rs photolabeled in the presence of bicuculline or GABA (Fig. 10A). In the presence of bicuculline, S-[ 3 H]mTFD-MPPB photoincorporation in 2Ser-280 accounts for 50% of the 3 H incorporated in the 56-kDa gel band containing the 1 and 2 subunits (Fig. 5A). In contrast, in the presence of GABA, photolabeling of 2Ser-280 is inhibited by 90%, and 3 H in the 56-kDa gel band results primarily from ...
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... further clarify the state-dependence of S-mTFD-MPPB binding at interface sites, we also determined its inhibition of R-[ 3 H]mTFD-MPAB photolabeling (Fig. 10B). In the presence of bicuculline, S-mTFD-MPPB inhibited R-[ 3 H]mTFD-MPAB photolabeling with low affinity (IC 50 130 60 M), not with the IC 50 of 1.7 M characteristic of its binding to the -site based upon inhibition of S-[ 3 H]mTFD-MPPB photolabeling. In contrast, R-mTFD-MPAB inhibition of R-[ 3 H]mTFD-MPAB photolabeling was ...
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... (IC 50 130 60 M), not with the IC 50 of 1.7 M characteristic of its binding to the -site based upon inhibition of S-[ 3 H]mTFD-MPPB photolabeling. In contrast, R-mTFD-MPAB inhibition of R-[ 3 H]mTFD-MPAB photolabeling was consistent with a single site model (n H 1) in the presence of bicuculline (IC 50 2.3 0.3 M) or GABA (IC 50 0.7 0.04 M) ( Fig. 10D and Table 2). The difference in IC 50 values for S-mTFD-MPPB inhibition of S-[ 3 H]mTFD-MPPB and R-[ 3 H]mTFD-MPAB photolabeling was unexpected. However, 3Met-227, the amino acid that dominates subunit photolabeling by R-[ 3 H]mTFD-MPAB in the presence of GABA or bicuculline (Table 1), is present at both the -and -interfaces (Fig. 7). ...
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... concentration dependence of S-mTFD-MPPB inhibition of R-[ 3 H]mTFD-MPAB photolabeling (Fig. 10B) in the presence of GABA was fit by a Hill coefficient, n H 0.6 ...
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... In the absence of GABA, 2Ser-280 (M2-15) is labeled 10-fold more efficiently than any other residue (Table 1). GABA reduces photolabeling of 2Ser-280 by 90% and enhances photolabeling of 3Thr-262 (M2-12) and 3Phe-289 in M3 by 3-5-fold. The locations of the residues are approximated based upon their locations in an 132 GABA A R homology model (see Fig. ...
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... as follows: 1) a high affinity component (IC 50, H 10 2 M) that reduced photolabeling to the level observed in the presence of bicuculline, and 2) a low affinity component with IC 50, L 310 130 M, similar to the affinity seen in the presence of bicuculline. In the presence of bicuculline, R-mTFD-MPAB inhibition of S-[ 3 H]mTFD-MPPB photolabeling (Fig. 10C) was consistent with a single-site model (n H 1) with an IC 50 (7.5 1.6 M) close to that seen for inhibition of R-[ 3 H]mTFD-MPAB photolabeling (Table 2). However, in the presence of GABA, inhibition was characterized by n H 0.6 0.1 (IC 50 38 6 M), a consequence of S-[ 3 H]mTFD-MPPB photolabeling of amino acids in M3 ( interface) and ...
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... of a homomeric 3 GABA A R (Fig. 12), 2Ser-280 is located at the -interface, lining a pocket in which R-[ 3 H]mTFD-MPAB binds and photolabels 3Met-227 in M1 and, at lower efficiency, 2Ser-301 in M3 (Fig. 12, C and E) ...
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... of a homomeric 3 GABA A R (Fig. 12), 2Ser-280 is located at the -interface, lining a pocket in which R-[ 3 H]mTFD-MPAB binds and photolabels 3Met-227 in M1 and, at lower efficiency, 2Ser-301 in M3 (Fig. 12, C and E) ...
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... seen for agonist binding at the nonequivalent transmitter-binding sites in the muscle-type nicotinic acetylcholine receptor (39 -41). S-mTFD-MPPB and R-mTFD-MPAB Bind in Different Orientations at the -Interface-S-mTFD-MPPB and R-mTFD-MPAB, which differ in structure only by chirality and the presence of either a 5-propyl or 5-allyl substituent (Fig. 1), bind with high affinity (IC 50 values of 3 M) at theinterface, in the presence of bicuculline or GABA, respectively. Therefore, the selective photolabeling of 2Ser-280 by S-[ 3 H]mTFD-MPPB compared with that of 3Met-227 by R-[ 3 H]mTFD-MPAB provides direct experimental evidence that the two drugs must bind in different orientations ...
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... of 2Ser-280 by S-[ 3 H]mTFD-MPPB compared with that of 3Met-227 by R-[ 3 H]mTFD-MPAB provides direct experimental evidence that the two drugs must bind in different orientations within this interface pocket. 2Ser-280 and 3Met-227 are on opposite surfaces of the pocket with a distance of 11 Å between -carbons in the GABA A R homology model (Fig. 12, C and E). S-[ 3 H]mTFD-MPPB and R-[ 3 H]mTFD-MPAB, with extended lengths of 10 Å, must bind in opposite but overlapping orientations with their diazirines oriented toward M2 and M1, respectively. In contrast, at the -interface both S-mTFD-MPPB and ...
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... with low affinity (enhanced by GABA) and photolabel the same amino acids. Based upon computational docking, S-mTFD-MPPB is predicted to bind stably and with similar energies in the pockets at each of the subunit interfaces. The predicted locations of bound S-mTFD-MPPB are shown in Fig. 12 as Connolly surface representations of the 10 lowest energy solutions, with S-mTFD-MPPB shown in stick representation in Fig. 12, C and E, ...
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... acids. Based upon computational docking, S-mTFD-MPPB is predicted to bind stably and with similar energies in the pockets at each of the subunit interfaces. The predicted locations of bound S-mTFD-MPPB are shown in Fig. 12 as Connolly surface representations of the 10 lowest energy solutions, with S-mTFD-MPPB shown in stick representation in Fig. 12, C and E, ...
Citations
... The receptors subunit composition affects the kinetics of chloride currents (I GABA ), the potency of their agonists and the pharmacological properties 20 . GABA A Rs are targets for many drugs (such as benzodiazepines, barbiturates, propofol and volatile anaesthetics) that change I GABA in a subunit-specific manner [21][22][23] . ...
Gamma-aminobutyric acid type A receptors (GABA A Rs) are ligand gated channels mediating inhibition in the central nervous system. Here, we identify a so far undescribed function of β-subunit homomers as proton-gated anion channels. Mutation of a single H267A in β3 subunits completely abolishes channel activation by protons. In molecular dynamic simulations of the β3 crystal structure protonation of H267 increased the formation of hydrogen bonds between H267 and E270 of the adjacent subunit leading to a pore stabilising ring formation and accumulation of Cl ⁻ within the transmembrane pore. Conversion of these residues in proton insensitive ρ1 subunits transfers proton-dependent gating, thus highlighting the role of this interaction in proton sensitivity. Activation of chloride and bicarbonate currents at physiological pH changes (pH 50 is in the range 6- 6.3) and kinetic studies suggest a physiological role in neuronal and non-neuronal tissues that express beta subunits, and thus as potential novel drug target.
... One class of sites selectively binds etomidate and is located at the two β + /α À subunit interfaces, whereas the other class of sites selectively binds the barbiturate R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB) and is located at the α + /β À and γ + /β À subunit interfaces. At each binding site, plus-faced M2-15 0 mutations (β 3 N265M at the two etomidate binding sites or γ 2 S280W and α 1 S270I at the R-mTFD-MPAB sites) significantly reduce or abolish GABA A receptor sensitivity to intravenous general anaesthetics and other allosteric modulators that act via that site (Jayakar et al., 2015;Nourmahnad et al., 2016;Siegwart et al., 2002;Walters et al., 2000). ...
... To assess the potential roles of the different transmembrane anaesthetic binding sites in mediating the GABA A receptor action described above, we introduced single subunit M2-15 0 amino acid mutations into the receptor that have previously been shown to selectively reduce or abolish etomidate or R-mTFD-MPAB modulation via these sites (Jayakar et al., 2015;Nourmahnad et al., 2016;Siegwart et al., 2002). We then evaluated the impact of each mutation on diazepam action in the presence of flumazenil. ...
... The goal of the current studies was to assess the functional conse- convulsants that act via each of these sites (Jayakar et al., 2015;Nourmahnad et al., 2016;Siegwart et al., 2002). Specifically, a β 3 N265M mutation at each of the two etomidate binding sites abolished the potentiating actions of diazepam and midazolam, whereas a γ 2 S280W mutation at the R-mTFD-MPAB binding site located at the γ + /β À subunit interface abolished their inhibitory actions. ...
Background and Purpose
In addition to binding to the classical high‐affinity extracellular benzodiazepine binding site of the GABAA receptor, some benzodiazepines occupy transmembrane inter‐subunit anaesthetic sites that bind etomidate (β⁺/α⁻ sites) or the barbiturate derivative R‐mTFD‐MPAB (α⁺/β⁻ and γ⁺/β⁻ sites). We aimed to define the functional effects of these interactions on GABAA receptor activity and animal behaviour.
Experimental Approach
With flumazenil blocking classical high‐affinity extracellular benzodiazepine site effects, modulation of GABA‐activated currents by diazepam, midazolam and flurazepam was measured electrophysiologically in wildtype and M2‐15′ mutant α1β3γ2L GABAA receptors. Zebrafish locomotive activity was also assessed in the presence of each benzodiazepine plus flumazenil.
Key Results
In the presence of flumazenil, micromolar concentrations of diazepam and midazolam both potentiated and inhibited wildtype GABAA receptor currents. β3N265M (M2‐15′ in the β⁺/α⁻ sites) and α1S270I (M2‐15′ in the α⁺/β⁻ site) mutations reduced or abolished potentiation by these drugs. In contrast, the γ2S280W mutation (M2‐15′ in the γ⁺/β⁻ site) abolished inhibition. Flurazepam plus flumazenil only inhibited wildtype receptor currents, an effect unaltered by M2‐15′ mutations. In the presence of flumazenil, zebrafish locomotion was enhanced by diazepam at concentrations up to 30 μM and suppressed at 100 μM, suppressed by midazolam and enhanced by flurazepam.
Conclusions and Implications
Benzodiazepine binding to transmembrane anaesthetic binding sites of the GABAA receptor can produce positive or negative modulation manifesting as decreases or increases in locomotion, respectively. Selectivity for these sites may contribute to the distinct GABAA receptor and behavioural actions of different benzodiazepines, particularly at high (i.e. anaesthetic) concentrations.
... The photolabeled amino acids were identified by protein microsequencing of fragments beginning near the N-termini of the 3M4, 3M3, and 3M1 helices that can be produced by digestion with endoproteinase Lys-C (EndoLys-C) and resolved by reversed-phase HPLC (rpHPLC) (13,32,34). When aliquots of the β subunit EndoLys-C digests were sequenced, peaks of 3 H release were seen in cycles 3/4 and 6/7 that were inhibitable by 35-P (Fig. 5A). ...
Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABA A receptors (GABA A Rs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analyses, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABA A R transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ GABA A Rs by photoaffinity labeling with [ ³ H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([ ³ H]21- p TFDBzox-AP), a potent GABA A R PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M3 (β3Pro-415, β3Leu-417, and β3Thr-418) and M4 (β3Arg-309) helices located at the base of a pocket in the β ⁺ –α – subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABA A R antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABA A R PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABA A R β ⁺ –α – subunit interface steroid binding site and identify several steroid PAMs that act via other sites.
... The pentameric ligand-gated receptor family retains a characteristic structural signature (Ernst et al., 2005). They possess a large extracellular domain (ECD) that incorporates the neurotransmitter (orthosteric) binding site (Lummis, 2009) located at interfaces between β + -αsubunits, and allosteric binding sites for modulators, such as the benzodiazepines at the α + -γsubunit interface (Sigel, 2002) and barbiturates at the γ + -αinterface (Jayakar et al., 2015) (Figure 2(a) and (c)). The signature Cys loop is evident in all receptors and appeared to interact with residues in the transmembrane domain (TMD) M2-M3 region ( Figure 2(b)). ...
γ-aminobutyric acid has become one of the most widely known neurotransmitter molecules in the brain over the last 50 years, recognised for its pivotal role in inhibiting neural excitability. It emerged from studies of crustacean muscle and neurons before its significance to the mammalian nervous system was appreciated. Now, after five decades of investigation, we know that most neurons are γ-aminobutyric-acid-sensitive, it is a cornerstone of neural physiology and dysfunction to γ-aminobutyric acid signalling is increasingly documented in a range of neurological diseases. In this review, we briefly chart the neurodevelopment of γ-aminobutyric acid and its two major receptor subtypes: the γ-aminobutyric acid A and γ-aminobutyric acid B receptors, starting from the humble invertebrate origins of being an ‘interesting molecule’ acting at a single γ-aminobutyric acid receptor type, to one of the brain’s most important neurochemical components and vital drug targets for major therapeutic classes of drugs. We document the period of molecular cloning and the explosive influence this had on the field of neuroscience and pharmacology up to the present day and the production of atomic γ-aminobutyric acid A and γ-aminobutyric acid B receptor structures. γ-Aminobutyric acid is no longer a humble molecule but the instigator of rich and powerful signalling processes that are absolutely vital for healthy brain function.
... Overall, our results are consistent with results collected via mutagenesis and photolabeling involving GABA A receptors over the past two decades, particularly those reported in Refs. (26,29,30,34,40,58,59,(67)(68)(69)(70). These studies have focused exclusively on protein-mediated mechanisms of anesthetic binding. ...
GABA(A) receptors are pentameric ligand-gated ion channels playing a critical role in the modulation of neuronal excitability. These inhibitory receptors, gated by γ -aminobutyric acid (GABA), can be potentiated and even directly activated by intravenous and inhalational anesthetics. Intersubunit cavities in the transmembrane domain have been consistently identified as putative binding sites by numerous experiment and simulation results. Synaptic GABA(A) receptors are predominantly found in a 2 α :2 β :1 γ stoichiometry, with four unique inter-subunit interfaces. Experimental and computational results have suggested a perplexing specificity, given that cavity-lining residues are highly conserved, and the functional effects of general anesthetics are only weakly sensitive to most mutations of cavity residues. Here we use Molecular Dynamics simulations and thermodynamically rigorous alchemical free energy perturbation (AFEP) techniques to calculate affinities of the intravenous anesthetic propofol and the inhaled anesthetic sevoflurane to all intersubunit sites in a heteromeric GABA(A) receptor. We find that the best predictor of general anesthetic affinity for the intersubunit cavity sites is water displacement: combinations of anesthetic and binding site that displace more water molecules have higher affinities than those that displace fewer. The amount of water displacement is, in turn, a function of size of the general anesthetic, successful competition of the general anesthetic with water for the few hydrogen bonding partners in the site, and inaccessibility of the site to lipid acyl chains. The latter explains the surprisingly low affinity of GAs for the γ − α intersubunit site, which is missing a bulky methionine residue at the cavity entrance and can be occupied by acyl chains in the unbound state. Simulations also identify sevoflurane binding sites in the β subunit centers and in the pore, but predict that these are lower affinity than the intersubunit sites.
Significance
After over a century of research, it is established that general anesthetics interact directly with hydrophobic cavities in proteins. We still do not know why not all small hydrophobic molecules can act as general anesthetics, or why not all hydrophobic cavities bind these molecules. General anesthetics can even select among homologous sites on one critical target, the GABA(A) heteropentamer, although the origins of selectivity are unknown. Here we used rigorous free energy calculations to find that binding affinity correlates with the number of released water molecules, which in turn depends upon the lipid content of the cavity without bound anesthetic. Results suggest a mechanism that reconciles lipid-centered and protein-centered theories, and which can directly inform design of new anesthetics.
... Interestingly, the TM2 F14′A mutation widens a hydrophilic passage between the intrasubunit and intersubunit cavities, suggesting a functional linkage between these two anesthetic binding regions. Recent structural evidence demonstrates binding of propofol to GLIC channels bearing mutations within the intersubunit site [11], and photolabeling studies show modification by barbiturates and isoflurane in this region [3,7]. Additionally, a distinct intersubunit region at the cytoplasmic end of the channel has been implicated by structural and photolabeling studies as a binding site for anesthetic neurosteroids [16,17], though neurosteroids have also been found to bind to an intrasubunit neurosteroid bindings site at the EC end of GLIC [30,31]. ...
Significant progress has been made in the 21st century towards a comprehensive understanding of the mechanisms of action of general anesthetics, coincident with progress in structural biology and molecular, cellular, and systems neuroscience. This review summarizes important new findings that include target identification through structural determination of anesthetic binding sites, details of receptors and ion channels involved in neurotransmission, and the critical roles of neuronal networks in anesthetic effects on memory and consciousness. These recent developments provide a comprehensive basis for conceptualizing pharmacological control of amnesia, unconsciousness, and immobility.
... The + and + sites are intrinsically non-equivalent due to differences in the amino acids contributed by M3 and M3 helices to the + side of interfaces, but in the presence of GABA, R-mTFD-MPAB binds with similar high affinity to both sites (Jayakar et al., 2015). ...
... we found in 21 independent GABA A R purifications that the ratio of specific incorporation of (Jayakar et al., 2015). Under our photolabeling conditions at ~1 M [ 3 H]R-mTFD-MPAB, it is the + -βsite that is occupied and photolabeled (B bic -B ns ), and the enhanced photolabeling in the presence of GABA or other modulator primarily reflects increased binding to the + - ...
... In the presence of GABA, [ 3 H]R-mTFD-MPAB photoincorporation in the subunit resulted from labeling of the and sites with similar efficiency (Chiara et al., 2013;Jayakar et al., 2015). ...
GABAA receptors (GABAARs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([3H]azietomidate) and mephobarbital [[3H]1-methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid ([3H]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the α1β3γ2 GABAAR transmembrane domain at β+-α- (β+ sites) and α+-β-/γ+-β- (β- sites) subunit interfaces. We now use competition photolabeling with [3H]azietomidate and [3H]R-mTFD-MPAB to identify para-substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to β+, while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to β- sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the α+-β- and γ+-β- sites. However, we discovered four compounds that bind with different affinities to the two β- interface sites. Two of these bind with higher affinity to one of the β- sites than to the β+ sites. We deduce that 4-benzoyl-propofol binds with >100-fold higher affinity to the γ+-β- site than to the α+-β- or β+-α- sites, whereas loreclezole, an anticonvulsant, binds with 5- and 100-fold higher affinity to the α+-β- site than to the β+ and γ+-β- sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABAAR transmembrane domain, a property that may facilitate the development of subtype selective GABAAR PAMs.
... They found the GluCl structure generated models that were more appropriate to perform this study because this receptor was crystallized with an ivermectin molecule bound to a homologous site to the one in question. Other contemporary models aided the identification of the binding sites for alcohol in α 2 β 2 [116] and ρ 1 [115] receptors, in the middle section of the M2 helix from the TMD, as well as the recognition of transmembrane inter-subunit binding sites for inhibition barbiturates [147], propofol and its analogs [148] and etomidate [149]. GABA A Rs are also modulated by hormones, such as the thyroidal T3 (L-3,5,3 -triiodothyronine). ...
The pentameric γ-aminobutyric acid type A receptors are ion channels activated by ligands, which intervene in the rapid inhibitory transmission in the mammalian CNS. Due to their rich pharmacology and therapeutic potential, it is essential to understand their structure and function thoroughly. This deep characterization was hampered by the lack of experimental structural information for many years. Thus, computational techniques have been extensively combined with experimental data, in order to undertake the study of γ-aminobutyric acid type A receptors and their interaction with drugs. Here, we review the exciting journey made to assess the structures of these receptors and outline major outcomes. Finally, we discuss the brand-new structure of the α1β2γ2 subtype and the amazing advances it brings to the field.
... Also, the blockade of gamma-aminobutyric acid (GABA) and glycine inhibitory chloride channels descend the depressant effect of anesthetics in the intact spinal cord (19,20). The GABA receptor and glycine receptor function could be potentiated with most of intravenous anesthetics (21). When local anesthetics show depressant effect, the GABA receptor complex conducts disinhibition of nervous conduction because of the blockade of sodium channels. ...
... For example, unlike etomidate, barbiturates (Feng et al., 2004;Feng and Macdonald, 2010) and the neurosteroid tetrahydrodeoxycorticosterone (Wohlfarth et al., 2002) increase the desensitization of a1b3d receptors. These general anesthetics act at different binding sites on a1b2/3g2 receptors (Jayakar et al., 2015;Feng and Forman, 2018) and probably also on a1b3d receptors. Thus, distinct anesthetic binding sites may be differentially coupled with receptor desensitization. ...
Central a4bd receptors are the most abundant isoform of d subunit-containing extrasynaptic GABAA receptors that mediate tonic inhibition. Although the amplitude of GABA-activated currents through a4bd receptors is modulated by multiple general anesthetics, the effects of general anesthetics on desensitization and deactivation of a4bd receptors remain unknown. In the current study, we investigated the effect of etomidate, a potent general anesthetic, on the kinetics and the pseudo steady-state current amplitude of a4b3d receptors inducibly expressed in human embryonic kidney 293 TetR cells. Etomidate directly activates a4b3d receptors in a concentration-dependent manner. Etomidate at a clinically relevant concentration (3.2 mM) enhances maximal response without altering the EC50 of GABA concentration response. Etomidate also increases the extent of desensitization and prolongs the deactivation of a4b3d receptors in the presence of maximally activating concentrations of GABA (1 mM). To mimic the modulatory effect of etomidate on tonic currents, long pulses (30-60 seconds) of a low GABA concentration (1 mM) were applied to activate a4b3d receptors in the absence and presence of etomidate. Although etomidate increases the desensitization of a4b3d receptors, the pseudo steady-state current amplitude at 1 mM GABA is augmented by etomidate. Our data demonstrate that etomidate enhances the pseudo steady-state current of a4b3d receptors evoked by a GABA concentration comparable to an ambient GABA level, suggesting that a4b3d receptors may mediate etomidate's anesthetic effect in the brain. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics
