International Union of Basic and Clinical Pharmacology. CVI: GABAA Receptor Subtype- and Function-selective Ligands: Key Issues in Translation to Humans

GABAA receptors are themajor inhibitory transmitter receptors in the brain. They are ligand-gated chloride channels and the site of action of benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and convulsants. GABAA receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 homologous subunits and their distinct regional, cellular, and subcellular distribution gives rise to a large number of GABAA receptor subtypes with distinct pharmacology, which modulate different functions of the brain. A variety of compounds have been identified that were claimed to modulate selectively individual GABAA receptor subtypes. However, many of these compounds have only incompletely been investigated or, in addition to a preferential modulation of a receptor subtype, also modulate other subtypes at similar concentrations. Although their differential efficacy at distinct receptor subtypes reduced side effects in behavioral experiments in rodents, the exact receptor subtypes mediating their behavioral effects cannot be unequivocally delineated. In addition, the discrepant in vivo effects of some of these compounds in rodents and man raised doubts on the applicability of the concept of receptor subtype selectivity as a guide for the development of clinically useful drugs. Here, we provide an up-to-date review on the currently available GABAA receptor subtype-selective ligands. We present data on their actual activity atGABAA receptor subtypes, discuss the translational aspect of subtype-selective drugs, and make proposals for the future development of ligands with better anxioselectivity in humans. Finally, we discuss possible ways to strengthen the conclusions of behavioral studies with the currently available drugs

Gephyrin, the enigmatic organizer at GABAergic synapses

GABAA receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows for an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABAA receptors have also been shown to directly interact with gephyrin and interaction sites have been identified and mapped within the intracellular loops of the GABAA receptor α1, α2, and α3 subunits. Gephyrin-binding to GABAA receptors seems to be at least one order of magnitude weaker than to glycine receptors (GlyRs) and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but also plays a crucial role in synaptic dynamics and is a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signaling proteins into close spatial proximity

GABAA receptors in GtoPdb v.2023.1

The GABAA receptor is a ligand-gated ion channel of the Cys-loop family that includes the nicotinic acetylcholine, 5-HT3 and strychnine-sensitive glycine receptors. GABAA receptor-mediated inhibition within the CNS occurs by fast synaptic transmission, sustained tonic inhibition and temporally intermediate events that have been termed 'GABAA, slow' [45]. GABAA receptors exist as pentamers of 4TM subunits that form an intrinsic anion selective channel. Sequences of six α, three β, three γ, one δ, three ρ, one ε, one π and one θ GABAA receptor subunits have been reported in mammals [281, 237, 238, 288]. The π-subunit is restricted to reproductive tissue. Alternatively spliced versions of many subunits exist (e.g. α4- and α6- (both not functional) α5-, β2-, β3- and γ2), along with RNA editing of the α3 subunit [71]. The three ρ-subunits, (ρ1-3) function as either homo- or hetero-oligomeric assemblies [365, 50]. Receptors formed from ρ-subunits, because of their distinctive pharmacology that includes insensitivity to bicuculline, benzodiazepines and barbiturates, have sometimes been termed GABAC receptors [365], but they are classified as GABAA receptors by NC-IUPHAR on the basis of structural and functional criteria [16, 237, 238].Many GABAA receptor subtypes contain α-, β- and γ-subunits with the likely stoichiometry 2α.2β.1γ [170, 237]. It is thought that the majority of GABAA receptors harbour a single type of α- and β -subunit variant. The α1β2γ2 hetero-oligomer constitutes the largest population of GABAA receptors in the CNS, followed by the α2β3γ2 and α3β3γ2 isoforms. Receptors that incorporate the α4- α5-or α6-subunit, or the β1-, γ1-, γ3-, δ-, ε- and θ-subunits, are less numerous, but they may nonetheless serve important functions. For example, extrasynaptically located receptors that contain α6- and δ-subunits in cerebellar granule cells, or an α4- and δ-subunit in dentate gyrus granule cells and thalamic neurones, mediate a tonic current that is important for neuronal excitability in response to ambient concentrations of GABA [211, 275, 84, 19, 293]. GABA binding occurs at the β+/α- subunit interface and the homologous γ+/α- subunits interface creates the benzodiazepine site. A second site for benzodiazepine binding has recently been postulated to occur at the α+/β- interface ([257]; reviewed by [287]). The particular α-and γ-subunit isoforms exhibit marked effects on recognition and/or efficacy at the benzodiazepine site. Thus, receptors incorporating either α4- or α6-subunits are not recognised by ‘classical’ benzodiazepines, such as flunitrazepam (but see [362]). The trafficking, cell surface expression, internalisation and function of GABAA receptors and their subunits are discussed in detail in several recent reviews [52, 141, 190, 322] but one point worthy of note is that receptors incorporating the γ2 subunit (except when associated with α5) cluster at the postsynaptic membrane (but may distribute dynamically between synaptic and extrasynaptic locations), whereas those incorporating the δ subunit appear to be exclusively extrasynaptic. NC-IUPHAR [16, 237, 3, 2] class the GABAA receptors according to their subunit structure, pharmacology and receptor function. Currently, eleven native GABAA receptors are classed as conclusively identified (i.e., α1β2γ2, α2βγ2, α3βγ2, α4βγ2, α4β2δ, α4β3δ, α5βγ2, α6βγ2, α6β2δ, α6β3δ and ρ) with further receptor isoforms occurring with high probability, or only tentatively [237, 238]. It is beyond the scope of this Guide to discuss the pharmacology of individual GABAA receptor isoforms in detail; such information can be gleaned in the reviews [16, 96, 170, 175, 144, 281, 218, 237, 238, 284, 9, 10]. Agents that discriminate between α-subunit isoforms are noted in the table and additional agents that demonstrate selectivity between receptor isoforms, for example via β-subunit selectivity, are indicated in the text below. The distinctive agonist and antagonist pharmacology of ρ receptors is summarised in the table and additional aspects are reviewed in [365, 50, 146, 225].Several high-resolution cryo-electron microscopy structures have been described in which the full-length human α1β3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam [200]

GABAA receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The GABAA receptor is a ligand-gated ion channel of the Cys-loop family that includes the nicotinic acetylcholine, 5-HT3 and strychnine-sensitive glycine receptors. GABAA receptor-mediated inhibition within the CNS occurs by fast synaptic transmission, sustained tonic inhibition and temporally intermediate events that have been termed ‘GABAA, slow’ [41]. GABAA receptors exist as pentamers of 4TM subunits that form an intrinsic anion selective channel. Sequences of six α, three β, three γ, one δ, three ρ, one ε, one π and one θ GABAA receptor subunits have been reported in mammals [273, 232, 231, 278]. The π-subunit is restricted to reproductive tissue. Alternatively spliced versions of many subunits exist (e.g. α4- and α6- (both not functional) α5-, β2-, β3- and γ2), along with RNA editing of the α3 subunit [67]. The three ρ-subunits, (ρ1-3) function as either homo- or hetero-oligomeric assemblies [354, 46]. Receptors formed from ρ-subunits, because of their distinctive pharmacology that includes insensitivity to bicuculline, benzodiazepines and barbiturates, have sometimes been termed GABAC receptors [354], but they are classified as GABAA receptors by NC-IUPHAR on the basis of structural and functional criteria [14, 232, 231].Many GABAA receptor subtypes contain α-, β- and γ-subunits with the likely stoichiometry 2α.2β.1γ [164, 232]. It is thought that the majority of GABAA receptors harbour a single type of α- and β -subunit variant. The α1β2γ2 hetero-oligomer constitutes the largest population of GABAA receptors in the CNS, followed by the α2β3γ2 and α3β3γ2 isoforms. Receptors that incorporate the α4- α5-or α6-subunit, or the β1-, γ1-, γ3-, δ-, ε- and θ-subunits, are less numerous, but they may nonetheless serve important functions. For example, extrasynaptically located receptors that contain α6- and δ-subunits in cerebellar granule cells, or an α4- and δ-subunit in dentate gyrus granule cells and thalamic neurones, mediate a tonic current that is important for neuronal excitability in response to ambient concentrations of GABA [205, 268, 79, 17, 283]. GABA binding occurs at the β+/α- subunit interface and the homologous γ+/α- subunits interface creates the benzodiazepine site. A second site for benzodiazepine binding has recently been postulated to occur at the α+/β- interface ([250]; reviewed by [277]). The particular α-and γ-subunit isoforms exhibit marked effects on recognition and/or efficacy at the benzodiazepine site. Thus, receptors incorporating either α4- or α6-subunits are not recognised by ‘classical’ benzodiazepines, such as flunitrazepam (but see [351]). The trafficking, cell surface expression, internalisation and function of GABAA receptors and their subunits are discussed in detail in several recent reviews [48, 136, 184, 311] but one point worthy of note is that receptors incorporating the γ2 subunit (except when associated with α5) cluster at the postsynaptic membrane (but may distribute dynamically between synaptic and extrasynaptic locations), whereas as those incorporating the d subunit appear to be exclusively extrasynaptic. NC-IUPHAR [14, 232] class the GABAA receptors according to their subunit structure, pharmacology and receptor function. Currently, eleven native GABAA receptors are classed as conclusively identified (i.e., α1β2γ2, α1βγ2, α3βγ2, α4βγ2, α4β2δ, α4β3δ, α5βγ2, α6βγ2, α6β2δ, α6β3δ and ρ) with further receptor isoforms occurring with high probability, or only tentatively [232, 231]. It is beyond the scope of this Guide to discuss the pharmacology of individual GABAA receptor isoforms in detail; such information can be gleaned in the reviews [14, 91, 164, 169, 140, 273, 212, 232, 231] and [8, 7]. Agents that discriminate between α-subunit isoforms are noted in the table and additional agents that demonstrate selectivity between receptor isoforms, for example via β-subunit selectivity, are indicated in the text below. The distinctive agonist and antagonist pharmacology of ρ receptors is summarised in the table and additional aspects are reviewed in [354, 46, 141, 219].Several high-resolution cryo-electron microscopy structures have been described in which the full-length human α1β3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam [194]

GABAA receptors in GtoPdb v.2021.3

The GABAA receptor is a ligand-gated ion channel of the Cys-loop family that includes the nicotinic acetylcholine, 5-HT3 and strychnine-sensitive glycine receptors. GABAA receptor-mediated inhibition within the CNS occurs by fast synaptic transmission, sustained tonic inhibition and temporally intermediate events that have been termed 'GABAA, slow' [45]. GABAA receptors exist as pentamers of 4TM subunits that form an intrinsic anion selective channel. Sequences of six α, three β, three γ, one δ, three ρ, one ε, one π and one θ GABAA receptor subunits have been reported in mammals [278, 235, 236, 283]. The π-subunit is restricted to reproductive tissue. Alternatively spliced versions of many subunits exist (e.g. α4- and α6- (both not functional) α5-, β2-, β3- and γ2), along with RNA editing of the α3 subunit [71]. The three ρ-subunits, (ρ1-3) function as either homo- or hetero-oligomeric assemblies [359, 50]. Receptors formed from ρ-subunits, because of their distinctive pharmacology that includes insensitivity to bicuculline, benzodiazepines and barbiturates, have sometimes been termed GABAC receptors [359], but they are classified as GABAA receptors by NC-IUPHAR on the basis of structural and functional criteria [16, 235, 236].Many GABAA receptor subtypes contain α-, β- and γ-subunits with the likely stoichiometry 2α.2β.1γ [168, 235]. It is thought that the majority of GABAA receptors harbour a single type of α- and β -subunit variant. The α1β2γ2 hetero-oligomer constitutes the largest population of GABAA receptors in the CNS, followed by the α2β3γ2 and α3β3γ2 isoforms. Receptors that incorporate the α4- α5-or α6-subunit, or the β1-, γ1-, γ3-, δ-, ε- and θ-subunits, are less numerous, but they may nonetheless serve important functions. For example, extrasynaptically located receptors that contain α6- and δ-subunits in cerebellar granule cells, or an α4- and δ-subunit in dentate gyrus granule cells and thalamic neurones, mediate a tonic current that is important for neuronal excitability in response to ambient concentrations of GABA [209, 272, 83, 19, 288]. GABA binding occurs at the β+/α- subunit interface and the homologous γ+/α- subunits interface creates the benzodiazepine site. A second site for benzodiazepine binding has recently been postulated to occur at the α+/β- interface ([254]; reviewed by [282]). The particular α-and γ-subunit isoforms exhibit marked effects on recognition and/or efficacy at the benzodiazepine site. Thus, receptors incorporating either α4- or α6-subunits are not recognised by ‘classical’ benzodiazepines, such as flunitrazepam (but see [356]). The trafficking, cell surface expression, internalisation and function of GABAA receptors and their subunits are discussed in detail in several recent reviews [52, 140, 188, 316] but one point worthy of note is that receptors incorporating the γ2 subunit (except when associated with α5) cluster at the postsynaptic membrane (but may distribute dynamically between synaptic and extrasynaptic locations), whereas as those incorporating the δ subunit appear to be exclusively extrasynaptic. NC-IUPHAR [16, 235, 3, 2] class the GABAA receptors according to their subunit structure, pharmacology and receptor function. Currently, eleven native GABAA receptors are classed as conclusively identified (i.e., α1β2γ2, α1βγ2, α3βγ2, α4βγ2, α4β2δ, α4β3δ, α5βγ2, α6βγ2, α6β2δ, α6β3δ and ρ) with further receptor isoforms occurring with high probability, or only tentatively [235, 236]. It is beyond the scope of this Guide to discuss the pharmacology of individual GABAA receptor isoforms in detail; such information can be gleaned in the reviews [16, 95, 168, 173, 143, 278, 216, 235, 236] and [9, 10]. Agents that discriminate between α-subunit isoforms are noted in the table and additional agents that demonstrate selectivity between receptor isoforms, for example via β-subunit selectivity, are indicated in the text below. The distinctive agonist and antagonist pharmacology of ρ receptors is summarised in the table and additional aspects are reviewed in [359, 50, 145, 223].Several high-resolution cryo-electron microscopy structures have been described in which the full-length human α1β3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam [198]

PWZ-029, a compound with moderate inverse agonist functional selectivity at GABA(A) receptors containing alpha 5 subunits, improves passive, but not active, avoidance learning in rats

Benzodiazepine (BZ) site ligands affect vigilance, anxiety, memory processes, muscle tone and epileptogenic propensity through modulation of neurotransmission at GABA(A) receptors containing alpha 1, alpha 2, alpha 3 or alpha 5 subunits, and may have numerous experimental and clinical applications. The ability of non-selective BZ site inverse agonists to enhance cognition, documented in animal models and human studies, is clinically not feasible due to potentially unacceptable psychomotor effects. Most investigations to date have proposed the alpha 1 and/or alpha 5 subunit-containing GABA(A) receptors as comprising the memory-modulating population of these receptors. The novel ligand PWZ-029, which we synthesized and characterized electrophysiologically, possesses in vitro binding selectivity and moderate inverse agonist functional selectivity at alpha 5-containing GABA(A) receptors. This ligand has also been examined in rats in the passive and active avoidance, spontaneous locomotor activity, elevated plus maze and grip strength tests, primarily predictive of the effects on the memory acquisition, basal locomotor activity, anxiety level and muscle tone, respectively. The improvement of task learning was detected at the dose of 5 mg/kg in the passive, but not active avoidance test. The inverse agonist PWZ-029 had no effect on anxiety or muscle tone, whereas at higher doses (10 and 20 mg/kg) it decreased locomotor activity. This effect was antagonized by flumazenil. and also by the lower (but not the higher) dose of an agonist (SH-053-R-CH3-2'F) selective for GABA(A) receptors containing the alpha 5 subunit. The hypolocomotor effect of PWZ-029 was not antagonized by the antagonist beta-CCt exhibiting a preferential affinity for alpha 1-subunit-containing receptors. These data suggest that moderate negative modulation at GABA(A) receptors containing the alpha 5 subunit is a sufficient condition for eliciting enhanced encoding/consolidation of declarative memory, while the influence of higher doses of modulators at these receptors on motor activity shows an intricate pattern whose relevance and mechanism await to be defined