CGP7930 - An allosteric modulator of GABABRs, GABAARs and inwardly-rectifying potassium channels.

Type-A and -B GABA receptors (GABAARs/GABABRs) control brain function and behaviour by fine tuning neurotransmission. Over-time these receptors have become important therapeutic targets for treating neurodevelopmental and neuropsychiatric disorders. Several positive allosteric modulators (PAMs) of GABARs have reached the clinic and selective targeting of receptor subtypes is crucial. For GABABRs, CGP7930 is a widely used PAM for in vivo studies, but its full pharmacological profile has not yet been established. Here, we reveal that CGP7930 has multiple effects not only on GABABRs but also GABAARs, which for the latter involves potentiation of GABA currents, direct receptor activation, and also inhibition. Furthermore, at higher concentrations, CGP7930 also blocks G protein-coupled inwardly-rectifying K+ (GIRK) channels diminishing GABABR signalling in HEK 293 cells. In male and female rat hippocampal neuron cultures, CGP7930 allosteric effects on GABAARs caused prolonged rise and decay times and reduced the frequency of inhibitory postsynaptic currents and potentiated GABAAR-mediated tonic inhibition. Additional comparison between predominant synaptic- and extrasynaptic-isoforms of GABAAR indicated no evident subtype selectivity for CGP7930. In conclusion, our study of CGP7930 modulation of GABAARs, GABABRs and GIRK channels, indicates this compound is unsuitable for use as a specific GABABR PAM.

Pregnenolone sulfate analogues differentially modulate GABAA receptor closed/desensitised states.

BACKGROUND AND PURPOSE: GABAA receptors are regulated by numerous classes of allosteric modulators. However, regulation of receptor macroscopic desensitisation remains largely unexplored and may offer new therapeutic opportunities. Here, we report the emerging potential for modulating desensitisation with analogues of the endogenous inhibitory neurosteroid, pregnenolone sulfate. EXPERIMENTAL APPROACH: New pregnenolone sulfate analogues were synthesised incorporating various heterocyclic substitutions located at the C-21 position on ring D. The pharmacological profiles of these compounds were assessed using electrophysiology and recombinant GABAA receptors together with mutagenesis, molecular dynamics simulations, structural modelling and kinetic simulations. KEY RESULTS: All seven analogues retained a negative allosteric modulatory capability whilst exhibiting diverse potencies. Interestingly, we observed differential effects on GABA current decay by compounds incorporating either a six- (compound 5) or five-membered heterocyclic ring (compound 6) on C-21, which was independent of their potencies as inhibitors. We propose that differences in molecular charges, and the targeted binding of analogues to specific states of the GABAA receptor, are the most likely cause of the distinctive functional profiles. CONCLUSIONS AND IMPLICATIONS: Our findings reveal that heterocyclic addition to inhibitory neurosteroids not only affected their potency and macroscopic efficacy but also affected innate receptor mechanisms that underlie desensitisation. Acute modulation of macroscopic desensitisation will determine the degree and duration of GABA inhibition, which are vital for the integration of neural circuit activity. Discovery of this form of modulation could present an opportunity for next-generation GABAA receptor drug design and development.

Pre- and postsynaptic modulation of hippocampal inhibitory synaptic transmission by pregnenolone sulphate.

Neurosteroids are important endogenous modulators of GABAA receptor-mediated neurotransmission within the CNS and play a vital role in maintaining normal healthy brain function. Research has mainly focussed on neurosteroids such as allopregnanolone and tetrahydro-deoxycorticosterone (THDOC) which are allosteric potentiators of GABAA receptors, whilst the sulphated steroids, including pregnenolone sulphate (PS), which inhibit GABAA receptor function, have been relatively neglected. Importantly, a full description of PS effects on inhibitory synaptic transmission, at concentrations that are expected to inhibit postsynaptic GABAA receptors, is lacking. Here, we address this deficit by recording inhibitory postsynaptic currents (IPSCs) from rat hippocampal neurons both in culture and in acute brain slices and explore the impact of PS at micromolar concentrations. We reveal that PS inhibits postsynaptic GABAA receptors, evident from reductions in IPSC amplitude and decay time. Concurrently, PS also causes an increase in synaptic GABA release which we discover is due to the activation of presynaptic TRPM3 receptors located close to presynaptic GABA release sites. Pharmacological blockade of TRPM3 receptors uncovers a PS-evoked reduction in IPSC frequency. This second presynaptic effect is caused by PS activation of inwardly-rectifying Kir2.3 channels on interneurons, which act to depress synaptic GABA release. Overall, we provide a comprehensive characterisation of pre- and postsynaptic modulation by PS of inhibitory synaptic transmission onto hippocampal neurons which elucidates the diverse mechanisms by which this understudied neurosteroid can modulate brain function.

Phosphorylation of neuroligin-2 by PKA regulates its cell surface abundance and synaptic stabilization.

The trans-synaptic adhesion molecule neuroligin-2 (NL2) is essential for the development and function of inhibitory synapses. NL2 recruits the postsynaptic scaffold protein gephyrin, which, in turn, stabilizes γ-aminobutyric acid type A receptors (GABAARs) in the postsynaptic domain. Thus, the amount of NL2 at the synapse can control synaptic GABAAR concentration to tune inhibitory neurotransmission efficacy. Here, using biochemistry, imaging, single-particle tracking, and electrophysiology, we uncovered a key role for cAMP-dependent protein kinase (PKA) in the synaptic stabilization of NL2. We found that PKA-mediated phosphorylation of NL2 at Ser714 caused its dispersal from the synapse and reduced NL2 surface amounts, leading to a loss of synaptic GABAARs. Conversely, enhancing the stability of NL2 at synapses by abolishing PKA-mediated phosphorylation led to increased inhibitory signaling. Thus, PKA plays a key role in regulating NL2 function and GABA-mediated synaptic inhibition.

Mechanisms of inhibition and activation of extrasynaptic αß GABAA receptors.

Type A GABA (γ-aminobutyric acid) receptors represent a diverse population in the mammalian brain, forming pentamers from combinations of α-, ß-, γ-, δ-, ε-, ρ-, θ- and π-subunits1. αß, α4ßδ, α6ßδ and α5ßγ receptors favour extrasynaptic localization, and mediate an essential persistent (tonic) inhibitory conductance in many regions of the mammalian brain1,2. Mutations of these receptors in humans are linked to epilepsy and insomnia3,4. Altered extrasynaptic receptor function is implicated in insomnia, stroke and Angelman and Fragile X syndromes1,5, and drugs targeting these receptors are used to treat postpartum depression6. Tonic GABAergic responses are moderated to avoid excessive suppression of neuronal communication, and can exhibit high sensitivity to Zn2+ blockade, in contrast to synapse-preferring α1ßγ, α2ßγ and α3ßγ receptor responses5,7-12. Here, to resolve these distinctive features, we determined structures of the predominantly extrasynaptic αß GABAA receptor class. An inhibited state bound by both the lethal paralysing agent α-cobratoxin13 and Zn2+ was used in comparisons with GABA-Zn2+ and GABA-bound structures. Zn2+ nullifies the GABA response by non-competitively plugging the extracellular end of the pore to block chloride conductance. In the absence of Zn2+, the GABA signalling response initially follows the canonical route until it reaches the pore. In contrast to synaptic GABAA receptors, expansion of the midway pore activation gate is limited and it remains closed, reflecting the intrinsic low efficacy that characterizes the extrasynaptic receptor. Overall, this study explains distinct traits adopted by αß receptors that adapt them to a role in tonic signalling.

AKAP79 enables calcineurin to directly suppress protein kinase A activity.

Interplay between the second messengers cAMP and Ca2+ is a hallmark of dynamic cellular processes. A common motif is the opposition of the Ca2+-sensitive phosphatase calcineurin and the major cAMP receptor, protein kinase A (PKA). Calcineurin dephosphorylates sites primed by PKA to bring about changes including synaptic long-term depression (LTD). AKAP79 supports signaling of this type by anchoring PKA and calcineurin in tandem. In this study, we discovered that AKAP79 increases the rate of calcineurin dephosphorylation of type II PKA regulatory subunits by an order of magnitude. Fluorescent PKA activity reporter assays, supported by kinetic modeling, show how AKAP79-enhanced calcineurin activity enables suppression of PKA without altering cAMP levels by increasing PKA catalytic subunit capture rate. Experiments with hippocampal neurons indicate that this mechanism contributes toward LTD. This non-canonical mode of PKA regulation may underlie many other cellular processes.

Structural determinants and regulation of spontaneous activity in GABAA receptors.

GABAA receptors are vital for controlling neuronal excitability and can display significant levels of constitutive activity that contributes to tonic inhibition. However, the mechanisms underlying spontaneity are poorly understood. Here we demonstrate a strict requirement for ß3 subunit incorporation into receptors for spontaneous gating, facilitated by α4, α6 and δ subunits. The crucial molecular determinant involves four amino acids (GKER) in the ß3 subunit's extracellular domain, which interacts with adjacent receptor subunits to promote transition to activated, open channel conformations. Spontaneous activity is further regulated by ß3 subunit phosphorylation and by allosteric modulators including neurosteroids and benzodiazepines. Promoting spontaneous activity reduced neuronal excitability, indicating that spontaneous currents will alter neural network activity. This study demonstrates how regional diversity in GABAA receptor isoform, protein kinase activity, and neurosteroid levels, can impact on tonic inhibition through the modulation of spontaneous GABAA receptor gating.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Ion channels.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15539. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Physiological role for GABAA receptor desensitization in the induction of long-term potentiation at inhibitory synapses.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels distributed throughout the brain where they mediate synaptic and tonic inhibition. Following activation, these receptors undergo desensitization which involves entry into long-lived agonist-bound closed states. Although the kinetic effects of this state are recognised and its structural basis has been uncovered, the physiological impact of desensitization on inhibitory neurotransmission remains unknown. Here we describe an enduring form of long-term potentiation at inhibitory synapses that elevates synaptic current amplitude for 24 h following desensitization of GABAARs in response to agonist exposure or allosteric modulation. Using receptor mutants and allosteric modulators we demonstrate that desensitization of GABAARs facilitates their phosphorylation by PKC, which increases the number of receptors at inhibitory synapses. These observations provide a physiological relevance to the desensitized state of GABAARs, acting as a signal to regulate the efficacy of inhibitory synapses during prolonged periods of inhibitory neurotransmission.

Differential Coassembly of α1-GABAARs Associated with Epileptic Encephalopathy.

GABAA receptors (GABAARs) are profoundly important for controlling neuronal excitability. Spontaneous and familial mutations to these receptors feature prominently in excitability disorders and neurodevelopmental deficits following disruption to GABA-mediated inhibition. Recent genotyping of an individual with severe epilepsy and Williams-Beuren syndrome identified a frameshifting de novo variant in a major GABAAR gene, GABRA1 This truncated the α1 subunit between the third and fourth transmembrane domains and introduced 24 new residues forming the mature protein, α1Lys374Serfs*25 Cell surface expression of mutant murine GABAARs is severely impaired compared with WT, due to retention in the endoplasmic reticulum. Mutant receptors were differentially coexpressed with ß3, but not with ß2, subunits in mammalian cells. Reduced surface expression was reflected by smaller IPSCs, which may underlie the induction of seizures. The mutant does not have a dominant-negative effect on native neuronal GABAAR expression since GABA current density was unaffected in hippocampal neurons, although mutant receptors exhibited limited GABA sensitivity. To date, the underlying mechanism is unique for epileptogenic variants and involves differential ß subunit expression of GABAAR populations, which profoundly affected receptor function and synaptic inhibition.SIGNIFICANCE STATEMENT GABAARs are critical for controlling neural network excitability. They are ubiquitously distributed throughout the brain, and their dysfunction underlies many neurologic disorders, especially epilepsy. Here we report the characterization of an α1-GABAAR variant that results in severe epilepsy. The underlying mechanism is structurally unusual, with the loss of part of the α1 subunit transmembrane domain and part-replacement with nonsense residues. This led to compromised and differential α1 subunit cell surface expression with ß subunits resulting in severely reduced synaptic inhibition. Our study reveals that disease-inducing variants can affect GABAAR structure, and consequently subunit assembly and cell surface expression, critically impacting on the efficacy of synaptic inhibition, a property that will orchestrate the extent and duration of neuronal excitability.

Optopharmacology reveals a differential contribution of native GABAA receptors to dendritic and somatic inhibition using azogabazine.

γ-aminobutyric acid type-A receptors (GABAARs) are inhibitory ligand-gated ion channels in the brain that are crucial for controlling neuronal excitation. To explore their physiological roles in cellular and neural network activity, it is important to understand why specific GABAAR isoforms are distributed not only to various brain regions and cell types, but also to specific areas of the membrane in individual neurons. To address this aim we have developed a novel photosensitive compound, azogabazine, that targets and reversibly inhibits GABAARs. The receptor selectivity of the compound is based on the competitive antagonist, gabazine, and photosensitivity is conferred by a photoisomerisable azobenzene group. Azogabazine can exist in either cis or trans conformations that are controlled by UV and blue light respectively, to affect receptor inhibition. We report that the trans-isomer preferentially binds and inhibits GABAAR function, whilst promotion of the cis-isomer caused unbinding of azogabazine from GABAARs. Using cultured cerebellar granule cells, azogabazine in conjunction with UV light applied to defined membrane domains, revealed higher densities of GABAARs at somatic inhibitory synapses compared to those populating proximal dendritic zones, even though the latter displayed a higher number of synapses per unit area of membrane. Azogabazine also revealed more pronounced GABA-mediated inhibition of action potential firing in proximal dendrites compared to the soma. Overall, azogabazine is a valuable addition to the photochemical toolkit that can be used to interrogate GABAAR function and inhibition.

GABAAR isoform and subunit structural motifs determine synaptic and extrasynaptic receptor localisation.

GABAA receptors (GABAARs) are the principal inhibitory neurotransmitter receptors in the central nervous system. They control neuronal excitability by synaptic and tonic forms of inhibition mostly mediated by different receptor subtypes located in specific cell membrane subdomains. A consensus suggests that α1-3ßγ comprise synaptic GABAARs, whilst extrasynaptic α4ßδ, α5ßγ and αß isoforms largely underlie tonic inhibition. Although some structural features that enable the spatial segregation of receptors are known, the mobility of key synaptic and extrasynaptic GABAARs are less understood, and yet this is a key determinant of the efficacy of GABA inhibition. To address this aspect, we have incorporated functionally silent α-bungarotoxin binding sites (BBS) into prominent hippocampal GABAAR subunits which mediate synaptic and tonic inhibition. Using single particle tracking with quantum dots we demonstrate that GABAARs that are traditionally considered to mediate synaptic or tonic inhibition are all able to access inhibitory synapses. These isoforms have variable diffusion rates and are differentially retained upon entering the synaptic membrane subdomain. Interestingly, α2 and α4 subunits reside longer at synapses compared to α5 and δ subunits. Furthermore, a high proportion of extrasynaptic δ-containing receptors exhibited slower diffusion compared to δ subunits at synapses. A chimera formed from δ-subunits, with the intracellular domain of γ2L, reversed this behaviour. In addition, we observed that receptor activation affected the diffusion of extrasynaptic, but not of synaptic GABAARs. Overall, we conclude that the differential mobility profiles of key synaptic and extrasynaptic GABAARs are determined by receptor subunit composition and intracellular structural motifs. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

Developing New 4-PIOL and 4-PHP Analogues for Photoinactivation of γ-Aminobutyric Acid Type A Receptors.

The critical roles played by GABAA receptors as inhibitory regulators of excitation in the central nervous system has been known for many years. Aberrant GABAA receptor function and trafficking deficits have also been associated with several diseases including anxiety, depression, epilepsy, and insomnia. As a consequence, important drug groups such as the benzodiazepines, barbiturates, and many general anesthetics have become established as modulators of GABAA receptor activity. Nevertheless, there is much we do not understand about the roles and mechanisms of GABAA receptors at neural network and systems levels. It is therefore crucial to develop novel technologies and especially chemical entities that can interrogate GABAA receptor function in the nervous system. Here, we describe the chemistry and characterization of a novel set of 4-PIOL and 4-PHP analogues synthesized with the aim of developing a toolkit of drugs that can photoinactivate GABAA receptors. Most of these new analogues show higher affinities/potencies compared with the respective lead compounds. This is indicative of cavernous areas being present near their binding sites that can be potentially associated with novel receptor interactions. The 4-PHP azide-analogue, 2d, possesses particularly impressive nanomolar affinity/potency and is an effective UV-inducible photoinhibitor of GABAA receptors with considerable potential for photocontrol of GABAA receptor function in situ.

A half century of γ-aminobutyric acid.

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 acidA and γ-aminobutyric acidB 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 acidA and γ-aminobutyric acidB 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.

Cell surface expression of homomeric GABAA receptors depends on single residues in subunit transmembrane domains.

Cell surface expression of type A GABA receptors (GABAARs) is a critical determinant of the efficacy of inhibitory neurotransmission. Pentameric GABAARs are assembled from a large pool of subunits according to precise co-assembly rules that limit the extent of receptor structural diversity. These rules ensure that particular subunits, such as ρ1 and ß3, form functional cell surface ion channels when expressed alone in heterologous systems, whereas other brain-abundant subunits, such as α and γ, are retained within intracellular compartments. Why some of the most abundant GABAAR subunits fail to form homomeric ion channels is unknown. Normally, surface expression of α and γ subunits requires co-assembly with ß subunits via interactions between their N-terminal sequences in the endoplasmic reticulum. Here, using molecular biology, imaging, and electrophysiology with GABAAR chimeras, we have identified two critical residues in the transmembrane domains of α and γ subunits, which, when substituted for their ρ1 counterparts, permit cell surface expression as homomers. Consistent with this, substitution of the ρ1 transmembrane residues for the α subunit equivalents reduced surface expression and altered channel gating, highlighting their importance for GABAAR trafficking and signaling. Although not ligand-gated, the formation of α and γ homomeric ion channels at the cell surface was revealed by incorporating a mutation that imparts the functional signature of spontaneous channel activity. Our study identifies two single transmembrane residues that enable homomeric GABAAR subunit cell surface trafficking and demonstrates that α and γ subunits can form functional ion channels.

Wnt Signaling Mediates LTP-Dependent Spine Plasticity and AMPAR Localization through Frizzled-7 Receptors.

The structural and functional plasticity of synapses is critical for learning and memory. Long-term potentiation (LTP) induction promotes spine growth and AMPAR accumulation at excitatory synapses, leading to increased synaptic strength. Glutamate initiates these processes, but the contribution from extracellular modulators is not fully established. Wnts are required for spine formation; however, their impact on activity-mediated spine plasticity and AMPAR localization is unknown. We found that LTP induction rapidly increased synaptic Wnt7a/b protein levels. Acute blockade of endogenous Wnts or loss of postsynaptic Frizzled-7 (Fz7) receptors impaired LTP-mediated synaptic strength, spine growth, and AMPAR localization at synapses. Live imaging of SEP-GluA1 and single-particle tracking revealed that Wnt7a rapidly promoted synaptic AMPAR recruitment and trapping. Wnt7a, through Fz7, induced CaMKII-dependent loss of SynGAP from spines and increased extrasynaptic AMPARs by PKA phosphorylation. We identify a critical role for Wnt-Fz7 signaling in LTP-mediated synaptic accumulation of AMPARs and spine plasticity.

Disease-associated missense mutations in GluN2B subunit alter NMDA receptor ligand binding and ion channel properties.

Genetic and bioinformatic analyses have identified missense mutations in GRIN2B encoding the NMDA receptor GluN2B subunit in autism, intellectual disability, Lennox Gastaut and West Syndromes. Here, we investigated several such mutations using a near-complete, hybrid 3D model of the human NMDAR and studied their consequences with kinetic modelling and electrophysiology. The mutants revealed reductions in glutamate potency; increased receptor desensitisation; and ablation of voltage-dependent Mg2+ block. In addition, we provide new views on Mg2+ and NMDA channel blocker binding sites. We demonstrate that these mutants have significant impact on excitatory transmission in developing neurons, revealing profound changes that could underlie their associated neurological disorders. Of note, the NMDAR channel mutant GluN2BV618G unusually allowed Mg2+ permeation, whereas nearby N615I reduced Ca2+ permeability. By identifying the binding site for an NMDAR antagonist that is used in the clinic to rescue gain-of-function phenotypes, we show that drug binding may be modified by some GluN2B disease-causing mutations.

Probing GABAA receptors with inhibitory neurosteroids.

γ-aminobutyric acid type A receptors (GABAARs) are important components of the central nervous system and they are functionally tasked with controlling neuronal excitability. These receptors are subject to post-translational modification and also to modulation by endogenous regulators, such as the neurosteroids. These modulators can either potentiate or inhibit GABAAR function. Whilst the former class of neurosteroids are considered to bind to and act from the transmembrane domain of the receptor, the domains that are important for the inhibitory neurosteroids remain less clear. In this study, we systematically compare a panel of recombinant synaptic-type and extrasynaptic-type GABAARs expressed in heterologous cell systems for their sensitivity to inhibition by the classic inhibitory neurosteroid, pregnenolone sulphate. Generally, peak GABA current responses were inhibited less compared to steady-state currents, implicating the desensitised state in inhibition. Moreover, pregnenolone sulphate inhibition increased with GABA concentration, but showed minimal voltage dependence. There was no strong dependence of inhibition on receptor subunit composition, the exception being the ρ1 receptor, which is markedly less sensitive. By using competition experiments with pregnenolone sulphate and the GABA channel blocker picrotoxinin, discrete binding sites are proposed. Furthermore, by assessing inhibition using site-directed mutagenesis and receptor chimeras comprising α, ß or γ subunits with ρ1 subunits, the receptor transmembrane domains are strongly implicated in mediating inhibition and most likely the binding location for pregnenolone sulphate in GABAARs. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.

γ-Aminobutyric acid receptors (GABAARs) are vital for controlling excitability in the brain. This is emphasized by the numerous neuropsychiatric disorders that result from receptor dysfunction. A critical component of most native GABAARs is the α subunit. Its transmembrane domain is the target for many modulators, including endogenous brain neurosteroids that impact anxiety, stress and depression, and for therapeutic drugs, such as general anesthetics. Understanding the basis for the modulation of GABAAR function requires high-resolution structures. Here we present the first atomic structures of a GABAAR chimera at 2.8-Å resolution, including those bound with potentiating and inhibitory neurosteroids. These structures define new allosteric binding sites for these modulators that are associated with the α-subunit transmembrane domain. Our findings will enable the exploitation of neurosteroids for therapeutic drug design to regulate GABAARs in neurological disorders.