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Structure of a human synaptic GABAA receptor

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Shaotong Zhu at University of Texas Southwestern Medical Center
Shaotong Zhu
Colleen M. Noviello
Colleen M. Noviello
Colleen M. Noviello
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Jinfeng Teng at University of Texas Southwestern Medical Center
Jinfeng Teng
Richard Walsh Jr at Harvard Medical School
Richard Walsh Jr
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Abstract and Figures

Fast inhibitory neurotransmission in the brain is principally mediated by the neurotransmitter GABA (γ-aminobutyric acid) and its synaptic target, the type A GABA receptor (GABAA receptor). Dysfunction of this receptor results in neurological disorders and mental illnesses including epilepsy, anxiety and insomnia. The GABAA receptor is also a prolific target for therapeutic, illicit and recreational drugs, including benzodiazepines, barbiturates, anaesthetics and ethanol. Here we present high-resolution cryo-electron microscopy structures of the human α1β2γ2 GABAA receptor, the predominant isoform in the adult brain, in complex with GABA and the benzodiazepine site antagonist flumazenil, the first-line clinical treatment for benzodiazepine overdose. The receptor architecture reveals unique heteromeric interactions for this important class of inhibitory neurotransmitter receptor. This work provides a template for understanding receptor modulation by GABA and benzodiazepines, and will assist rational approaches to therapeutic targeting of this receptor for neurological disorders and mental illness.
Alignment of GABAA and other Cys-loop receptor subunits Cryo-EM constructs (γ2 affinity tag not shown) are numbered starting with the first residue of the mature protein. Sequences aligned (UniProt or PDB accession codes): Homo sapiens α1 GABAA (HS, P14867), H. sapiens β2 GABAA (P47870), H. sapiens γ2 GABAA (P18507), H. sapiens GABAA β3 (4COF), H. sapiens glycine α3 (5CFB), Danio rerio glycine α1 (DR, 3JAE), Caenorhabditis elegans α (CE, 3RHW), H. sapiens α4 nAChR (5KXI), H. sapiens β2 nAChR (5KXI) and Mus musculus 5-HT3 receptor (MM, 4PIR). α-helices (cylinders), β-strands (arrows), and inserted linker (cyan) are indicated.
Alignment of GABAA and other Cys-loop receptor subunits Cryo-EM constructs (γ2 affinity tag not shown) are numbered starting with the first residue of the mature protein. Sequences aligned (UniProt or PDB accession codes): Homo sapiens α1 GABAA (HS, P14867), H. sapiens β2 GABAA (P47870), H. sapiens γ2 GABAA (P18507), H. sapiens GABAA β3 (4COF), H. sapiens glycine α3 (5CFB), Danio rerio glycine α1 (DR, 3JAE), Caenorhabditis elegans α (CE, 3RHW), H. sapiens α4 nAChR (5KXI), H. sapiens β2 nAChR (5KXI) and Mus musculus 5-HT3 receptor (MM, 4PIR). α-helices (cylinders), β-strands (arrows), and inserted linker (cyan) are indicated.
… 
Transmembrane domain flexibility and comparison with reference structures a, b, Top and side view of the TMD of conformation A with density for the γ2-subunit shown. c, d, As in a, but for conformation B. e, Transmembrane domain superposition of conformation A (subunits in colour) over conformation B (grey). α-Helices are represented as cylinders. f–j, Superposition of single subunit TMD in conformation A (coloured) with its corresponding subunit in conformation B (grey). k–r, Superpositions of the four non-γ-subunits. Top and bottom rows contain the same superpositions in different representations. Conformation B is shown in all panels with α-subunits in green and β-subunits in blue. Reference structures include the glycine receptor with ivermectin bound (3JAF)⁷⁷, glutamate-gated chloride channel with ivermectin bound (3RHW)⁷⁸ and the GABAA β3 homopentamer (4COF)¹⁶.
Transmembrane domain flexibility and comparison with reference structures a, b, Top and side view of the TMD of conformation A with density for the γ2-subunit shown. c, d, As in a, but for conformation B. e, Transmembrane domain superposition of conformation A (subunits in colour) over conformation B (grey). α-Helices are represented as cylinders. f–j, Superposition of single subunit TMD in conformation A (coloured) with its corresponding subunit in conformation B (grey). k–r, Superpositions of the four non-γ-subunits. Top and bottom rows contain the same superpositions in different representations. Conformation B is shown in all panels with α-subunits in green and β-subunits in blue. Reference structures include the glycine receptor with ivermectin bound (3JAF)⁷⁷, glutamate-gated chloride channel with ivermectin bound (3RHW)⁷⁸ and the GABAA β3 homopentamer (4COF)¹⁶.
… 
Biochemistry and binding assay a, FSEC of GABAA receptor with and without Fab bound, and SDS–PAGE analysis of a representative purification (from n > 10 purifications). b, Saturation binding assay with [³H]-flumazenil. Single-site binding fits for receptor alone and receptor plus Fab both exhibited a Hill slope of ~1 (0.97 and 0.89, respectively). Plotted results are from a representative experiment performed in triplicate. n = 3 independent experiments. Data are shown as mean ± s.d. for a representative triplicate measurement. c, Competition of 10 nM [³H]-flumazenil with diazepam. Calculated Ki for diazepam assumes a Kd for [³H]-flumazenil of 7.7 nM. n = 2 independent experiments in triplicate. Data are shown as mean ± s.d. for a representative triplicate measurement. d, Dose–response experiments in the presence or absence of Fab. HEK cells were transfected with cryo-EM constructs and patch-clamped with or without pretreatment with 1 µM Fab for 1 min. Hill slopes are 1.7 and 1.4 with and without Fab, respectively. Published EC50 values for GABA range from 6.6 µM–107 µM71–74. n = 3 experiments from different cells. Data are plotted as mean ± s.d. e, Whole-cell patch-clamp recording of long application of cryo-EM ligands at concentrations used in cryo-EM samples to assess conformational state at equilibrium. The two traces shown are from one continuous recording; in between the two responses, Fab was added to 1 µM for one minute to saturate all receptor sites before second application of GABA and flumazenil (including Fab). n = 3 independent experiments. f–g, Docking of diazepam at the benzodiazepine-binding site based on superposition of benzodiazepine rings. The phenyl ring of diazepam would orient towards the membrane, possibly forming π–π-stacking interactions with Y58 on the complementary subunit. Similar to flumazenil, the halogen of diazepam could interact with H102, suggesting that this contact is conserved broadly among benzodiazepines and flumazenil. This orientation is largely consistent with predictions from a modelling and docking study⁷⁵, and distinct from that suggested by affinity labelling⁷⁶. In this latter prediction, the diazepam phenyl group orients away from the membrane and would require local reorganization of side chains to avoid atomic clashes. h–j, Structural details of Fab–α1 interaction. Labelled residues are on the α-subunit. i, Top view. j, Side view.
Biochemistry and binding assay a, FSEC of GABAA receptor with and without Fab bound, and SDS–PAGE analysis of a representative purification (from n > 10 purifications). b, Saturation binding assay with [³H]-flumazenil. Single-site binding fits for receptor alone and receptor plus Fab both exhibited a Hill slope of ~1 (0.97 and 0.89, respectively). Plotted results are from a representative experiment performed in triplicate. n = 3 independent experiments. Data are shown as mean ± s.d. for a representative triplicate measurement. c, Competition of 10 nM [³H]-flumazenil with diazepam. Calculated Ki for diazepam assumes a Kd for [³H]-flumazenil of 7.7 nM. n = 2 independent experiments in triplicate. Data are shown as mean ± s.d. for a representative triplicate measurement. d, Dose–response experiments in the presence or absence of Fab. HEK cells were transfected with cryo-EM constructs and patch-clamped with or without pretreatment with 1 µM Fab for 1 min. Hill slopes are 1.7 and 1.4 with and without Fab, respectively. Published EC50 values for GABA range from 6.6 µM–107 µM71–74. n = 3 experiments from different cells. Data are plotted as mean ± s.d. e, Whole-cell patch-clamp recording of long application of cryo-EM ligands at concentrations used in cryo-EM samples to assess conformational state at equilibrium. The two traces shown are from one continuous recording; in between the two responses, Fab was added to 1 µM for one minute to saturate all receptor sites before second application of GABA and flumazenil (including Fab). n = 3 independent experiments. f–g, Docking of diazepam at the benzodiazepine-binding site based on superposition of benzodiazepine rings. The phenyl ring of diazepam would orient towards the membrane, possibly forming π–π-stacking interactions with Y58 on the complementary subunit. Similar to flumazenil, the halogen of diazepam could interact with H102, suggesting that this contact is conserved broadly among benzodiazepines and flumazenil. This orientation is largely consistent with predictions from a modelling and docking study⁷⁵, and distinct from that suggested by affinity labelling⁷⁶. In this latter prediction, the diazepam phenyl group orients away from the membrane and would require local reorganization of side chains to avoid atomic clashes. h–j, Structural details of Fab–α1 interaction. Labelled residues are on the α-subunit. i, Top view. j, Side view.
… 
Cryo-EM image processing procedure a, Representative cryo-electron micrograph of the GABAA receptor–Fab complex. n = 5,594 images. b, Images of selected 2D classes from reference-free 2D classification by Relion. c, Overview of the image processing procedure (see Methods).
Cryo-EM image processing procedure a, Representative cryo-electron micrograph of the GABAA receptor–Fab complex. n = 5,594 images. b, Images of selected 2D classes from reference-free 2D classification by Relion. c, Overview of the image processing procedure (see Methods).
… 
Three-dimensional reconstructions of the two GABAA receptor conformations a, Angular distribution histogram of GABAA receptor conformation A particle images. b, Fourier shell correlation (FSC) of conformation A maps before (black) and after (blue) masking. c, Local resolution of the GABAA receptor estimated by ResMap. d–f, as in a–c but for GABAA receptor conformation B.
+11
Three-dimensional reconstructions of the two GABAA receptor conformations a, Angular distribution histogram of GABAA receptor conformation A particle images. b, Fourier shell correlation (FSC) of conformation A maps before (black) and after (blue) masking. c, Local resolution of the GABAA receptor estimated by ResMap. d–f, as in a–c but for GABAA receptor conformation B.
… 
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ARTICLE https://doi.org/10.1038/s41586-018-0255-3
Structure of a human synaptic GABAA
receptor
Shaotong Zhu1, Colleen M. Noviello1, Jinfeng Teng1, Richard M. Walsh Jr1, Jeong Joo Kim1 & Ryan E. Hibbs1*
Fast inhibitory neurotransmission in the brain is principally mediated by the neurotransmitter GABA (γ-aminobutyric
acid) and its synaptic target, the type A GABA receptor (GABAA receptor). Dysfunction of this receptor results in
neurological disorders and mental illnesses including epilepsy, anxiety and insomnia. The GABAA receptor is also a
prolific target for therapeutic, illicit and recreational drugs, including benzodiazepines, barbiturates, anaesthetics and
ethanol. Here we present high-resolution cryo-electron microscopy structures of the human α1β2γ2 GABA
A
receptor,
the predominant isoform in the adult brain, in complex with GABA and the benzodiazepine site antagonist flumazenil,
the first-line clinical treatment for benzodiazepine overdose. The receptor architecture reveals unique heteromeric
interactions for this important class of inhibitory neurotransmitter receptor. This work provides a template for
understanding receptor modulation by GABA and benzodiazepines, and will assist rational approaches to therapeutic
targeting of this receptor for neurological disorders and mental illness.
The function of the nervous system is governed by a balance of excit-
atory and inhibitory signalling. GABA is the major inhibitory neuro-
transmitter in the central nervous system (CNS) and acts through the
GABA
A
and GABA
B
receptors. GABA
A
receptors, found at 20–50%
of synapses in the brain1, react on a millisecond timescale to bind-
ing of GABA by opening a transmembrane channel that is permeable
to chloride, which suppresses neuronal activity in the adult brain2.
Dysfunction of these channels results in anxiety disorders, epilepsy,
and neurodevelopmental disorders, including autism3–5.
GABAA receptors are the targets of a remarkably diverse array of
drugs that act through distinct binding sites. GABA was discovered
in 19506,7, and was soon followed by the discovery of benzodiaze-
pines
8
, allosteric modulators of GABA
A
receptors that are widely used
in the treatment of epilepsy, insomnia, anxiety and panic disorder9,10.
Flumazenil is a competitive antagonist of the benzodiazepine-binding
site; it is used clinically to reverse benzodiazepine-induced anaesthesia
and is the principal antidote for benzodiazepine overdose
11
. Allosteric
potentiation of the GABA
A
receptor for therapeutic (or recreational)
purposes extends far beyond benzodiazepines: barbiturates, volatile
and intravenous anesthetics, neurosteroids and ethanol are all allosteric
modulators that act on GABAA receptors12,13.
The rich pharmacology of the GABAA receptor derives in part from
its complex subunit assembly. A total of 19 subunits assemble in lim-
ited combinations to make functional receptors
14
. The predominant
synaptic isoform comprises two α1-subunits, two β2-subunits and
one γ2-subunit. The general architecture of the receptor is known
from structural studies of the pentameric ligand-gated ion channel
superfamily15 and from the structure of a homopentameric GABAA
receptor
16
. In the physiological assembly, GABA binds at β–α-subunit
interfaces, and benzodiazepines bind at the α–γ interface10,17.
Mutagenesis and functional studies have approximated the loci for
these and many other compounds on GABAA receptors10,17–19, but
currently there is no structural information regarding a physiolog-
ical GABA
A
receptor. Here we present high-resolution structures of
the α1β2γ2 GABA
A
receptor, which illuminate atomic mechanisms
of GABA and flumazenil recognition and features of the assembly of
this heteromeric receptor.
Biochemistry and structure determination
We optimized receptor constructs and expression conditions to pro-
duce and purify the receptor assembly comprising the α1, β2 and
γ2-subunits (Methods, Extended Data Fig.1). We raised monoclonal
antibodies to the receptor and purified a receptor–Fab complex to
disrupt the low-resolution pseudo-symmetry and facilitate particle
alignment
20
(Extended Data Fig.2a). The purified GABA
A
receptor
construct (henceforth the ‘cryo-EM construct’) retained the ability
to bind [
3
H]-flumazenil with low nanomolar affinity
13,21
(Extended
Data Fig.2b). We observed a small positive effect of Fab on GABA
potency, and found that binding of Fab did not affect affinity for
[
3
H]-flumazenil. Fab had no effect on the functional response to GABA
and flumazenil applied at the concentrations used for cryo-electron
microscopy (cryo-EM) (Extended Data Fig.2).
Processing of cryo-EM images of the sample containing GABAA
receptor, GABA, flumazenil and Fab revealed a homogeneous complex
with two bound Fabs (Extended Data Fig.3). Classification yielded
reconstructions with two distinct transmembrane domain (TMD)
arrangements, which we call conformation A and conformation B.
Refinement of the two reconstructions yielded density maps, both
at overall resolutions of approximately 3.9Å (Extended Data Fig.4).
Cryo-EM density maps were of sufficient quality to allow modelling
of almost the entire receptor and the variable domains of the Fabs
(Methods and Extended Data Fig.5–7). The density map shows clear
sidechain densities and resolution of 3Å or better in the extracellular
ligand binding sites, whereas the TMD (3–4Å) and the Fab fragments
(4–4.5Å) are resolved at lower resolutions. The γ2-subunit in
conformation B, and in particular its TMD, was comparatively more
disordered than the rest of the receptor but still exhibited secondary
structural features.
Overall architecture
The GABAA receptor–Fab complex is a cylinder-shaped receptor
assembly, with two Fab fragments extending radially from the extra-
cellular domain (ECD) of the receptor (Fig.1). Five receptor subunits
assemble in a pseudo-symmetrical fashion around an extracellular
vestibule and integral ion channel. The two Fab fragments interact
1Departments of Neuroscience and Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA. *e-mail: ryan.hibbs@utsouthwestern.edu
5 JULY 2018 | VOL 559 | NATURE | 67
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Supplementary resources (2)

Supplementary Material 1
Data
July 2018
Shaotong Zhu · Colleen M. Noviello · Jinfeng Teng · Richard Walsh Jr · Ryan E. Hibbs
Supplementary Material 2
Data
July 2018
Shaotong Zhu · Colleen M. Noviello · Jinfeng Teng · Richard Walsh Jr · Ryan E. Hibbs
... GABA A Rs are heteropentameric complexes assembled from various subunits combinations, including α (1-6), β (1-3), γ (1-3), δ, ε, π, θ, and ρ (1-3). The most common isoform of GABA A Rs in the mature brain comprises two α1, two β2/3, and one γ2 subunit [6,7]. These receptors mediate inhibitory neurotransmission by facilitating chloride ion influx upon GABA binding, thereby maintaining the excitatory-inhibitory (E-I) balance [8]. ...
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... 40 The formation of H-bonds is important in protein/ligand interaction to maintain the stability of the complex. 41 In our study, diazepam, gingerols, and shogaols have built several hydrogen bonds (H-bonds) with hGABA A R. Shogaols α,β-unsaturated ketone side chain builds H-bonds to α1 his102, which indicates that the side chain was crucial for the formation of H-bonds and may contribute to the stability of the ligandprotein complex. ...
Gingerols and Shogaols of Zingiber officinale var. sunti Valeton as Potential Allosteric Agonists of Human GABAA Receptor by in silico Pharmacology Approach
Article
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Background Gingerols and shogaols are the main active constituents in Zingiber officinale var. sunti Valeton has been reported for its anti-anxiety activity. Anti-anxiety drugs, such as benzodiazepines, alleviate anxiety disorders by enhancing the activity of gamma-aminobutyric acid type A (GABAA) receptors through positive allosteric regulation at the α1/γ2 interface extracellular domain. Purpose To elucidate the binding energy and stability of gingerols and shogaols toward human GABAA receptor (hGABAAR), compared to their known allosteric agonist (diazepam), and further phytochemical analysis in the ethanol extract of Z. officinale var. sunti Valeton rhizome (EEZO). Methods The ligands, namely gingerols (6-, 8-, and 10-gingerol) and shogaols (6-, 8-, and 10-shogaol), were evaluated by pre-ADMET screening tools and molecular docking simulation towards hGABAAR α1/γ2 subtype (PDB ID: 6X3X). Compounds with the best pre-ADMET profile and affinity were subjected to a 200 ns molecular dynamics (MD) simulation. The UPLC analysis was performed to detect and quantify gingerol and shogaol in EEZO. Results The best pre-ADMET prediction was shown by 6-gingerol, whereas the molecular docking simulations revealed that the best binding affinity and stability were shown by 6-gingerol (−7.41 kcal/mol) and 10-shogaol (−8.24 kcal/mol), which are comparable to that of diazepam. They build hydrogen bonds with α1 Ser206 and pi interaction with γ2 Phe77. The MD simulation confirmed that the stability of the 10-shogaol/hGABAAR and 6-gingerol/hGABAAR complexes is equal to that of diazepam/hGABAAR. The UPLC analysis resulted in a level of 44.98 µg/mL for 6-gingerol and 2.52 µg/mL for 10-shogaol. Conclusion 6-Gingerol and 10-shogaol of EEZO may have the potential to be developed as novel allosteric agonists of human GABAA receptors, thus explaining their anti-anxiety activity. However, the activity towards the human GABAA receptor is lower than diazepam, its known allosteric agonist.
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... El receptor GABA-A es un receptor iónico permeable al cloruro, formado por dos subunidades α1, dos subunidades β2 y una subunidad γ2 o δ, que media la transmisión inhibitoria rápida en el sistema nervioso central, por lo que juega un papel importante en la regulación de la excitabilidad cerebral. Estudios en ratones han demostrado que la pérdida de la subunidad β3 provoca convulsiones y déficit de aprendizaje y memoria (Ohkawa et al., 2014;Petit-Pedrol et al., 2014;Spatola et al., 2017;Zhu et al., 2018). La autoinmunidad puede desencadenarse ante la liberación de antígenos debido a la degradación neuronal después de una infección viral del tejido nervioso (Guo et al., 2020;Valle et al., 2021). ...
Actualidades en encefalitis autoinmunes mediadas por anticuerpos en pediatría
Article
Full-text available
  • Jan 2024
La encefalitis autoinmune (EA) en pediatría presentan un cuadro clínico de difícil diagnóstico, frecuentemente son confundidas con enfermedades psiquiátricas. Estas se caracterizan por provocar una inflamación mediada por la respuesta inmune humoral y en menor medida por una respuesta celular citotóxica. Se reporta una incidencia mínima de 10,5 pacientes pediátricos (1 mes-18 años) con encefalitis por cada 100,000 casos al año en países occidentales. Son padecimientos caracterizados por una inflamación del parénquima cerebral que causa síntomas similares a infecciones, síntomas psiquiátricos y manifestaciones neurológicas. El objetivo del presente trabajo fue desarrollar una actualización de la información a través de una revisión bibliográfica clínica que contribuya al diagnóstico oportuno de estos padecimientos. La identificación de autoanticuerpos específicos en la EA en la etapa pediátrica, contribuyen al desarrollo de nuevas técnicas de diagnóstico temprano de la enfermedad con mayor sensibilidad y especificidad.
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... Additionally, the α4β3δ GABA A R tolerates an agonist (Az-INP-G) that carries a guanidine group at its amino end. Receptor structures show that GABA's ammonium group sits deeply in the orthosteric site, where very little space would be available for a bulkier group (Masiulis et al., 2019;Sente et al., 2022;Zhu et al., 2018). The strong photo-agonism by Az-INP-G implies that the orthosteric site in α4β3δ might be larger or more adaptive. ...
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Full-text available
Background and Purpose Neuronal inhibition is largely mediated by type‐A GABA receptors (GABAARs), a family of ligand‐gated chloride‐permeable channels, which can be sub‐classified by their subunit composition. Unravelling the function and distribution of each GABAAR subtype is essential for a holistic understanding of GABAergic inhibition in health and diseases. Photopharmacology, a technique that utilises light‐sensitive compounds to precisely manipulate endogenous proteins, is powerful for this purpose. To resolve the molecular complexity of neuronal inhibition, we aimed to develop subtype‐selective photoswitchable agonists for GABAARs. Experimental Approach Inspired by THIP (gaboxadol), an agonist selective for δ subunit‐containing GABAARs (δ‐GABAARs), we merged a photoswitch moiety (azobenzene) with an analogue of THIP (isoguvacine) to construct Az‐IGU. Using whole‐cell voltage‐clamp recording, Az‐IGU was tested on 13 GABAAR subtypes expressed in human embryonic kidney (HEK) cells. Optical activation of endogenous GABAARs was examined via electrophysiology in cultured cortical neurons. Key Results In HEK cells, Az‐IGU exerted reversible photo‐agonism selectively for α4β3δ and α6β3δ GABAARs, two major mediators of tonic inhibition. Pharmacological and mutagenesis studies suggested that activation of the α4β3δ GABAAR involves interaction between Az‐IGU and the GABA‐binding pocket and is strongly correlated with the spontaneous activity of the receptor. In cultured cortical neurons, photoisomerisation of Az‐IGU triggered responses that enabled reversible control of action potential firing. Conclusions and Implications GABAARs are potential therapeutic targets for many disorders. However, their physiological and pathophysiological roles remain largely unexplored. Az‐IGU may enable photopharmacological studies of α4/6β3δ GABAARs, providing new opportunities for biomedical and neurobiological applications.
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Understanding the effects of functionally unknown variants in epilepsy associated genes is crucial for elucidating disease pathophysiology and developing personalized therapeutics. With a multiscale framework, spanning from DNA sequence to protein function and neural behavior, we describe a novel approach for predicting and investigating pathogenic mutations, hypothesizing that epileptogenic mutations in the GABA A receptor subunit and nearby predicted mutations may produce similar effects on the CA1 pyramidal neuron model. By exploring the characteristic relationships between predicted pathogenic mutations and proximal epileptogenic mutations, the study aims to estimate the effects of predicted mutations based on the effects of epileptogenic mutations on hippocampal pyramidal neuron simulations. The methodology begins with the collection of GABA A receptor γ2 subunit genetic data, followed by data cleaning and formatting performed in R using a custom script. Next, ensemble predictors will be applied to identify and prioritize the pathogenic missense variants of the γ2 subunit. Mapping a specific pathogenic variant (predicted) to the subunit structural domains shared by epileptogenic mutations will be illustrated, accompanied by molecular modeling of their effects and consideration of evolutionary conservation. Then, variant-specific meta-analysis and parameter normalization will be performed, followed by correlation analysis to identify any significant relationships between predicted mutations and proximal epileptogenic mutations. Using a Python-based neural simulator, multi-compartmental conductance-based neuron model, reflecting the effect of wild-type and epileptogenic mutants will be described. Simulation of neural responses generated by epileptogenic GABA A receptor subtype will be considered for the rough estimation of the predicted pathogenic variants' effect on neural response. To our knowledge, this is the first protocol exploring a multiscale framework to estimate the effects of GABA A receptor variants on neuronal behavior, June 2025 • 220 • e67833 • Page 2 of 38 crucial for epilepsy research. This protocol can serve as a foundation for enhancing predictions of cellular phenotypes caused by potentially pathogenic variants of GABA A receptors associated with epilepsy.
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Mechanism of human α3β GlyR regulation by intracellular M3/M4 loop phosphorylation and 2,6-di-tert-butylphenol interaction
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Fast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic acetylcholine receptor. As with most ligand-gated ion channels, nicotinic receptors assemble as oligomers of subunits, usually as hetero-oligomers and often with variable stoichiometries 1 . This intrinsic heterogeneity in protein composition provides fine tunability in channel properties, which is essential to brain function, but frustrates structural and biophysical characterization. The α4β2 subtype of the nicotinic acetylcholine receptor is the most abundant isoform in the human brain and is the principal target in nicotine addiction. This pentameric ligand-gated ion channel assembles in two stoichiometries of α- and β-subunits (2α:3β and 3α:2β). Both assemblies are functional and have distinct biophysical properties, and an imbalance in the ratio of assemblies is linked to both nicotine addiction2,3 and congenital epilepsy4,5. Here we leverage cryo-electron microscopy to obtain structures of both receptor assemblies from a single sample. Antibody fragments specific to β2 were used to 'break' symmetry during particle alignment and to obtain high-resolution reconstructions of receptors of both stoichiometries in complex with nicotine. The results reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the two different stoichiometries of this receptor.
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Full-text available
  • Apr 2018
Coot is a graphics application that is used to build or manipulate macromolecular models; its particular forte is manipulation of the model at the residue level. The model-building tools of Coot have been combined and extended to assist or automate the building of N-linked glycans. The model is built by the addition of monosaccharides, placed by variation of internal coordinates. The subsequent model is refined by real-space refinement, which is stabilized with modified and additional restraints. It is hoped that these enhanced building tools will help to reduce building errors of N-linked glycans and improve our knowledge of the structures of glycoproteins.
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Genetic and Molecular Regulation of Extrasynaptic GABA-A Receptors in the Brain: Therapeutic Insights for Epilepsy
Article
Full-text available
  • Nov 2017
GABA-A receptors play a pivotal role in many brain diseases. Epilepsy is caused by acquired conditions and genetic defects in GABA receptor channels regulating neuronal excitability in the brain. The latter is referred to as GABA channelopathies. In the last two decades, major advances have been made in the genetics of epilepsy. The presence of specific GABAergic genetic abnormalities leading to some of the classical epileptic syndromes has been identified. Advances in molecular cloning and recombinant systems have helped characterize mutations in GABA-A receptor subunit genes in clinical neurology. GABA-A receptors are the prime targets for neurosteroids. However, GABA-A receptors are not static, but undergo rapid changes in their number or composition in response to neuroendocrine milieu. This review describes the recent advances in the genetic and neuroendocrine control of extrasynaptic and synaptic GABA-A receptors in epilepsy and its impact on neurological conditions. It highlights the current knowledge of GABA genetics in epilepsy, with an emphasis on the neuroendocrine regulation of extrasynaptic GABA-A receptors in network excitability and seizure susceptibility. Recent advances in molecular regulation of extrasynaptic GABA-A receptor-mediated tonic inhibition are providing unique new therapeutic approaches for epilepsy, status epilepticus, and certain brain disorders. The discovery of an extrasynaptic molecular mechanism represents a milestone for developing novel therapies such as neurosteroid replacement therapy for catamenial epilepsy.
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α subunits in GABAA receptors are dispensable for GABA and diazepam action
Article
Full-text available
  • Nov 2017
The major isoform of the GABAA receptor is α1β2γ2. The binding sites for the agonist GABA are located at the β2+/α1− subunit interfaces and the modulatory site for benzodiazepines at α1+/γ2−. In the absence of α1 subunits, a receptor was formed that was gated by GABA and modulated by diazepam similarly. This indicates that alternative subunits can take over the role of the α1 subunits. Point mutations were introduced in β2 or γ2 subunits at positions homologous to α1− benzodiazepine binding and GABA binding positions, respectively. From this mutation work we conclude that the site for GABA is located at a β2+/β2− subunit interface and that the diazepam site is located at the β2+/γ2− subunit interface. Computational docking leads to a structural hypothesis attributing this non-canonical interaction to a binding mode nearly identical with the one at the α1+/γ2− interface. Thus, the β2 subunit can take over the role of the α1 subunit for the formation of both sites, its minus side for the GABA binding site and its plus side for the diazepam binding site.
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Structural basis for GABAA receptor potentiation by neurosteroids
Article
Full-text available
  • Nov 2017
  • NAT STRUCT MOL BIOL
Type A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of inhibitory neurotransmission in the human brain. Endogenous neurosteroids interact with GABAARs to regulate acute and chronic anxiety and are potent sedative, analgesic, anticonvulsant and anesthetic agents. Their mode of binding and mechanism of receptor potentiation, however, remain unknown. Here we report crystal structures of a chimeric GABAAR construct in apo and pregnanolone-bound states. The neurosteroid-binding site is mechanically coupled to the helices lining the ion channel pore and modulates the desensitization-gate conformation. We demonstrate that the equivalent site is responsible for physiological, heteromeric GABAAR potentiation and explain the contrasting modulatory properties of 3a versus 3b neurosteroid epimers. These results illustrate how peripheral lipid ligands can regulate the desensitization gate of GABAARs, a process of broad relevance to pentameric ligand-gated ion channels.
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Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites
Article
Full-text available
  • Nov 2017
  • NAT STRUCT MOL BIOL
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.
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Predicting Secretory Proteins with SignalP
Chapter
  • Apr 2017
  • Meth Mol Biol
SignalP is the currently most widely used program for prediction of signal peptides from amino acid sequences. Proteins with signal peptides are targeted to the secretory pathway, but are not necessarily secreted. After a brief introduction to the biology of signal peptides and the history of signal peptide prediction, this chapter will describe all the options of the current version of SignalP and the details of the output from the program. The chapter includes a case study where the scores of SignalP were used in a novel way to predict the functional effects of amino acid substitutions in signal peptides.
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Processing of Structurally Heterogeneous Cryo-EM Data in RELION
Chapter
  • May 2016
This chapter describes algorithmic advances in the RELION software, and how these are used in high-resolution cryo-electron microscopy (cryo-EM) structure determination. Since the presence of projections of different three-dimensional structures in the dataset probably represents the biggest challenge in cryo-EM data processing, special emphasis is placed on how to deal with structurally heterogeneous datasets. As such, this chapter aims to be of practical help to those who wish to use RELION in their cryo-EM structure determination efforts.
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