Distinct neurodevelopmental and epileptic phenotypes associated with gain- and loss-of-function GABRB2 variants.

BACKGROUND: Variants in GABRB2, encoding the ß2 subunit of the γ-aminobutyric acid type A (GABAA) receptor, can result in a diverse range of conditions, ranging from febrile seizures to severe developmental and epileptic encephalopathies. However, the mechanisms underlying the risk of developing milder vs more severe forms of disorder remain unclear. In this study, we conducted a comprehensive genotype-phenotype correlation analysis in a cohort of individuals with GABRB2 variants. METHODS: Genetic and electroclinical data of 42 individuals harbouring 26 different GABRB2 variants were collected and accompanied by electrophysiological analysis of the effects of the variants on receptor function. FINDINGS: Electrophysiological assessments of α1ß2γ2 receptors revealed that 25/26 variants caused dysfunction to core receptor properties such as GABA sensitivity. Of these, 17 resulted in gain-of-function (GOF) while eight yielded loss-of-function traits (LOF). Genotype-phenotype correlation analysis revealed that individuals harbouring GOF variants suffered from severe developmental delay/intellectual disability (DD/ID, 74%), movement disorders such as dystonia or dyskinesia (59%), microcephaly (50%) and high risk of early mortality (26%). Conversely, LOF variants were associated with milder disease manifestations. Individuals with these variants typically exhibited fever-triggered seizures (92%), milder degrees of DD/ID (85%), and maintained ambulatory function (85%). Notably, severe movement disorders or microcephaly were not reported in individuals with loss-of-function variants. INTERPRETATION: The data reveals that genetic variants in GABRB2 can lead to both gain and loss-of-function, and this divergence is correlated with distinct disease manifestations. Utilising this information, we constructed a diagnostic flowchart that aids in predicting the pathogenicity of recently identified variants by considering clinical phenotypes. FUNDING: This work was funded by the Australian National Health & Medical Research Council, the Novo Nordisk Foundation and The Lundbeck Foundation.

Correlations of receptor desensitization of gain-of-function GABRB3 variants with clinical severity.

Genetic variants associated with developmental and epileptic encephalopathies have been identified in the GABRB3 gene that encodes the ß3 subunit of GABAA receptors. Typically, variants alter receptor sensitivity to GABA resulting in either gain- or loss-of-function, which correlates with patient phenotypes. However, it is unclear how another important receptor property, desensitization, contributes to the greater clinical severity of gain-of-function variants. Desensitization properties of 20 gain-of-function GABRB3 variant receptors were evaluated using two-electrode voltage-clamp electrophysiology. The parameters measured included current decay rates and steady-state currents. Selected variants with increased or reduced desensitization were also evaluated using whole-cell electrophysiology in transfected mammalian cell lines. Of the 20 gain-of-function variants assessed, 13 were found to alter receptor desensitization properties. Seven variants reduced desensitization at equilibrium, which acts to worsen gain-of-function traits. Six variants accelerated current decay kinetics, which limits gain-of-function traits. All affected patients displayed severe clinical phenotypes with intellectual disability and difficult-to-treat epilepsy. Nevertheless, variants that reduced desensitization at equilibrium were associated with more severe clinical outcomes. This included younger age of first seizure onset (median 0.5 months), movement disorders (dystonia and dyskinesia), epilepsy of infancy with migrating focal seizures (EIMFS) and risk of early mortality. Variants that accelerated current decay kinetics were associated with slightly milder phenotypes with later seizure onset (median 4 months), unclassifiable developmental and epileptic encephalopathies or Lennox-Gastaut syndrome and no movement disorders. Our study reveals that gain-of-function GABRB3 variants can increase or decrease receptor desensitization properties and that there is a correlation with the degree of disease severity. Variants that reduced the desensitization at equilibrium were clustered in the transmembrane regions that constitute the channel pore and correlated with greater disease severity, while variants that accelerated current decay were clustered in the coupling loops responsible for receptor activation and correlated with lesser severity.

GABAA receptors in epilepsy: Elucidating phenotypic divergence through functional analysis of genetic variants.

Normal brain function requires a tightly regulated balance between excitatory and inhibitory neurotransmissions. γ-Aminobutyric acid type A (GABAA ) receptors represent the major class of inhibitory ion channels in the mammalian brain. Dysregulation of these receptors and/or their associated pathways is strongly implicated in the pathophysiology of epilepsy. To date, hundreds of different GABAA receptor subunit variants have been associated with epilepsy, making them a prominent cause of genetically linked epilepsy. While identifying these genetic variants is crucial for accurate diagnosis and effective genetic counselling, it does not necessarily lead to improved personalised treatment options. This is because the identification of a variant does not reveal how the function of GABAA receptors is affected. Genetic variants in GABAA receptor subunits can cause complex changes to receptor properties resulting in various degrees of gain-of-function, loss-of-function or a combination of both. Understanding how variants affect the function of GABAA receptors therefore represents an important first step in the ongoing development of precision therapies. Furthermore, it is important to ensure that functional data are produced using methodologies that allow genetic variants to be classified using clinical guidelines such as those developed by the American College of Medical Genetics and Genomics. This article will review the current knowledge in the field and provide recommendations for future functional analysis of genetic GABAA receptor variants.

GABRA1-Related Disorders: From Genetic to Functional Pathways.

OBJECTIVE: Variants in GABRA1 have been associated with a broad epilepsy spectrum, ranging from genetic generalized epilepsies to developmental and epileptic encephalopathies. However, our understanding of what determines the phenotype severity and best treatment options remains inadequate. We therefore aimed to analyze the electroclinical features and the functional effects of GABRA1 variants to establish genotype-phenotype correlations. METHODS: Genetic and electroclinical data of 27 individuals (22 unrelated and 2 families) harboring 20 different GABRA1 variants were collected and accompanied by functional analysis of 19 variants. RESULTS: Individuals in this cohort could be assigned into different clinical subgroups based on the functional effect of their variant and its structural position within the GABRA1 subunit. A homogenous phenotype with mild cognitive impairment and infantile onset epilepsy (focal seizures, fever sensitivity, and electroencephalographic posterior epileptiform discharges) was described for variants in the extracellular domain and the small transmembrane loops. These variants displayed loss-of-function (LoF) effects, and the patients generally had a favorable outcome. A more severe phenotype was associated with variants in the pore-forming transmembrane helices. These variants displayed either gain-of-function (GoF) or LoF effects. GoF variants were associated with severe early onset neurodevelopmental disorders, including early infantile developmental and epileptic encephalopathy. INTERPRETATION: Our data expand the genetic and phenotypic spectrum of GABRA1 epilepsies and permit delineation of specific subphenotypes for LoF and GoF variants, through the heterogeneity of phenotypes and variants. Generally, variants in the transmembrane helices cause more severe phenotypes, in particular GoF variants. These findings establish the basis for a better understanding of the pathomechanism and a precision medicine approach in GABRA1-related disorders. Further studies in larger populations are needed to provide a conclusive genotype-phenotype correlation. ANN NEUROL 2023.

Vinpocetine improved neuropsychiatric and epileptic outcomes in a patient with a GABRA1 loss-of-function variant.

Vinpocetine is a synthetic derivative of the alkaloid vincamine and has been used as a dietary supplement for decades. Following a positive report of the use of vinpocetine in a patient with a loss-of-function GABRB3 variant, we here describe another patient with a loss-of-function GABRA1 variant (p.(Arg112Gln)) who benefited from vinpocetine treatment. This patient was diagnosed with autism spectrum disorder, psychiatric complications, and therapy-resistant focal epilepsy. Upon add-on treatment with 40 mg vinpocetine daily for 16 months, the patient experienced an overall improved quality of life as well as seizure freedom. Our findings corroborate that vinpocetine can attenuate epilepsy-associated behavioral issues in patients with loss-of-function GABAA receptor gene variants.

Gain-of-function and loss-of-function GABRB3 variants lead to distinct clinical phenotypes in patients with developmental and epileptic encephalopathies.

Many patients with developmental and epileptic encephalopathies present with variants in genes coding for GABAA receptors. These variants are presumed to cause loss-of-function receptors leading to reduced neuronal GABAergic activity. Yet, patients with GABAA receptor variants have diverse clinical phenotypes and many are refractory to treatment despite the availability of drugs that enhance GABAergic activity. Here we show that 44 pathogenic GABRB3 missense variants segregate into gain-of-function and loss-of-function groups and respective patients display distinct clinical phenotypes. The gain-of-function cohort (n = 27 patients) presented with a younger age of seizure onset, higher risk of severe intellectual disability, focal seizures at onset, hypotonia, and lower likelihood of seizure freedom in response to treatment. Febrile seizures at onset are exclusive to the loss-of-function cohort (n = 47 patients). Overall, patients with GABRB3 variants that increase GABAergic activity have more severe developmental and epileptic encephalopathies. This paradoxical finding challenges our current understanding of the GABAergic system in epilepsy and how patients should be treated.

Pharmacological Effect of GABA Analogues on GABA-ϱ2 Receptors and Their Subtype Selectivity.

GABAϱ receptors are distinctive GABAergic receptors from other ionotropic GABAA and metabotropic GABAB receptors in their pharmacological, biochemical, and electrophysiological properties. Although GABA-ϱ1 receptors are the most studied in this subfamily, GABA-ϱ2 receptors are widely distributed in the brain and are considered a potential target for treating neurological disorders such as stroke. The structure of GABA-ϱ2 receptors and their pharmacological features are poorly studied. We generated the first homology model of GABA-ϱ2 channel, which predicts similar major interactions of GABA with the binding-site residues in GABA-ϱ1 and GABA-ϱ2 channels. We also investigated the pharmacological properties of several GABA analogues on the activity of GABA-ϱ2 receptors. In comparison to their pharmacological effect on GABA-ϱ1 receptors, the activation effect of these ligands and their potentiation/inhibition impact on GABA response have interestingly shown inter-selectivity between the two GABA-ϱ receptors. Our results suggest that several GABA analogues can be used as research tools to study the distinctive physiology of GABA-ϱ1 and GABA-ϱ2 receptors. Furthermore, their partial agonist effect may hold promise for the future discovery of selective modulatory agents on GABAA receptors.

Gain-of-function variants in GABRD reveal a novel pathway for neurodevelopmental disorders and epilepsy.

A potential link between GABRD encoding the δ subunit of extrasynaptic GABAA receptors and neurodevelopmental disorders has largely been disregarded due to conflicting conclusions from early studies. However, we identified seven heterozygous missense GABRD variants in 10 patients with neurodevelopmental disorders and generalized epilepsy. One variant occurred in two sibs of healthy parents with presumed somatic mosaicism, another segregated with the disease in three affected family members, and the remaining five occurred de novo in sporadic patients. Electrophysiological measurements were used to determine the functional consequence of the seven missense δ subunit variants in receptor combinations of α1ß3δ and α4ß2δ GABAA receptors. This was accompanied by analysis of electroclinical phenotypes of the affected individuals. We determined that five of the seven variants caused altered function of the resulting α1ß3δ and α4ß2δ GABAA receptors. Surprisingly, four of the five variants led to gain-of-function effects, whereas one led to a loss-of-function effect. The stark differences between the gain-of-function and loss-of function effects were mirrored by the clinical phenotypes. Six patients with gain-of-function variants shared common phenotypes: neurodevelopmental disorders with behavioural issues, various degrees of intellectual disability, generalized epilepsy with atypical absences and generalized myoclonic and/or bilateral tonic-clonic seizures. The EEG showed qualitative analogies among the different gain-of-function variant carriers consisting of focal slowing in the occipital regions often preceding irregular generalized epileptiform discharges, with frontal predominance. In contrast, the one patient carrying a loss-of-function variant had normal intelligence and no seizure history, but has a diagnosis of autism spectrum disorder and suffers from elevated internalizing psychiatric symptoms. We hypothesize that increase in tonic GABA-evoked current levels mediated by δ-containing extrasynaptic GABAA receptors lead to abnormal neurotransmission, which represent a novel mechanism for severe neurodevelopmental disorders. In support of this, the electroclinical findings for the gain-of-function GABRD variants resemble the phenotypic spectrum reported in patients with missense SLC6A1 (GABA uptake transporter) variants. This also indicates that the phenomenon of extrasynaptic receptor overactivity is observed in a broader range of patients with neurodevelopmental disorders, because SLC6A1 loss-of-function variants also lead to overactive extrasynaptic δ-containing GABAA receptors. These findings have implications when selecting potential treatment options, as a substantial portion of available antiseizure medication act by enhancing GABAergic function either directly or indirectly, which could exacerbate symptoms in patients with gain-of-function GABRD variants.

Roles of hydrophilic residues in GABA binding site of GABA-ρ1 receptor explain the addition/inhibition effects of competitive ligands.

The orthosteric binding site of GABA-gated ion channels has been widely explored. Many residues in the binding site of GABA were studied. The interactions due to the binding of GABA into the binding site drive channel activation and determine the potency and efficacy of GABA response. The combined effect of a competitive ligand and GABA on GABA-ρ1 receptors has been poorly studied. Here, we used point mutations, molecular modeling, and electrophysiological studies to explore the role of two hydrophilic residues (Serine 168 and Serine 243) of the GABA-ρ1 receptors in response to the binding of GABA and other studied ligands. Our results suggested that Ser168 residue stabilizes either closed state or open conformation depending on the other determinant interactions of each state. On the other hand, Ser243 residue is predicted to form different inter-subunit interactions with residues in the adjacent subunit at different states of the channel. Our current findings enlighten us to reasonably explain the additive/inhibitive effects of applying a competitive ligand with GABA simultaneously. Understanding the mixed effect of potentiation and inhibition would facilitate the discovery of new drugs to work as a direct GABA's activity modulators with more selectivity at various subunits forming GABA-gated ion channels.

Novel methyllycaconitine analogues selective for the α4ß2 over α7 nicotinic acetylcholine receptors.

Analogues of methyllycaconitine (MLA) based on a (3-ethyl-9-methylidene-3-azabicyclo[3.3.1]nonan-1-yl)methanol template have been designed and synthesised that incorporate the modified ester sidechains distinct from that present in the natural product. Electrophysiology experiments using Xenopus oocytes expressing nicotinic acetylcholine receptors (nAChRs) revealed selected analogues served as non-competitive inhibitors that showed selectivity for the α4ß2 over α7 nAChR subtypes, and selectivity for the (α4)3(ß2)2 over (α4)2(ß2)3 stoichiometry. This study more clearly defines the biological effects of MLA analogues and identifies strategies for the development of MLA analogues as selective ligands for the α4ß2 nAChR subtype.

Cannabigerolic acid, a major biosynthetic precursor molecule in cannabis, exhibits divergent effects on seizures in mouse models of epilepsy.

BACKGROUND AND PURPOSE: Cannabis has been used to treat epilepsy for millennia, with such use validated by regulatory approval of cannabidiol (CBD) for Dravet syndrome. Unregulated artisanal cannabis-based products used to treat children with intractable epilepsies often contain relatively low doses of CBD but are enriched in other phytocannabinoids. This raises the possibility that other cannabis constituents might have anticonvulsant properties. EXPERIMENTAL APPROACH: We used the Scn1a+/- mouse model of Dravet syndrome to investigate the cannabis plant for phytocannabinoids with anticonvulsant effects against hyperthermia-induced seizures. The most promising, cannabigerolic acid (CBGA), was further examined against spontaneous seizures and survival in Scn1a+/- mice and in electroshock seizure models. Pharmacological effects of CBGA were surveyed across multiple drug targets. KEY RESULTS: The initial screen identified three phytocannabinoids with novel anticonvulsant properties: CBGA, cannabidivarinic acid (CBDVA) and cannabigerovarinic acid (CBGVA). CBGA was most potent and potentiated the anticonvulsant effects of clobazam against hyperthermia-induced and spontaneous seizures, and was anticonvulsant in the MES threshold test. However, CBGA was proconvulsant in the 6-Hz threshold test and a high dose increased spontaneous seizure frequency in Scn1a+/- mice. CBGA was found to interact with numerous epilepsy-relevant targets including GPR55, TRPV1 channels and GABAA receptors. CONCLUSION AND IMPLICATIONS: These results suggest that CBGA, CBDVA and CBGVA may contribute to the effects of cannabis-based products in childhood epilepsy. Although these phytocannabinoids have anticonvulsant potential and could be lead compounds for drug development programmes, several liabilities would need to be overcome before CBD is superseded by another in this class.

Delta-containing GABAA receptors in pain management: Promising targets for novel analgesics.

Communication between nerve cells depends on the balance between excitatory and inhibitory circuits. GABA, the major inhibitory neurotransmitter, regulates this balance and insufficient GABAergic activity is associated with numerous neuropathological disorders including pain. Of the various GABAA receptor subtypes, the δ-containing receptors are particularly interesting drug targets in management of chronic pain. These receptors are pentameric ligand-gated ion channels composed of α, ß and δ subunits and can be activated by ambient levels of GABA to generate tonic conductance. However, only a few ligands preferentially targeting δ-containing GABAA receptors have so far been identified, limiting both pharmacological understanding and drug-discovery efforts, and more importantly, understanding of how they affect pain pathways. Here, we systemically review and discuss the known drugs and ligands with analgesic potential targeting δ-containing GABAA receptors and further integrate the biochemical nature of the receptors with clinical perspectives in pain that might generate interest among researchers and clinical physicians to encourage analgesic discovery efforts leading to more efficient therapies.

Targeting GABAC Receptors Improves Post-Stroke Motor Recovery.

Ischemic stroke remains a leading cause of disability worldwide, with limited treatment options available. This study investigates GABAC receptors as novel pharmacological targets for stroke recovery. The expression of ρ1 and ρ2 mRNA in mice were determined in peri-infarct tissue following photothrombotic motor cortex stroke. (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (R)-4-ACPBPA and (S)-4-ACPBPA were assessed using 2-elecotrode voltage electrophysiology in Xenopus laevis oocytes. Stroke mice were treated for 4 weeks with either vehicle, the α5-selective negative allosteric modulator, L655,708, or the ρ1/2 antagonists, (R)-4-ACPBPA and (S)-4-ACPBPA respectively from 3 days post-stroke. Infarct size and expression levels of GAT3 and reactive astrogliosis were determined using histochemistry and immunohistochemistry respectively, and motor function was assessed using both the grid-walking and cylinder tasks. After stroke, significant increases in ρ1 and ρ2 mRNAs were observed on day 3, with ρ2 showing a further increase on day 7. (R)- and (S)-4-ACPBPA are both potent antagonists at ρ2 and only weak inhibitors of α5ß2γ2 receptors. Treatment with either L655,708, (S)-4-ACPBPA (ρ1/2 antagonist; 5 mM only), or (R)-4-ACPBPA (ρ2 antagonist; 2.5 and 5 mM) from 3 days after stroke resulted in a significant improvement in motor recovery on the grid-walking task, with L655,708 and (R)-4-ACPBPA also showing an improvement in the cylinder task. Infarct size was unaffected, and only (R)-4-ACPBPA significantly increased peri-infarct GAT3 expression and decreased the level of reactive astrogliosis. Importantly, inhibiting GABAC receptors affords significant improvement in motor function after stroke. Targeting the ρ-subunit could provide a novel delayed treatment option for stroke recovery.

Efficient expression of concatenated α1ß2δ and α1ß3δ GABAA receptors, their pharmacology and stoichiometry.

BACKGROUND AND PURPOSE: GABAA receptors containing δ-subunits are notorious for being difficult to study in vitro due to heterogeneity of expressed receptor populations and low GABA-evoked current amplitudes. Thus, there are some published misconceptions and contradictory conclusions made regarding the pharmacology and stoichiometry of δ-containing receptors. The aim of this study was to obtain robust homogenous expression of α1ßδ receptors for in-depth investigation. EXPERIMENTAL APPROACH: Novel δ-containing pentameric concatenated constructs were designed. The resulting α1ß2δ and α1ß3δ GABAA receptor concatemers were investigated by two-electrode voltage-clamp electrophysiology using Xenopus laevis oocytes. KEY RESULTS: First, while homogenous α1ßδ GABAA receptor pools could not be obtained by manipulating the ratio of injected cRNAs of free α1, ß2/3, and δ subunits, concatenated pentameric α1ß2δ and α1ß3δ constructs resulted in robust expression levels of concatemers. Second, by using optimised constructs that give unidirectional assembly of concatemers, we found that the δ subunit cannot directly participate in GABA binding and receptor activation. Hence, functional δ-containing receptors are likely to all have a conventional 2α:2ß:1δ stoichiometry arranged as ßαßαδ when viewed counterclockwise from the extracellular side. Third, α1ß2/3δ receptors were found to express efficiently in X. laevis oocytes but have a low estimated open probability of ~0.5% upon GABA activation. Because of this, these receptors are uniquely susceptible to positive allosteric modulation by, for example, neurosteroids. CONCLUSION AND IMPLICATIONS: Our data answer important outstanding questions regarding the pharmacology and stoichiometry of α1δ-containing GABAA receptors and pave the way for future analysis and drug discovery efforts.

The anticonvulsant zonisamide positively modulates recombinant and native glycine receptors at clinically relevant concentrations.

GABAA and glycine receptors mediate fast synaptic inhibitory neurotransmission. Despite studies showing that activation of cerebral glycine receptors could be a potential strategy in the treatment of epilepsy, few studies have assessed the effects of existing anticonvulsant therapies on recombinant or native glycine receptors. We, therefore, evaluated the actions of a series of anticonvulsants at recombinant human homo-oligomeric glycine receptor α1, α2 and α3 subtypes expressed in Xenopus oocytes using two-electrode voltage-clamp methods, and then assessed the most effective drug at native glycine receptors from entorhinal cortex neurons using whole-cell voltage-clamp recordings. Ganaxolone, tiagabine and zonisamide positively modulated glycine induced currents at recombinant homomeric glycine receptors. Of these, zonisamide was the most efficacious and exhibited an EC50 value ranging between 450 and 560 µM at α1, α2 and α3 subtypes. These values were not significantly different indicating a non-selective modulation of glycine receptors. Using a therapeutic concentration of zonisamide (100 µM), the potency of glycine was significantly shifted from 106 to 56 µM at α1, 185 to 112 µM at α2, and 245 to 91 µM at α3 receptors. Furthermore, zonisamide (100 µM) potentiated exogenous homomeric and heteromeric glycine mediated currents from layer II pyramidal cells of the lateral or medial entorhinal cortex. As therapeutic concentrations of zonisamide positively modulate recombinant and native glycine receptors, we propose that the anticonvulsant effects of zonisamide may, at least in part, be mediated via this action.

The Z-Drugs Zolpidem, Zaleplon, and Eszopiclone Have Varying Actions on Human GABA A Receptors Containing γ1, γ2, and γ3 Subunits.

γ-Aminobutyric-acid type A (GABA A ) receptors expressing the γ1 or γ3 subunit are only found within a few regions of the brain, some of which are involved in sleep. No known compounds have been reported to selectively target γ1- or γ3-containing GABA A receptors. Pharmacological assessments of this are conflicting, possibly due to differences in experimental models, conditions, and exact protocols when reporting efficacies and potencies. In this study, we evaluated the modulatory properties of five non-benzodiazepine Z-drugs (zaleplon, indiplon, eszopiclone, zolpidem, and alpidem) used in sleep management and the benzodiazepine, diazepam on human α1ß2γ receptors using all three γ subtypes. This was accomplished using concatenated GABA A pentamers expressed in Xenopus laevis oocytes and measured via two-electrode voltage clamp. This approach removes the potential for single subunits to form erroneous receptors that could contribute to the pharmacological assessment of these compounds. No compound tested had significant effects on γ1-containing receptors below 10 µM. Interestingly, zaleplon and indiplon were found to modulate γ3-containing receptors equally as efficacious as γ2-containing receptors. Furthermore, zaleplon had a higher potency for γ3- than for γ2-containing receptors, indicating certain therapeutic effects could occur via these γ3-containing receptors. Eszopiclone modulated γ3-containing receptors with reduced efficacy but no reduction in potency. These data demonstrate that the imidazopyridines zaleplon and indiplon are well suited to further investigate potential γ3 effects on sleep in vivo.

Ligand-gated ion channels in genetic disorders and the question of efficacy.

Whole-genome sequencing has unearthed a substantial number of individual variants in ion channels associated with genetic disorders. Ligand-gated ion channels, including glycine, γ-aminobutyric acid type A and nicotinic acetylcholine receptors, have long been known to harbour genetic variants associated with hyperekplexia and different forms of epilepsy. In some of these cases, missense variants enhance or impair the intrinsic ability of the receptor to convert ligand binding to channel opening, or the efficacy of receptor activation. We review the current understanding of how ligand-gated ion channels are activated and the properties that define the efficacy of an agonist, and how these properties can be altered by disease-causing variants. Additionally, we consider the mechanisms defining drug modulation of receptors and consider how this may differ in genetic variants. This fundamental knowledge is likely to be essential in understanding how effective treatments will be for patients with genetic variants in ligand-gated ion channels.

AE Succinimide, an Analogue of Methyllycaconitine, When Bound Generates a Nonconducting Conformation of the α4ß2 Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine (nACh) receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission. The α4ß2 nACh receptor is highly expressed in the brain and exists in two functional stoichiometries: the (α4)2(ß2)3 and (α4)3(ß2)2 that differ by an ACh-binding site at the α4-α4 interface of (α4)3(ß2)2 receptors. Methyllycaconitine (MLA) is an nACh receptor antagonist, and while potent at both α7 and α4ß2 nACh receptors, it has a higher selectivity for the α7 nACh receptor. The anthranilate-succinimide ester side-chain is important for its activity and selectivity. Here we identify a simplified MLA analogue that contains only the A and E ring skeleton of MLA, AE succinimide, that binds close to the channel lumen to display insurmountable inhibition at α4ß2 nACh receptors. Although inhibition by AE succinimide was found to be voltage-dependent indicating a possible pore channel blocker, substituted-cysteine accessibility experiments indicated it did not bind between 2'-16' region of the channel pore. Instead, we found that upon binding and in the presence of ACh, there is a conformational change to the channel membrane that was identified when the compound was assessed against (α4 V13'C)ß2 nACh receptors. It was found that in the 3:2 stoichiometry the two adjacent α4 subunits containing 13' cysteine mutations formed a disulfide bond and occluded ion conductance. This was reversed by treatment with the reducing agent, dithiothreitol. Thus, AE succinimide has a different mechanism of inhibition to both MLA and other AE analogues, such as AE bicyclic alcohol, in that upon binding to an as yet unidentified site, AE succinimide in the presence of ACh induces a conformational change to the channel that generates a ligand-bound closed state.

Gain-of-function GABRB3 variants identified in vigabatrin-hypersensitive epileptic encephalopathies.

Variants in the GABRB3 gene encoding the ß3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.