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.

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.

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.

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.

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.

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.

Coadministered cannabidiol and clobazam: Preclinical evidence for both pharmacodynamic and pharmacokinetic interactions.

OBJECTIVE: Cannabidiol (CBD) has been approved by the US Food and Drug Administration (FDA) to treat intractable childhood epilepsies, such as Dravet syndrome and Lennox-Gastaut syndrome. However, the intrinsic anticonvulsant activity of CBD has been questioned due to a pharmacokinetic interaction between CBD and a first-line medication, clobazam. This recognized interaction has led to speculation that the anticonvulsant efficacy of CBD may simply reflect CBD augmenting clobazam exposure. The present study aimed to address the nature of the interaction between CBD and clobazam. METHODS: We examined whether CBD inhibits human CYP3A4 and CYP2C19 mediated metabolism of clobazam and N-desmethylclobazam (N-CLB), respectively, and performed studies assessing the effects of CBD on brain and plasma pharmacokinetics of clobazam in mice. We then used the Scn1a+/- mouse model of Dravet syndrome to examine how CBD and clobazam interact. We compared anticonvulsant effects of CBD-clobazam combination therapy to monotherapy against thermally-induced seizures, spontaneous seizures and mortality in Scn1a+/- mice. In addition, we used Xenopus oocytes expressing γ-aminobutyric acid (GABA)A receptors to investigate the activity of GABAA receptors when treated with CBD and clobazam together. RESULTS: CBD potently inhibited CYP3A4 mediated metabolism of clobazam and CYP2C19 mediated metabolism of N-CLB. Combination CBD-clobazam treatment resulted in greater anticonvulsant efficacy in Scn1a+/- mice, but only when an anticonvulsant dose of CBD was used. It is important to note that a sub-anticonvulsant dose of CBD did not promote greater anticonvulsant effects despite increasing plasma clobazam concentrations. In addition, we delineated a novel pharmacodynamic mechanism where CBD and clobazam together enhanced inhibitory GABAA receptor activation. SIGNIFICANCE: Our study highlights the involvement of both pharmacodynamic and pharmacokinetic interactions between CBD and clobazam that may contribute to its efficacy in Dravet syndrome.

Functional genomics of epilepsy-associated mutations in the GABAA receptor subunits reveal that one mutation impairs function and two are catastrophic.

A number of epilepsy-causing mutations have recently been identified in the genes of the α1, ß3, and γ2 subunits comprising the γ-aminobutyric acid type A (GABAA) receptor. These mutations are typically dominant, and in certain cases, such as the α1 and ß3 subunits, they may lead to a mix of receptors at the cell surface that contain no mutant subunits, a single mutated subunit, or two mutated subunits. To determine the effects of mutations in a single subunit or in two subunits on receptor activation, we created a concatenated protein assembly that links all five subunits of the α1ß3γ2 receptor and expresses them in the correct orientation. We created nine separate receptor variants with a single-mutant subunit and four receptors containing two subunits of the γ2R323Q, ß3D120N, ß3T157M, ß3Y302C, and ß3S254F epilepsy-causing mutations. We found that the singly mutated γ2R323Q subunit impairs GABA activation of the receptor by reducing GABA potency. A single ß3D120N, ß3T157M, or ß3Y302C mutation also substantially impaired receptor activation, and two copies of these mutants within a receptor were catastrophic. Of note, an effect of the ß3S254F mutation on GABA potency depended on the location of this mutant subunit within the receptor, possibly because of the membrane environment surrounding the transmembrane region of the receptor. Our results highlight that precise functional genomic analyses of GABAA receptor mutations using concatenated constructs can identify receptors with an intermediate phenotype that contribute to epileptic phenotypes and that are potential drug targets for precision medicine approaches.

Revisiting autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) mutations in the nicotinic acetylcholine receptor reveal an increase in efficacy regardless of stochiometry.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a genetic form of epilepsy that is caused by mutations in several genes, including genes encoding for the α4 and ß2 subunits of the nicotinic acetylcholine (nACh) receptor. Pentameric α4ß2 nACh receptors are the most abundant nicotinic receptor in the mammalian brain and form two stoichiometries, the (α4)3(ß2)2 and (α4)2(ß2)3 receptors that differ in their physiological and pharmacological properties. The purpose of this study was to investigate how ADNFLE mutations ß2V287M, ß2V287L or α4T293I manifest themselves in different receptor stoichiometries. We expressed wild-type and mutant receptors in Xenopus oocytes and measured the response to ACh and other agonists at both receptor stoichiometries. For all three mutations, the efficacy of ACh at (α4)2(ß2)3 receptors was increased. At (α4)3(ß2)2 receptors, the efficacy of activation was increased both when two molecules of agonist, either ACh or the site-selective agonist sazetidine-A, were bound at the α4-ß2 interfaces, and when a third ACh molecule was bound at the α4-α4 site. Regardless of stoichiometry, the mutations increased the current elicited by low concentrations of ACh. Further, the smoking cessation agents, nicotine, varenicline and cytisine increased activation of mutant (α4)3(ß2)2 receptors, while only nicotine increased activation of mutant (α4)2(ß2)3 receptors. Chronic exposure of all agonists reduced ACh-activation levels at low and high ACh concentrations. From this, we concluded that mutations that cause ADNFLE manifest themselves in a change in efficacy regardless of the stoichiometry of the receptor.

Ligand Binding at the 4-4 Agonist-Binding Site of the 42 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is the most abundant subtype in the brain and exists in two functional stoichiometries: (α4)3(ß2)2 and (α4)2(ß2)3. A distinct feature of the (α4)3(ß2)2 receptor is the biphasic activation response to the endogenous agonist acetylcholine, where it is activated with high potency and low efficacy when two α4-ß2 binding sites are occupied and with low potency/high efficacy when a third α4-α4 binding site is occupied. Further, exogenous ligands can bind to the third α4-α4 binding site and potentiate the activation of the receptor by ACh that is bound at the two α4-ß2 sites. We propose that perturbations of the recently described pre-activation step when a third binding site is occupied are a key driver of these distinct activation properties. To investigate this, we used a combination of simple linear kinetic models and voltage clamp electrophysiology to determine whether transitions into the pre-activated state were increased when three binding sites were occupied. We separated the binding at the two different sites with ligands selective for the α4-ß2 site (Sazetidine-A and TC-2559) and the α4-α4 site (NS9283) and identified that when a third binding site was occupied, changes in the concentration-response curves were best explained by an increase in transitions into a pre-activated state. We propose that perturbations of transitions into a pre-activated state are essential to explain the activation properties of the (α4)3(ß2)2 receptor by acetylcholine and other ligands. Considering the widespread clinical use of benzodiazepines, this discovery of a conserved mechanism that benzodiazepines and ACh potentiate receptor activation via a third binding site can be exploited to develop therapeutics with similar properties at other cys-loop receptors.

Kavain, the Major Constituent of the Anxiolytic Kava Extract, Potentiates GABAA Receptors: Functional Characteristics and Molecular Mechanism.

Extracts of the pepper plant kava (Piper methysticum) are effective in alleviating anxiety in clinical trials. Despite the long-standing therapeutic interest in kava, the molecular target(s) of the pharmacologically active constituents, kavalactones have not been established. γ-Aminobutyric acid type A receptors (GABAARs) are assumed to be the in vivo molecular target of kavalactones based on data from binding assays, but evidence in support of a direct interaction between kavalactones and GABAARs is scarce and equivocal. In this study, we characterised the functional properties of the major anxiolytic kavalactone, kavain at human recombinant α1ß2, ß2γ2L, αxß2γ2L (x = 1, 2, 3 and 5), α1ßxγ2L (x = 1, 2 and 3) and α4ß2δ GABAARs expressed in Xenopus oocytes using the two-electrode voltage clamp technique. We found that kavain positively modulated all receptors regardless of the subunit composition, but the degree of enhancement was greater at α4ß2δ than at α1ß2γ2L GABAARs. The modulatory effect of kavain was unaffected by flumazenil, indicating that kavain did not enhance GABAARs via the classical benzodiazepine binding site. The ß3N265M point mutation which has been previously shown to profoundly decrease anaesthetic sensitivity, also diminished kavain-mediated potentiation. To our knowledge, this study is the first report of the functional characteristics of a single kavalactone at distinct GABAAR subtypes, and presents the first experimental evidence in support of a direct interaction between a kavalactone and GABAARs.

High and low GABA sensitivity α4ß2δ GABAA receptors are expressed in Xenopus laevis oocytes with divergent stoichiometries.

GABAA receptors that contain the α4 and δ subunits are thought to be located extrasynaptically, mediating tonic currents elicited by low concentrations of GABA. These α4ßδ receptors are modulated by neurosteroids and certain anesthetics, identifying them as important drug targets in research. However, pharmacological studies on these receptors have often yielded variable results, possibly due to the expression of receptors in different stoichiometries or arrangements. In this study, we injected different ratios of α4, ß2 and δ cRNA into Xenopus oocytes and measured the sensitivity to GABA and DS2 activation of the resulting receptor populations. By creating a matrix of RNA injection ratios from stock RNA concentrations, we were able to compare the changes in pharmacology between injection ratios where the ratio of only one subunit was altered. We identified two distinct populations of receptors, the first with an EC50 value of approximately 100 nM to GABA, a low Hill slope of approximately 0.3 and substantial direct activation by DS2. The second population had an EC50 value of approximately 1 µM to GABA, a steeper Hill slope of 1 and little direct activation, but substantial potentiation, by DS2. The second population was formed with high α4 ratios and low ß2 ratios, but altering the ratio of δ subunit injected had little effect. We propose that receptors with high sensitivity to GABA and direct activation by DS2 are the result of a greater number of ß2 subunits being incorporated into the receptor.

The Direct Actions of GABA, 2'-Methoxy-6-Methylflavone and General Anaesthetics at ß3γ2L GABAA Receptors: Evidence for Receptors with Different Subunit Stoichiometries.

2'-Methoxy-6-methylflavone (2'MeO6MF) is an anxiolytic flavonoid which has been shown to display GABAA receptor (GABAAR) ß2/3-subunit selectivity, a pharmacological profile similar to that of the general anaesthetic etomidate. Electrophysiological studies suggest that the full agonist action of 2'MeO6MF at α2ß3γ2L GABAARs may mediate the flavonoid's in vivo effects. However, we found variations in the relative efficacy of 2'MeO6MF (2'MeO6MF-elicited current responses normalised to the maximal GABA response) at α2ß3γ2L GABAARs due to the presence of mixed receptor populations. To understand which receptor subpopulation(s) underlie the variations observed, we conducted a systematic investigation of 2'MeO6MF activity at all receptor combinations that could theoretically form (α2, ß3, γ2L, α2ß3, α2γ2L, ß3γ2L and α2ß3γ2L) in Xenopus oocytes using the two-electrode voltage clamp technique. We found that 2'MeO6MF activated non-α-containing ß3γ2L receptors. In an attempt to establish the optimal conditions to express a uniform population of these receptors, we found that varying the relative amounts of ß3:γ2L subunit mRNAs resulted in differences in the level of constitutive activity, the GABA concentration-response relationships, and the relative efficacy of 2'MeO6MF activation. Like 2'MeO6MF, general anaesthetics such as etomidate and propofol also showed distinct levels of relative efficacy across different injection ratios. Based on these results, we infer that ß3γ2L receptors may form with different subunit stoichiometries, resulting in the complex pharmacology observed across different injection ratios. Moreover, the discovery that GABA and etomidate have direct actions at the α-lacking ß3γ2L receptors raises questions about the structural requirements for their respective binding sites at GABAARs.

Presence of multiple binding sites on α9α10 nAChR receptors alludes to stoichiometric-dependent action of the α-conotoxin, Vc1.1.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in fast synaptic transmission. nAChRs are pentameric receptors formed from a combination of different or similar subunits to produce heteromeric or homomeric channels. The heteromeric, α9α10 nAChR subtype is well-known for its role in the auditory system, being expressed in cochlear hair cells. These nAChRs have also been shown to be involved in immune-modulation. Antagonists of α9α10 nAChRs, like the α-conotoxin Vc1.1, have analgesic effects in neuropathic pain. Unlike other nAChR subtypes there is no evidence that functional receptor stoichiometries of α9α10 exist. By using 2-electrode voltage clamp methods and maintaining a constant intracellular Ca(2+) concentration, we observed a biphasic activation curve for ACh that is dependent on receptor stoichiometry. Vc1.1, but not the α9α10 antagonists RgIA or atropine, inhibits ACh-evoked currents in a biphasic manner. Characteristics of the ACh and Vc1.1 activation and inhibition curves can be altered by varying the ratio of α9 and α10 mRNA injected into oocytes, changing the curves from biphasic to monophasic when an excess of α10 mRNA is used. These results highlight the difference in the pharmacological profiles of at least two different α9α10 nAChR stoichiometries, possibly (α9)3(α10)2 and (α9)2(α10)3. As a result, we infer that there is an additional binding site for ACh and Vc1.1 at the α9-α9 interface on the hypothesized (α9)3(α10)2 nAChR, in addition to the α10-α9 and or α9-α10 interfaces that are common to both stoichiometries. This study provides further evidence that receptor stoichiometry contributes another layer of complexity in understanding Cys-loop receptors.

Mutations in Mll2, an H3K4 methyltransferase, result in insulin resistance and impaired glucose tolerance in mice.

We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5-14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.

Covalent trapping of methyllycaconitine at the α4-α4 interface of the α4ß2 nicotinic acetylcholine receptor: antagonist binding site and mode of receptor inhibition revealed.

The α4ß2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4ß2 nAChR, (α4)2(ß2)3 and (α4)3(ß2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and α4ß2 nAChRs in the Xenopus oocyte expression system, we demonstrate that inhibition by MLA occurs via two different mechanisms; that is, a direct competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4ß2 receptor containing an α4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (α4)3(ß2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(ß2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(ß2)2 nAChRs.

Conformational changes in extracellular loop 2 associated with signal transduction in the glycine receptor.

Ligand-gated ion channels efficiently couple neurotransmitter binding to the opening of an intrinsic ion channel to generate the post-synaptic potentials that are characteristic of fast synaptic transmission. In the Cys-loop family of ligand-gated ion channels, the ligand-binding site is approximately 60 Å above the channel gate. Structural modelling of related proteins and mutagenesis studies led to the hypothesis that loops 2 and 7 of the extracellular domain may couple ligand binding to receptor activation. Mutating loop 2 residues of the glycine receptor to cysteine reveals an alternating pattern of effect upon receptor function. Mutations A52C, T54C and M56C produced a threefold right-shift in EC(50) . In contrast, a 30-fold right-shift was seen for mutations E53C, T55C and D57C. Loop 2 conformational changes associated with ligand binding were assessed by measuring the rate of covalent modification of substituted cysteines by charged methane thiosulfonate reagents. We show for the first time state-dependent differences in the rate of reaction. A52C and T54C are more accessible in the resting state and M56C is more accessible in the activated state. These results demonstrate that loop 2 does undergo a conformational change as part of the mechanism that couples ligand binding to channel opening.