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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.
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Epilepsia Generalizada , Epilepsia , Trastornos del Movimiento , Animales , Humanos , Recién Nacido , Mutación con Ganancia de Función , Mutación/genética , Epilepsia/genética , Convulsiones , Mamíferos/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMEN
Erythromelalgia (EM), is a familial pain syndrome characterized by episodic 'burning' pain, warmth, and erythema. EM is caused by monoallelic variants in SCN9A, which encodes the voltage-gated sodium channel (NaV) NaV1.7. Over 25 different SCN9A mutations attributed to EM have been described to date, all identified in the SCN9A transcript utilizing exon 6N. Here we report a novel SCN9A missense variant identified in seven related individuals with stereotypic episodes of bilateral lower limb pain presenting in childhood. The variant, XM_011511617.3:c.659G>C;p.(Arg220Pro), resides in the exon 6A of SCN9A, an exon previously shown to be selectively incorporated by developmentally regulated alternative splicing. The mutation is located in the voltage-sensing S4 segment of domain I, which is important for regulating channel activation. Functional analysis showed the p.Arg220Pro mutation altered voltage-dependent activation and delayed channel inactivation, consistent with a NaV1.7 gain-of-function molecular phenotype. These results demonstrate that alternatively spliced isoforms of SCN9A should be included in all genomic testing of EM.
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Eritromelalgia , Humanos , Eritromelalgia/genética , Mutación Missense/genética , Canal de Sodio Activado por Voltaje NAV1.7/genética , Dolor/genética , Mutación , Exones/genéticaRESUMEN
OBJECTIVE: N-methyl-d-aspartate (NMDA) receptors are expressed at synaptic sites, where they mediate fast excitatory neurotransmission. NMDA receptors are critical to brain development and cognitive function. Natural variants to the GRIN1 gene, which encodes the obligatory GluN1 subunit of the NMDA receptor, are associated with severe neurological disorders that include epilepsy, intellectual disability, and developmental delay. Here, we investigated the pathogenicity of three missense variants to the GRIN1 gene, p. Ile148Val (GluN1-3b[I481V]), p.Ala666Ser (GluN1-3b[A666S]), and p.Tyr668His (GluN1-3b[Y668H]). METHODS: Wild-type and variant-containing NMDA receptors were expressed in HEK293 cells and primary hippocampal neurons. Patch-clamp electrophysiology and pharmacology were used to profile the functional properties of the receptors. Receptor surface expression was evaluated using fluorescently tagged receptors and microscopy. RESULTS: Our data demonstrate that the GluN1(I481V) variant is inhibited by the open pore blockers ketamine and memantine with reduce potency but otherwise has little effect on receptor function. By contrast, the other two variants exhibit gain-of-function molecular phenotypes. Glycine sensitivity was enhanced in receptors containing the GluN1(A666S) variant and the potency of pore block by memantine and ketamine was reduced, whereas that for MK-801 was increased. The most pronounced functional deficits, however, were found in receptors containing the GluN1(Y668H) variant. GluN1(Y668H)/2A receptors showed impaired surface expression, were more sensitive to glycine and glutamate by an order of magnitude, and exhibited impaired block by extracellular magnesium ions, memantine, ketamine, and MK-801. These variant receptors were also activated by either glutamate or glycine alone. Single-receptor recordings revealed that this receptor variant opened to several conductance levels and activated more frequently than wild-type GluN1/2A receptors. SIGNIFICANCE: Our study reveals a critical functional locus of the receptor (GluN1[Y668]) that couples receptor gating to ion channel conductance, which when mutated may be associated with neurological disorder.
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Ketamina , Trastornos del Neurodesarrollo , Humanos , Memantina/farmacología , Maleato de Dizocilpina/farmacología , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Células HEK293 , Glutamatos , Trastornos del Neurodesarrollo/genética , Glicina , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Stings of certain ant species (Hymenoptera: Formicidae) can cause intense, long-lasting nociception. Here we show that the major contributors to these symptoms are venom peptides that modulate the activity of voltage-gated sodium (NaV) channels, reducing their voltage threshold for activation and inhibiting channel inactivation. These peptide toxins are likely vertebrate-selective, consistent with a primarily defensive function. They emerged early in the Formicidae lineage and may have been a pivotal factor in the expansion of ants.
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Venenos de Hormiga , Hormigas , Toxinas Biológicas , Animales , Dolor , Canales de Sodio , VertebradosRESUMEN
GABAA receptors (GABAARs) mediate the majority of fast inhibitory transmission throughout the brain. Although it is widely known that pore-forming subunits critically determine receptor function, it is unclear whether their single-channel properties are modulated by GABAAR-associated transmembrane proteins. We previously identified Shisa7 as a GABAAR auxiliary subunit that modulates the trafficking, pharmacology, and deactivation properties of these receptors. However, whether Shisa7 also regulates GABAAR single-channel properties has yet to be determined. Here, we performed single-channel recordings of α2ß3γ2L GABAARs cotransfected with Shisa7 in HEK293T cells and found that while Shisa7 does not change channel slope conductance, it reduced the frequency of receptor openings. Importantly, Shisa7 modulates GABAAR gating by decreasing the duration and open probability within bursts. Through kinetic analysis of individual dwell time components, activation modeling, and macroscopic simulations, we demonstrate that Shisa7 accelerates GABAAR deactivation by governing the time spent between close and open states during gating. Together, our data provide a mechanistic basis for how Shisa7 controls GABAAR gating and reveal for the first time that GABAAR single-channel properties can be modulated by an auxiliary subunit. These findings shed light on processes that shape the temporal dynamics of GABAergic transmission.SIGNIFICANCE STATEMENT Although GABAA receptor (GABAAR) single-channel properties are largely determined by pore-forming subunits, it remains unknown whether they are also controlled by GABAAR-associated transmembrane proteins. Here, we show that Shisa7, a recently identified GABAAR auxiliary subunit, modulates GABAAR activation by altering single-channel burst kinetics. These results reveal that Shisa7 primarily decreases the duration and open probability of receptor burst activity during gating, leading to accelerated GABAAR deactivation. These experiments are the first to assess the gating properties of GABAARs in the presence of an auxiliary subunit and provides a kinetic basis for how Shisa7 modifies temporal attributes of GABAergic transmission at the single-channel level.
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Proteínas de la Membrana , Receptores de GABA-A , Humanos , Receptores de GABA-A/metabolismo , Cinética , Células HEK293 , Proteínas de la Membrana/metabolismo , Proteínas Portadoras/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Genetic sequencing is identifying an expanding number of variants of GABAA receptors associated with human epilepsies. We identified a new de novo variant of the ß2 subunit (ß2L51M) of the inhibitory GABAA receptor associated with seizures. Our analysis determined the pathogenicity of the variant and the effects of anti-seizure medications. Our data demonstrates that the variant reduced cell surface trafficking and peak GABA-gated currents. Synaptic currents mediated by variant-containing receptors decayed faster than wild-type and single receptor currents showed that the variant shortened the duration of receptor activity by decreasing receptor open times. We tested the effects of the anti-seizure medications, midazolam, carbamazepine and valproate and found that all three enhance variant receptor surface expression. Additionally, midazolam restored receptor function by increasing single receptor active periods and synaptic current decay times towards wild-type levels. By contrast, valproate increased synaptic peak currents, event frequency and promoted synaptic bursting. Our study identifies a new disease-causing variant to the GABAA receptor, profiles its pathogenic effects and demonstrates how anti-seizure drugs correct its functional deficits.
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Epilepsia , Receptores de GABA-A , Humanos , Receptores de GABA-A/metabolismo , Ácido Valproico/farmacología , Ácido Valproico/uso terapéutico , Midazolam/farmacología , Midazolam/uso terapéutico , Epilepsia/tratamiento farmacológico , Ácido gamma-Aminobutírico/uso terapéuticoRESUMEN
BACKGROUND: Genetic variants in the subunits of the gamma-aminobutyric acid type A (GABAA) receptors are implicated in the onset of multiple pathologic conditions including genetic epilepsy. Previous work showed that pathogenic GABAA subunits promote misfolding and inefficient assembly of the GABAA receptors, limiting receptor expression and activity at the plasma membrane. However, GABAA receptors containing variant subunits can retain activity, indicating that enhancing the folding, assembly, and trafficking of these variant receptors offers a potential opportunity to mitigate pathology associated with genetic epilepsy. RESULTS: Here, we demonstrate that pharmacologically enhancing endoplasmic reticulum (ER) proteostasis using small molecule activators of the ATF6 (Activating Transcription Factor 6) signaling arm of the unfolded protein response (UPR) increases the assembly, trafficking, and surface expression of variant GABAA receptors. These improvements are attributed to ATF6-dependent remodeling of the ER proteostasis environment, which increases protein levels of pro-folding ER proteostasis factors including the ER chaperone BiP (Immunoglobulin Binding Protein) and trafficking receptors, such as LMAN1 (Lectin Mannose-Binding 1) and enhances their interactions with GABAA receptors. Importantly, we further show that pharmacologic ATF6 activators increase the activity of GABAA receptors at the cell surface, revealing the potential for this strategy to restore receptor activity to levels that could mitigate disease pathogenesis. CONCLUSIONS: These results indicate that pharmacologic ATF6 activators offer an opportunity to restore GABAA receptor activity in diseases including genetic epilepsy and point to the potential for similar pharmacologic enhancement of ER proteostasis to improve trafficking of other disease-associated variant ion channels implicated in etiologically-diverse diseases.
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Ants (Hymenoptera: Formicidae) are familiar inhabitants of most terrestrial environments. Although we are aware of the ability of many species to sting, knowledge of ant venom chemistry remains limited. Herein, we describe the discovery and characterization of an O-linked glycopeptide (Mg7a) as a major component of the venom of the ant Myrmecia gulosa. Electron transfer dissociation and higher-energy collisional dissociation tandem mass spectrometry were used to localize three α-N-acetylgalactosaminyl residues (α-GalNAc) present on the 63-residue peptide. To allow for functional studies, we synthesized the full-length glycosylated peptide via solid-phase peptide synthesis, combined with diselenide-selenoester ligation-deselenization chemistry. We show that Mg7a is paralytic and lethal to insects, and triggers pain behavior and inflammation in mammals, which it achieves through a membrane-targeting mode of action. Deglycosylation of Mg7a renders it insoluble in aqueous solution, suggesting a key solubilizing role of the O-glycans.
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NMDA receptor (NMDAR)-dependent Ca2+ influx underpins multiple forms of synaptic plasticity. Most synaptic NMDAR currents in the adult forebrain are mediated by GluN2A-containing receptors, which are rapidly inserted into synapses during long-term potentiation (LTP); however, the underlying molecular mechanisms remain poorly understood. In this study, we show that GluN2A is phosphorylated at Ser-1459 by Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) in response to glycine stimulation that mimics LTP in primary neurons. Phosphorylation of Ser-1459 promotes GluN2A interaction with the sorting nexin 27 (SNX27)-retromer complex, thereby enhancing the endosomal recycling of NMDARs. Loss of SNX27 or CaMKIIα function blocks the glycine-induced increase in GluN2A-NMDARs on the neuronal membrane. Interestingly, mutations of Ser-1459, including the rare S1459G human epilepsy variant, prolong the decay times of NMDAR-mediated synaptic currents in heterosynapses by increasing the duration of channel opening. These findings not only identify a critical role of Ser-1459 phosphorylation in regulating the function of NMDARs, but they also explain how the S1459G variant dysregulates NMDAR function.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Activación del Canal Iónico , Subunidades de Proteína/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Glicina , Células HEK293 , Humanos , Modelos Biológicos , Mutación/genética , Proteínas del Tejido Nervioso , Fosforilación , Fosfoserina/metabolismo , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Sinapsis/metabolismoRESUMEN
Proteostasis deficiency in mutated ion channels leads to a variety of ion channel diseases that are caused by excessive endoplasmic reticulum-associated degradation (ERAD) and inefficient membrane trafficking. We investigated proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors, the primary mediators of neuronal inhibition in the mammalian central nervous system. We screened a structurally diverse, Food and Drug Administration-approved drug library and identified dinoprost (DNP) and dihydroergocristine (DHEC) as highly efficacious enhancers of surface expression of four epilepsy-causing trafficking-deficient mutant receptors. Furthermore, DNP and DHEC restore whole-cell and synaptic currents by incorporating mutated subunits into functional receptors. Mechanistic studies revealed that both drugs reduce subunit degradation by attenuating the Grp94/Hrd1/Sel1L/VCP-mediated ERAD pathway and enhance the subunit folding by promoting subunit interactions with major GABAA receptors-interacting chaperones, BiP and calnexin. In summary, we report that DNP and DHEC remodel the endoplasmic reticulum proteostasis network to restore the functional surface expression of mutant GABAA receptors.
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Dihidroergocristina/farmacología , Dinoprost/farmacología , Epilepsia/tratamiento farmacológico , Proteostasis/efectos de los fármacos , Receptores de GABA-A/metabolismo , Línea Celular , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Epilepsia/metabolismo , Femenino , Humanos , Masculino , Receptores de GABA-A/genéticaRESUMEN
Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRß subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.
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Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Síndrome de la Persona Rígida/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Animales , Humanos , Mutación , Unión Proteica/genética , Estructura Cuaternaria de Proteína , Transporte de Proteínas/genética , Receptores de Glicina/químicaRESUMEN
Mutations in synaptic NMDA receptors (NMDARs) are associated with epilepsy and neurodevelopmental disorders. The effects of several such mutations have been investigated in recombinantly-expressed NMDARs under conditions of steady-state activation. Such experiments provide only limited insight into how mutations affect NMDAR-mediated excitatory synaptic currents (EPSCs). The present study aimed to characterize the effects of the GluN2AN615K, GluN2BN615I and GluN2BV618G gain-of-function mutations on EPSCs mediated by diheteromeric GluN1/2A and GluN1/2B receptors and triheteromeric GluN1/2A/2B receptors, as these are the most abundant synaptic NMDARs in vivo. Subunit composition was controlled by studying 'artificial' synapses formed between cultured neurons (which provide presynaptic terminals) and HEK293 cells that express the NMDAR subunits of interest plus the synapse-promoting molecule, neuroligin-1B. When incorporated into diheteromeric receptors, all three mutations ablated voltage-dependent Mg2+ block of EPSCs, as previously shown. In addition, we were surprised to find that increasing external Mg2+ from 0 to 1 mM strongly enhanced the magnitude of EPSCs mediated by mutant diheteromers. In contrast, triheteromeric receptors exhibited normal voltage-dependent Mg2+ block. The GluN2AN615K mutation also slowed the decay of GluN1/2A/2B- but not GluN1/2A-mediated EPSCs. The GluN2BN615I mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs. The GluN2BV618G mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs, although these effects were partly compensated by a faster EPSC decay rate. The mutations also diminished the potency of the anti-epileptic pore-blocker, memantine, thus explaining the lack of memantine efficacy in patients with GluN2BN615I or GluN2BV618G mutations. Given these effects, the three mutations would be expected to enhance the cation influx rate and thereby contribute to epilepsy phenotypes.
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Epilepsia/genética , Mutación con Ganancia de Función , Receptores de N-Metil-D-Aspartato/genética , Sinapsis/fisiología , Animales , Femenino , Células HEK293 , Humanos , Masculino , Neuronas/fisiología , Técnicas de Placa-Clamp , RatasRESUMEN
BACKGROUND AND PURPOSE: Between half to 1 million people die annually from malaria. Anopheles gambiae mosquitoes are major malaria vectors. Unfortunately, resistance has emerged to the agents currently used to control A. gambiae, creating a demand for novel control measures. The pentameric glutamate-gated chloride channel (GluCl) expressed in the muscle and nerve cells of these organisms are a potentially important biological target for malaria control. The pharmacological properties of Anophiline GluCl receptors are, however, largely unknown. Accordingly, we compared the efficacy of four insecticides (lindane, fipronil, picrotoxin, and ivermectin) on two A. gambiae GluCl receptor splice variants with the aim of providing a molecular basis for designing novel anti-malaria treatments. EXPERIMENTAL APPROACH: The A. gambiae GluCl receptor b1 and c splice variants were expressed homomerically in Xenopus laevis oocytes and studied with electrophysiological techniques, using two-electrode voltage-clamp. KEY RESULTS: The b1 and c GluCl receptors were activated with similar potencies by glutamate and ivermectin. Fipronil was more potent than picrotoxin and lindane at inhibiting glutamate- and ivermectin-gated currents. Importantly, b1 GluCl receptors exhibited reduced sensitivity to picrotoxin and lindane. They also recovered from these effects to a greater extent than c GluCl receptors CONCLUSIONS AND IMPLICATIONS: The two splice variant subunits exhibited differential sensitivities to multiple, structurally divergent insecticides, without accompanying changes in the sensitivity to the endogenous neurotransmitter, glutamate, implying that drug resistance may be caused by alterations in relative subunit expression levels, without affecting physiological function. Our results strongly suggest that it should be feasible to develop novel subunit-specific pharmacological agents.
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Anopheles/genética , Canales de Cloruro/genética , Insecticidas/farmacología , Mosquitos Vectores/genética , Isoformas de Proteínas/genética , Secuencia de Aminoácidos , Animales , Anopheles/metabolismo , Canales de Cloruro/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Ácido Glutámico/farmacología , Ivermectina/farmacología , Mosquitos Vectores/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Isoformas de Proteínas/metabolismo , Xenopus laevisRESUMEN
Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor.
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Canales de Cloruro/metabolismo , Haemonchus/efectos de los fármacos , Ivermectina/farmacología , Animales , Canales de Cloruro/antagonistas & inhibidores , Antagonistas de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/farmacología , Células HEK293 , Haemonchus/metabolismo , Humanos , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Receptores de Glutamato/metabolismo , Xenopus laevis/embriologíaRESUMEN
The GABAA receptor (GABAAR) α1 subunit A295D epilepsy mutation reduces the surface expression of α1A295Dß2γ2 GABAARs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat) was recently shown to correct the misfolding of α1A295D subunits and thereby enhance the functional surface expression of α1A295Dß2γ2 GABAARs. Here we investigated whether SAHA can also restore the surface expression of γ2 GABAAR subunits that incorporate epilepsy mutations (N40S, R43Q, P44S, R138G) known to reduce surface expression via ER-associated protein degradation. As a control, we also investigated the γ2 K289M epilepsy mutation that impairs gating without reducing surface expression. Effects of mutations were evaluated on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic α1ß2γ2 GABAAR isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABAARs of undefined subunit composition. Transfection with α1ß2γ2 N40S , α1ß2γ2 R43Q , α1ß2γ2 P44S and α1ß2γ2 R138G subunits produced IPSCs with decay times slower than those of unmutated α1ß2γ2 GABAARs due to the low expression of mutant γ2 subunits and the correspondingly high expression of slow-decaying α1ß2 GABAARs. SAHA pre-treatment significantly accelerated the decay time constants of IPSCs consistent with the upregulation of mutant γ2 subunit expression. This increase in surface expression was confirmed by immunohistochemistry. SAHA had no effect on either the IPSC kinetics or surface expression levels of α1ß2γ2 K289M GABAARs, confirming its specificity for ER-retained mutant γ2 subunits. We also found that α1ß2γ2 K289M GABAARs and SAHA-treated α1ß2γ2 R43Q , α1ß2γ2 P44S and α1ß2γ2 R138G GABAARs all mediated IPSCs that decayed at significantly faster rates than wild type receptors as temperature was increased from 22 to 40°C. This may help explain why these mutations cause febrile seizures (FS). Given that SAHA is approved by therapeutic regulatory agencies for human use, we propose that it may be worth investigating as a treatment for epilepsies caused by the N40S, R43Q, P44S and R138G mutations. Although SAHA has already been proposed as a therapeutic for patients harbouring the α1A295D epilepsy mutation, the present study extends its potential utility to a new subunit and four new mutations.
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Inhibitory glycine receptors (GlyRs) are pentameric ligand-gated anion channels with major roles in startle disease/hyperekplexia (GlyR α1), cortical neuronal migration/autism spectrum disorder (GlyR α2), and inflammatory pain sensitization/rhythmic breathing (GlyR α3). However, the role of the GlyR α4 subunit has remained enigmatic, because the corresponding human gene (GLRA4) is thought to be a pseudogene due to an in-frame stop codon at position 390 within the fourth membrane-spanning domain (M4). Despite this, a recent genetic study has implicated GLRA4 in intellectual disability, behavioral problems and craniofacial anomalies. Analyzing data from sequenced genomes, we found that GlyR α4 subunit genes are predicted to be intact and functional in the majority of vertebrate species-with the exception of humans. Cloning of human GlyR α4 cDNAs excluded alternative splicing and RNA editing as mechanisms for restoring a full-length GlyR α4 subunit. Moreover, artificial restoration of the missing conserved arginine (R390) in the human cDNA was not sufficient to restore GlyR α4 function. Further bioinformatic and mutagenesis analysis revealed an additional damaging substitution at K59 that ablates human GlyR α4 function, which is not present in other vertebrate GlyR α4 sequences. The substitutions K59 and X390 were also present in the genome of an ancient Denisovan individual, indicating that GLRA4 has been a pseudogene for at least 30,000-50,000 years. In artificial synapses, we found that both mouse and gorilla α4ß GlyRs mediate synaptic currents with unusually slow decay kinetics. Lastly, to gain insights into the biological role of GlyR α4 function, we studied the duplicated genes glra4a and glra4b in zebrafish. While glra4b expression is restricted to the retina, using a novel tol2-GAL4FF gene trap line (SAIGFF16B), we found that the zebrafish GlyR α4a subunit gene (glra4a) is strongly expressed in spinal cord and hindbrain commissural neurones. Using gene knockdown and a dominant-negative GlyR α4aR278Q mutant, we found that GlyR α4a contributes to touch-evoked escape behaviors in zebrafish. Thus, although GlyR α4 is unlikely to be involved in human startle responses or disease states, this subtype may contribute to escape behaviors in other organisms.
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Ivermectin (IVM) is a widely-used anthelmintic that works by binding to and activating glutamate-gated chloride channel receptors (GluClRs) in nematodes. The resulting chloride flux inhibits the pharyngeal muscle cells and motor neurons of nematodes, causing death by paralysis or starvation. IVM resistance is an emerging problem in many pest species, necessitating the development of novel drugs. However, drug optimisation requires a quantitative understanding of GluClR activation and modulation mechanisms. Here we investigated the biophysical properties of homomeric α (avr-14b) GluClRs from the parasitic nematode, H. contortus, in the presence of glutamate and IVM. The receptor proved to be highly responsive to low nanomolar concentrations of both compounds. Analysis of single receptor activations demonstrated that the GluClR oscillates between multiple functional states upon the binding of either ligand. The G36'A mutation in the third transmembrane domain, which was previously thought to hinder access of IVM to its binding site, was found to decrease the duration of active periods and increase receptor desensitisation. On an ensemble macropatch level the mutation gave rise to enhanced current decay and desensitisation rates. Because these responses were common to both glutamate and IVM, and were observed under conditions where agonist binding sites were likely saturated, we infer that G36'A affects the intrinsic properties of the receptor with no specific effect on IVM binding mechanisms. These unexpected results provide new insights into the activation and modulatory mechanisms of the H. contortus GluClRs and provide a mechanistic framework upon which the actions of drugs can be reliably interpreted.
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Antihelmínticos/farmacología , Canales de Cloruro/metabolismo , Haemonchus , Ivermectina/farmacología , Animales , Caenorhabditis elegans/genética , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Mutación/genéticaRESUMEN
Epilepsy is a spectrum of neurological disorders with many causal factors. The GABA type-A receptor (GABAAR) is a major genetic target for heritable human epilepsies. Here we examine the functional effects of three epilepsy-causing mutations to the α1 subunit (α1T10'I, α1D192N and α1A295D) on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic GABAAR isoform, α1ß2γ2L. We employed a neuron - HEK293 cell heterosynapse preparation to record IPSCs mediated by mutant-containing GABAARs in isolation from other GABAAR isoforms. IPSCs were recorded in the presence of the anticonvulsant drugs, carbamazepine and midazolam, and at elevated temperatures (22, 37 and 40°C) to gain insight into mechanisms of febrile seizures. The mutant subunits were also transfected into cultured cortical neurons to investigate changes in synapse formation and neuronal morphology using fluorescence microscopy. We found that IPSCs mediated by α1T10'Iß2γ2L, α1D192Nß2γ2L GABAARs decayed faster than those mediated by α1ß2γ2L receptors. IPSCs mediated by α1D192Nß2γ2L and α1A295Dß2γ2L receptors also exhibited a heightened temperature sensitivity. In addition, the α1T10'Iß2γ2L GABAARs were refractory to modulation by carbamazepine or midazolam. In agreement with previous studies, we found that α1A295Dß2γ2L GABAARs were retained intracellularly in HEK293 cells and neurons. However, pre-incubation with 100nM suberanilohydroxamic acid (SAHA) induced α1A295Dß2γ2L GABAARs to mediate IPSCs that were indistinguishable in magnitude and waveform from those mediated by α1ß2γ2L receptors. Finally, mutation-specific changes to synaptic bouton size, synapse number and neurite branching were also observed. These results provide new insights into the mechanisms of epileptogenesis of α1 epilepsy mutations and suggest possible leads for improving treatments for patients harbouring these mutations.
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Epilepsia/metabolismo , Inhibición Neural/fisiología , Neuronas/metabolismo , Receptores de GABA-A/metabolismo , Sinapsis/metabolismo , Animales , Anticonvulsivantes/farmacología , Carbamazepina/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Técnicas de Cocultivo , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Epilepsia/patología , Células HEK293 , Humanos , Ácidos Hidroxámicos/farmacología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Midazolam/farmacología , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Técnicas de Placa-Clamp , Pliegue de Proteína/efectos de los fármacos , Ratas , Receptores de GABA-A/genética , Sinapsis/efectos de los fármacos , Sinapsis/patología , Temperatura , VorinostatRESUMEN
α5-containing GABAARs are potential therapeutic targets for clinical conditions including age-related dementia, stroke, schizophrenia, Down syndrome, anaesthetic-induced amnesia, anxiety and pain. α5-containing GABAARs are expressed in layer 5 cortical neurons and hippocampal pyramidal neurons where they mediate both tonic currents and slow inhibitory postsynaptic currents (IPSCs). A range of drugs has been developed to specifically modulate these receptors. The main α5-containing GABAARs that are likely to exist in vivo are the α5ß1γ2, α5ß2γ2 and α5ß3γ2 isoforms. We currently have few clues as to how these isoforms are distributed between synaptic and extrasynaptic compartments or their relative roles in controlling neuronal excitability. Accordingly, the aim of this study was to define the basic biophysical and pharmacological properties of IPSCs mediated by the three isoforms in a hippocampal neuron-HEK293 cell co-culture assay. The IPSC decay time constants were slow (α5ß1γ2L: 45 ms; α5ß1γ2L: 80 ms; α5ß3γ2L: 184 ms) and were largely dominated by the intrinsic channel deactivation rates. By comparing IPSC rise times, we inferred that α5ß1γ2L GABAARs are located postsynaptically whereas the other two are predominantly perisynaptic. α5ß3γ2L GABAARs alone mediated tonic currents. We quantified the effects of four α5-specific inverse agonists (TB-21007, MRK-016, α5IA and L-655708) on IPSCs mediated by the three isoforms. All compounds selectively inhibited IPSC amplitudes and accelerated IPSC decay rates, albeit with distinct isoform specificities. MRK-016 also significantly accelerated IPSC rise times. These results provide a reference for future studies seeking to identify and characterize the properties of IPSCs mediated by α5-containing GABAAR isoforms in neurons.
Asunto(s)
Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Receptores de GABA-A/metabolismo , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Agonistas de Receptores de GABA-A/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Técnicas de Placa-Clamp , Isoformas de Proteínas , Ratas , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
GABA-A receptors (GABAARs) are pentameric ligand-gated ion channels that are assembled mainly from α (α1-6), ß (ß1-3) and γ (γ1-3) subunits. Although GABAARs containing γ2L subunits mediate most of the inhibitory neurotransmission in the brain, significant expression of γ1 subunits is seen in the amygdala, pallidum and substantia nigra. However, the location and function of γ1-containing GABAARs in these regions remains unclear. In "artificial" synapses, where the subunit composition of postsynaptic receptors is specifically controlled, γ1 incorporation slows the synaptic current decay rate without affecting channel deactivation, suggesting that γ1-containing receptors are not clustered and therefore activated by diffuse neurotransmitter. However, we show that γ1-containing receptors are localized at neuronal synapses and form clusters in both synaptic and extrasynaptic regions. In addition, they exhibit rapid membrane diffusion and a higher frequency of exchange between synaptic and perisynaptic populations compared to γ2L-containing GABAARs. A point mutation in the large intracellular domain and a pharmacological analysis reveal that when a single non-conserved γ2L residue is mutated to its γ1 counterpart (T349L), the synaptic current decay is slowed from γ2L- to γ1-like without changing the clustering or diffusion properties of the receptors. In addition, previous fast perfusion and single channel kinetic experiments revealed no difference in the intrinsic closing rates of γ2L- and γ1-containing receptors when expressed in HEK293 cells. These observations together with Monte Carlo simulations of synaptic function confirm that decreased clustering does not control γ1-containing GABAAR kinetics. Rather, they suggest that γ1- and γ2L-containing receptors exhibit differential synaptic current decay rates due to differential gating dynamics when localized at the synapse.