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1.
Eur J Med Chem ; 261: 115832, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-37837674

RESUMO

Alzheimer's disease (AD) is a global health problem in the medical sector that will increase over time. The limited treatment of AD leads to the search for a new clinical candidate. Considering the multifactorial nature of AD, a strategy targeting number of regulatory proteins involved in the development of the disease is an effective approach. Here, we present a discovery of new multi-target-directed ligands (MTDLs), purposely designed as GABA transporter (GAT) inhibitors, that successfully provide the inhibitory activity against butyrylcholinesterase (BuChE), ß-secretase (BACE1), amyloid ß aggregation and calcium channel blockade activity. The selected GAT inhibitors, 19c and 22a - N-benzylamide derivatives of 4-aminobutyric acid, displayed the most prominent multifunctional profile. Compound 19c (mGAT1 IC50 = 10 µM, mGAT4 IC50 = 12 µM and BuChE IC50 = 559 nM) possessed the highest hBACE1 and Aß40 aggregation inhibitory activity (IC50 = 1.57 µM and 99 % at 10 µM, respectively). Additionally, it showed a decrease in both the elongation and nucleation constants of the amyloid aggregation process. In contrast compound 22a represented the highest activity and a mixed-type of eqBuChE inhibition (IC50 = 173 nM) with hBACE1 (IC50 = 9.42 µM), Aß aggregation (79 % at 10 µM) and mGATs (mGAT1 IC50 = 30 µM, mGAT4 IC50 = 25 µM) inhibitory activity. Performed molecular docking studies described the mode of interactions with GATs and enzymatic targets. In ADMET in vitro studies both compounds showed acceptable metabolic stability and low neurotoxicity. Successfully, compounds 19c and 22a at the dose of 30 mg/kg possessed statistically significant antiamnesic properties in a mouse model of amnesia caused by scopolamine and assessed in the novel object recognition (NOR) task or the passive avoidance (PA) task.


Assuntos
Doença de Alzheimer , Butirilcolinesterase , Camundongos , Animais , Butirilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Inibidores da Colinesterase/metabolismo , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Desenho de Fármacos , Ácido Aspártico Endopeptidases/metabolismo , Acetilcolinesterase/metabolismo
2.
Brain ; 146(12): 5198-5208, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-37647852

RESUMO

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10-3, 95% confidence interval: 1.5-15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).


Assuntos
Proteínas da Membrana Plasmática de Transporte de GABA , Estudos de Associação Genética , Mutação de Sentido Incorreto , Humanos , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Fenótipo
4.
Epilepsia ; 64(10): e214-e221, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37501613

RESUMO

The solute carrier family 6 member 1 (SLC6A1) gene encodes GAT-1, a γ-aminobutyric acid transporter expressed on astrocytes and inhibitory neurons. Mutations in SLC6A1 are associated with epilepsy and developmental disorders, including motor and social impairments, but variant-specific animal models are needed to elucidate mechanisms. Here, we report electrocorticographic (ECoG) recordings and clinical data from a patient with a variant in SLC6A1 that encodes GAT-1 with a serine-to-leucine substitution at amino acid 295 (S295L), who was diagnosed with childhood absence epilepsy. Next, we show that mice bearing the S295L mutation (GAT-1S295L/+ ) have spike-and-wave discharges with motor arrest consistent with absence-type seizures, similar to GAT-1+/- mice. GAT-1S295L/+ and GAT-1+/- mice follow the same pattern of pharmacosensitivity, being bidirectionally modulated by ethosuximide (200 mg/kg ip) and the GAT-1 antagonist NO-711 (10 mg/kg ip). By contrast, GAT-1-/- mice were insensitive to both ethosuximide and NO-711 at the doses tested. In conclusion, ECoG findings in GAT-1S295L/+ mice phenocopy GAT-1 haploinsufficiency and provide a useful preclinical model for drug screening and gene therapy investigations.


Assuntos
Epilepsia Tipo Ausência , Etossuximida , Humanos , Camundongos , Animais , Criança , Etossuximida/uso terapêutico , Haploinsuficiência/genética , Ácidos Nipecóticos/uso terapêutico , Epilepsia Tipo Ausência/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo
5.
Glia ; 71(11): 2527-2540, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37431178

RESUMO

Gamma-aminobutyric acid (GABA), the principal inhibitory neurotransmitter in the brain, affects numerous immune cell functions. Microglia, the brain's resident innate immune cells, regulate GABA signaling through GABA receptors and express the complete GABAergic machinery for GABA synthesis, uptake, and release. Here, the use of primary microglial cell cultures and ex vivo brain tissue sections allowed for demonstrating that treatment with lipopolysaccharide (LPS) increased microglial GABA uptake as well as GABA transporter (GAT)-1 trafficking. This effect was not entirely abolished by treatment with GAT inhibitors (GAT-Is). Notably, LPS also induced microglial upregulation of bestrophin-1 (BEST-1), a Ca2+ -activated Cl- channel permeable to GABA. Combined administration of GAT-Is and a BEST-1 inhibitor completely abolished LPS-induced microglial GABA uptake. Interestingly, increased microglial GAT-1 membrane turnover via syntaxin 1A was detected in LPS-treated cultures after BEST-1 blockade. Altogether, these findings provided evidence for a novel mechanism through which LPS may trigger the inflammatory response by directly altering microglial GABA clearance and identified the GAT-1/BEST-1 interplay as a potential novel mechanism involved in brain inflammation.


Assuntos
Lipopolissacarídeos , Microglia , Microglia/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Bestrofinas/metabolismo , Ácido gama-Aminobutírico/metabolismo
6.
Nat Struct Mol Biol ; 30(7): 1023-1032, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37400654

RESUMO

The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.


Assuntos
Cloretos , Ácido gama-Aminobutírico , Ratos , Animais , Proteínas da Membrana Plasmática de Transporte de GABA/química , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Microscopia Crioeletrônica , Cloretos/metabolismo , Cloretos/farmacologia , Ácido gama-Aminobutírico/metabolismo , Sítios de Ligação
7.
Nat Struct Mol Biol ; 30(7): 1012-1022, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37400655

RESUMO

γ-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in the central nervous system, is recycled through specific GABA transporters (GATs). GAT1, which is mainly expressed in the presynaptic terminals of axons, is a potential drug target of neurological disorders due to its essential role in GABA transport. Here we report four cryogenic electron microscopy structures of human GAT1, at resolutions of 2.2-3.2 Å. GAT1 in substrate-free form or in complex with the antiepileptic drug tiagabine exhibits an inward-open conformation. In the presence of GABA or nipecotic acid, inward-occluded structures are captured. The GABA-bound structure reveals an interaction network bridged by hydrogen bonds and ion coordination for GABA recognition. The substrate-free structure unwinds the last helical turn of transmembrane helix TM1a to release sodium ions and substrate. Complemented by structure-guided biochemical analyses, our studies reveal detailed mechanism of GABA recognition and transport, and elucidate mode of action of the inhibitors, nipecotic acid and tiagabine.


Assuntos
Ácido gama-Aminobutírico , Humanos , Tiagabina , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Conformação Molecular
9.
PeerJ ; 11: e15275, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37159834

RESUMO

Sunflower is an important oilseed crop across the world. It is considered as a moderately drought tolerant plant, however, its yield is still negatively affected by drought stress. Improving drought tolerance is of the outmost important for breeding. Although several studies have documented the relationship between the sunflower phenotype and genotype under drought stress, but relatively few studies have simultaneously investigated the molecular mechanisms of drought tolerance in the sunflower at different growth stages. In this study, we conducted quantitative trait locus (QTL) analysis for different sunflower traits during the germination and seedling stages. Eighteen phenotypic traits were evaluated under well-watered and drought stress conditions. We determined that the germination rate, germination potential, germination index, and root-to-shoot ratio can be used as effective indexes for drought tolerance selection and breeding. A total of 33 QTLs were identified on eight chromosomes (PVE: 0.016%-10.712% with LOD: 2.017-7.439). Within the confidence interval of the QTL, we identified 60 putative drought-related genes. Four genes located on chromosome 13 may function in both germination and seedling stages for drought response. Genes LOC110898128, LOC110898092, LOC110898071, and LOC110898072 were annotated as aquaporin SIP1-2-like, cytochrome P450 94C1, GABA transporter 1-like, and GABA transporter 1-like isoform X2, respectively. These genes will be used for further functional validation. This study provides insight into the molecular mechanisms of the sunflower's in response to drought stress. At the same time, it lays a foundation for sunflower drought tolerance breeding and genetic improvement.


Assuntos
Asteraceae , Helianthus , Helianthus/genética , Plântula/genética , Germinação/genética , Secas , Proteínas da Membrana Plasmática de Transporte de GABA , Melhoramento Vegetal , Aquaporina 2
10.
Proc Natl Acad Sci U S A ; 120(21): e2301330120, 2023 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-37186824

RESUMO

The hypothalamic suprachiasmatic nucleus (SCN) is the master mammalian circadian clock. Its cell-autonomous timing mechanism, a transcriptional/translational feedback loop (TTFL), drives daily peaks of neuronal electrical activity, which in turn control circadian behavior. Intercellular signals, mediated by neuropeptides, synchronize and amplify TTFL and electrical rhythms across the circuit. SCN neurons are GABAergic, but the role of GABA in circuit-level timekeeping is unclear. How can a GABAergic circuit sustain circadian cycles of electrical activity, when such increased neuronal firing should become inhibitory to the network? To explore this paradox, we show that SCN slices expressing the GABA sensor iGABASnFR demonstrate a circadian oscillation of extracellular GABA ([GABA]e) that, counterintuitively, runs in antiphase to neuronal activity, with a prolonged peak in circadian night and a pronounced trough in circadian day. Resolving this unexpected relationship, we found that [GABA]e is regulated by GABA transporters (GATs), with uptake peaking during circadian day, hence the daytime trough and nighttime peak. This uptake is mediated by the astrocytically expressed transporter GAT3 (Slc6a11), expression of which is circadian-regulated, being elevated in daytime. Clearance of [GABA]e in circadian day facilitates neuronal firing and is necessary for circadian release of the neuropeptide vasoactive intestinal peptide, a critical regulator of TTFL and circuit-level rhythmicity. Finally, we show that genetic complementation of the astrocytic TTFL alone, in otherwise clockless SCN, is sufficient to drive [GABA]e rhythms and control network timekeeping. Thus, astrocytic clocks maintain the SCN circadian clockwork by temporally controlling GABAergic inhibition of SCN neurons.


Assuntos
Relógios Circadianos , Ritmo Circadiano , Animais , Ritmo Circadiano/genética , Relógios Circadianos/genética , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Núcleo Supraquiasmático/metabolismo , Ácido gama-Aminobutírico/metabolismo , Mamíferos/metabolismo
11.
Epilepsia Open ; 8(3): 834-845, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37052238

RESUMO

OBJECTIVE: As of 2022, 36 anti-seizure medications (ASMs) have been licensed for the treatment of epilepsy, however, adverse effects (AEs) are commonly reported. Therefore, ASMs with a wide margin between therapeutic effects and AEs are preferred over ASMs that are associated with a narrow margin between efficacy and risk of AEs. E2730 was discovered using in vivo phenotypic screening and characterized as an uncompetitive, yet selective, inhibitor of γ-aminobutyric acid (GABA) transporter 1 (GAT1). Here, we describe the preclinical characteristics of E2730. METHODS: Anti-seizure effects of E2730 were evaluated in several animal models of epilepsy: corneal kindling, 6 Hz-44 mA psychomotor seizure, amygdala kindling, Fragile X syndrome, and Dravet syndrome models. Effects of E2730 on motor coordination were assessed in accelerating rotarod tests. The mechanism of action of E2730 was explored by [3 H]E2730 binding assay. The GAT1-selectivity over other GABA transporters was examined by GABA uptake assay of GAT1, GAT2, GAT3, or betaine/GABA transporter 1 (BGT-1) stably expressing HEK293 cells. To further investigate the mechanism for E2730-mediated inhibition of GAT1, in vivo microdialysis and in vitro GABA uptake assays were conducted under conditions of different GABA concentrations. RESULTS: E2730 showed anti-seizure effects in the assessed animal models with an approximately >20-|fold margin between efficacy and motor incoordination. [3 H]E2730 binding on brain synaptosomal membrane was abolished in GAT1-deficient mice, and E2730 selectively inhibited GAT1-mediated GABA uptake over other GABA transporters. In addition, results of GABA uptake assays showed that E2730-mediated inhibition of GAT1 positively correlated to the level of ambient GABA in vitro. E2730 also increased extracellular GABA concentration in hyperactivated conditions but not under basal levels in vivo. SIGNIFICANCE: E2730 is a novel, selective, uncompetitive GAT1 inhibitor, which acts selectively under the condition of increasing synaptic activity, contributing to a wide margin between therapeutic effect and motor incoordination.


Assuntos
Anticonvulsivantes , Epilepsia , Proteínas da Membrana Plasmática de Transporte de GABA , Animais , Humanos , Camundongos , Ataxia , Epilepsia/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de GABA/administração & dosagem , Ácido gama-Aminobutírico/farmacologia , Ácido gama-Aminobutírico/metabolismo , Células HEK293 , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico
12.
Cell Rep ; 42(4): 112294, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-36947544

RESUMO

Stroke is a leading cause of adult disability worldwide, and better drugs are needed to promote functional recovery after stroke. Growing evidence suggests the critical role of network excitability during the repair phase for stroke recovery. Here, we show that ß-hydroxybutyrate (ß-HB), an essential ketone body (KB) component, is positively correlated with improved outcomes in patients with stroke and promotes functional recovery in rodents with stroke during the repair phase. These beneficial effects of ß-HB depend on HDAC2/HDAC3-GABA transporter 1 (GAT-1) signaling-mediated enhancement of excitability and phasic GABA inhibition in the peri-infarct cortex and structural and functional plasticity in the ipsilateral cortex, the contralateral cortex, and the corticospinal tract. Together with available clinical approaches to elevate KB levels, our results offer a clinically translatable means to promote stroke recovery. Furthermore, GAT-1 can serve as a pharmacological target for developing drugs to promote functional recovery after stroke.


Assuntos
Corpos Cetônicos , Acidente Vascular Cerebral , Humanos , Proteínas da Membrana Plasmática de Transporte de GABA
13.
Brain Dev ; 45(7): 395-400, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36966012

RESUMO

INTRODUCTION: Epilepsy with myoclonic atonic seizures (EMAtS) was previously thought to occur in normally developing children. We report a female case of EMAtS and mild developmental delay before onset. Importantly, a de novo balanced chromosomal translocation was recognized in the patient. CASE PRESENTATION: The patient was a 4-year-old girl. Mild developmental delay was observed during infancy. At the age of one and a half years, she developed atonic seizures once a month. At 4 years of age, her seizures increased to more than 10 times per hour. An ictal electroencephalogram (EEG) showed a 3-4-Hz spike-and-wave complex, which was consistent with atonic and myoclonic seizures of the trunk, eyelids, and lips. Therefore, EMAtS was diagnosed based on the symptoms and EEG findings. After administration of valproic acid (VPA), the epileptic seizures disappeared immediately. At the age of 5 years and 2 months, the seizures recurred but disappeared again when the dose of VPA was increased. Subsequently, no recurrence was observed until 6 years and 3 months of age on VPA and lamotrigine. Chromosome analysis of the patient disclosed 46,XX,t(3;11)(p25;q13.1)dn. Long-read sequencing of the the patient's genomic DNA revealed that the 3p25.3 translocation breakpoint disrupted the intron 7 of the SLC6A1 gene. CONCLUSION: The SLC6A1 disruption by chromosome translocation well explains the clinical features of this patient. Long-read sequencing is a powerful technique to determine genomic abnormality at the nucleotide level for disease-associated chromosomal abnormality.


Assuntos
Epilepsias Mioclônicas , Translocação Genética , Humanos , Criança , Feminino , Lactente , Pré-Escolar , Translocação Genética/genética , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/tratamento farmacológico , Mutação , Convulsões/genética , Convulsões/tratamento farmacológico , Anticonvulsivantes/uso terapêutico , Ácido Valproico/uso terapêutico , Eletroencefalografia , Proteínas da Membrana Plasmática de Transporte de GABA/genética
14.
Neurochem Int ; 165: 105522, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36966820

RESUMO

The GABA transporter GAT1 regulates brain inhibitory neurotransmission and it is considered a potential therapeutic target for the treatment of wide spectrum of neurological diseases including epilepsy, stroke and autism. Syntenin-1 binds to syntaxin 1A, which is known to regulate the plasma membrane insertion of several neurotransmitter transporters. Previously, a direct interaction of syntenin-1 with the glycine transporter GlyT2 was reported. Here, we show that the GABA transporter GAT1 also directly interacts with syntenin-1, involving both unidentified protein interaction interface and the GAT1 C-terminal PDZ binding motif interacting mainly with syntenin-1 PDZ domain 1. The PDZ interaction was eliminated by the mutation of GAT1 isoleucine 599 and tyrosine 598 located in PDZ positions 0 and -1, respectively. This indicates an unconventional PDZ interaction and possible regulation of the transporter PDZ motif via tyrosine phosphorylation. Whole syntenin-1 protein fused to GST protein and immobilised on glutathione resin coprecipitated intact GAT1 transporter from an extract of GAT1 transfected neuroblastoma N2a cells. This coprecipitation was inhibited by tyrosine phosphatases inhibitor pervanadate. The fluorescence tagged GAT1 and syntenin-1 colocalized upon coexpression in N2a cells. The above results show that syntenin-1 might be, in addition to GlyT2, directly involved in the trafficking of GAT1 transporter.


Assuntos
Proteínas da Membrana Plasmática de Transporte de GABA , Proteínas do Tecido Nervoso , Sinteninas , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Sinteninas/genética , Sinteninas/metabolismo , Tirosina/metabolismo , Animais , Camundongos , Linhagem Celular
15.
Front Immunol ; 14: 1114350, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36825000

RESUMO

Rheumatoid arthritis (RA) is a highly disabling chronic autoimmune disease. Multiple factors contribute to the complex pathological process of RA, in which an abnormal autoimmune response, high survival of inflammatory cells, and excessive release of inflammatory factors lead to a severe chronic inflammatory response. Clinical management of RA remains limited; therefore, exploring and discovering new mechanisms of action could enhance clinical benefits for patients with RA. Important bidirectional communication occurs between the brain and immune system in inflammatory diseases such as RA, and circulating immune complexes can cause neuroinflammatory responses in the brain. The gamma-aminobutyric acid (GABA)ergic system is a part of the nervous system that primarily comprises GABA, GABA-related receptors, and GABA transporter (GAT) systems. GABA is an inhibitory neurotransmitter that binds to GABA receptors in the presence of GATs to exert a variety of pathophysiological regulatory effects, with its predominant role being neural signaling. Nonetheless, the GABAergic system may also have immunomodulatory effects. GABA/GABA-A receptors may inhibit the progression of inflammation in RA and GATs may promote inflammation. GABA-B receptors may also act as susceptibility genes for RA, regulating the inflammatory response of RA via immune cells. Furthermore, the GABAergic system may modulate the abnormal pain response in RA patients. We also summarized the latest clinical applications of the GABAergic system and provided an outlook on its clinical application in RA. However, direct studies on the GABAergic system and RA are still lacking; therefore, we hope to provide potential therapeutic options and a theoretical basis for RA treatment by summarizing any potential associations.


Assuntos
Artrite Reumatoide , Ácido gama-Aminobutírico , Humanos , Artrite Reumatoide/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Ácido gama-Aminobutírico/metabolismo , Inflamação , Receptores de GABA/metabolismo
16.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36674476

RESUMO

In this article, we identified a novel epileptogenic variant (G307R) of the gene SLC6A1, which encodes the GABA transporter GAT-1. Our main goal was to investigate the pathogenic mechanisms of this variant, located near the neurotransmitter permeation pathway, and compare it with other variants located either in the permeation pathway or close to the lipid bilayer. The mutants G307R and A334P, close to the gates of the transporter, could be glycosylated with variable efficiency and reached the membrane, albeit inactive. Mutants located in the center of the permeation pathway (G297R) or close to the lipid bilayer (A128V, G550R) were retained in the endoplasmic reticulum. Applying an Elastic Network Model, to these and to other previously characterized variants, we found that G307R and A334P significantly perturb the structure and dynamics of the intracellular gate, which can explain their reduced activity, while for A228V and G362R, the reduced translocation to the membrane quantitatively accounts for the reduced activity. The addition of a chemical chaperone (4-phenylbutyric acid, PBA), which improves protein folding, increased the activity of GAT-1WT, as well as most of the assayed variants, including G307R, suggesting that PBA might also assist the conformational changes occurring during the alternative access transport cycle.


Assuntos
Epilepsias Mioclônicas , Proteínas da Membrana Plasmática de Transporte de GABA , Bicamadas Lipídicas , Humanos , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Epilepsias Mioclônicas/metabolismo , Epilepsias Mioclônicas/patologia
17.
Trends Pharmacol Sci ; 44(1): 4-6, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-35985875

RESUMO

The γ-aminobutyric acid transporter 1 (GAT1) is a transporter which clears the inhibitory neurotransmitter γ-aminobutyric acid (GABA) from the synaptic cleft. The paper by Motiwala et al. documents a structure of GAT1 in complex with the antiepileptic drug tiagabine. This study will enable structure-based docking of large chemical libraries for the discovery of novel antiepileptics.


Assuntos
Anticonvulsivantes , Ácido gama-Aminobutírico , Humanos , Proteínas da Membrana Plasmática de Transporte de GABA/química , Anticonvulsivantes/farmacologia , Tiagabina
18.
Biomolecules ; 12(11)2022 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-36359013

RESUMO

The recently obtained cryo-electron microscopy structure (PDB code: 7SK2) of the human γ-aminobutyric acid transporter type 1 (hGAT-1) in complex with the antiepileptic drug, tiagabine, revealed a rather unexpected binding mode for this inhibitor in an inward-open state of the transporter. The simultaneously released crystal structures of the modified dopamine transporter with mutations mimicking hGAT-1 indicated an alternative binding mode for the tiagabine analogues that were found to block the transporter in an outward-open state, which is more consistent with the results of previous biological and molecular modeling studies. In view of the above discrepancies, our study compares different hypothetical tiagabine binding modes using classical and accelerated molecular dynamics simulations, as well as MM-GBSA free binding energy (dG) calculations. The results indicate that the most stable and energetically favorable binding mode of tiagabine is the one where the nipecotic acid fragment is located in the main binding site (S1) and the aromatic rings are arranged within the S2 site of the hGAT-1 transporter in an outward-open state, confirming the previous molecular modelling findings. The position of tiagabine bound to hGAT-1 in an inward-open state, partially within the intracellular release pathway, was significantly less stable and the dG values calculated for this complex were higher. Furthermore, analysis of the cryo-electron map for the 7SK2 structure shows that the model does not appear to fit into the map optimally at the ligand binding site. These findings suggest that the position of tiagabine found in the 7SK2 structure is rather ambiguous and requires further experimental verification. The identification of the main, high-affinity binding site for tiagabine and its analogues is crucial for the future rational design of the GABA transporter inhibitors.


Assuntos
Anticonvulsivantes , Simulação de Dinâmica Molecular , Humanos , Tiagabina , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Microscopia Crioeletrônica , Anticonvulsivantes/farmacologia
19.
Cells ; 11(19)2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36231112

RESUMO

The concept of the tripartite synapse describes the close interaction of pre- and postsynaptic elements and the surrounding astrocyte processes. For glutamatergic synapses, it is established that the presence of astrocytic processes and their structural arrangements varies considerably between and within brain regions and between synapses of the same neuron. In contrast, less is known about the organization of astrocytic processes at GABAergic synapses although bi-directional signaling is known to exist at these synapses too. Therefore, we established super-resolution expansion microscopy of GABAergic synapses and nearby astrocytic processes in the stratum radiatum of the mouse hippocampal CA1 region. By visualizing the presynaptic vesicular GABA transporter and the postsynaptic clustering protein gephyrin, we documented the subsynaptic heterogeneity of GABAergic synaptic contacts. We then compared the volume distribution of astrocytic processes near GABAergic synapses between individual synapses and with glutamatergic synapses. We made two novel observations. First, astrocytic processes were more abundant at the GABAergic synapses with large postsynaptic gephyrin clusters. Second, astrocytic processes were less abundant in the vicinity of GABAergic synapses compared to glutamatergic, suggesting that the latter may be selectively approached by astrocytes. Because of the GABA transporter distribution, we also speculate that this specific arrangement enables more efficient re-uptake of GABA into presynaptic terminals.


Assuntos
Receptores de GABA-A , Ácido gama-Aminobutírico , Animais , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Camundongos , Terminações Pré-Sinápticas/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo
20.
J Mol Biol ; 434(17): 167747, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35870651

RESUMO

The transporter BetP in C. glutamicum is essential in maintaining bacterial cell viability during hyperosmotic stress and functions by co-transporting betaine and Na+ into bacterial cells. Hyperosmotic stress leads to increased intracellular K+ concentrations which in turn promotes betaine binding. While structural details of multiple end state conformations of BetP have provided high resolution snapshots, how K+ sensing by the C-terminal domain is allosterically relayed to the betaine binding site is not well understood. In this study, we describe conformational dynamics in solution of BetP using amide hydrogen/deuterium exchange mass spectrometry. These reveal how K+ alters conformation of the disordered C- and N-terminal domains to allosterically reconfigure transmembrane helices 3, 8, and 10 to enhance betaine interactions. A map of the betaine binding site, at near single amino acid resolution, reveals a critical extrahelical H-bond mediated by TM3 with betaine.


Assuntos
Proteínas de Bactérias , Betaína , Corynebacterium glutamicum , Proteínas da Membrana Plasmática de Transporte de GABA , Pressão Osmótica , Proteínas de Bactérias/química , Betaína/química , Sítios de Ligação , Corynebacterium glutamicum/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA/química , Ligação de Hidrogênio , Ligação Proteica , Estrutura Secundária de Proteína
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