RESUMEN
INTRODUCTION: The main inhibitory neurotransmitter in the central nervous system (CNS), γ-aminobutyric acid (GABA), is involved in a multitude of neurological and psychiatric disorders characterized by an imbalance in excitatory and inhibitory signaling. Regulation of extracellular levels of GABA is maintained by the four GABA transporters (GATs; GAT1, GAT2, GAT3, and BGT1), Na+/Cl--coupled transporters of the solute carrier 6 (SLC6) family. Despite mounting evidence for the involvement of the non-GAT1 GABA transporters in diseases, only GAT1 has successfully been translated into clinical practice via the drug tiagabine. AREAS COVERED: In this review, all four GATs will be described in terms of their involvement in disease, and the most recent data on structure, function, expression, and localization discussed in relation to their potential role as drug targets. This includes an overview of various ways to modulate the GATs in relation to treatment of diseases caused by imbalances in the GABAergic system. EXPERT OPINION: The recent publication of various GAT1 structures is an important milestone for future development of compounds targeting the GATs. Such information can provide much needed insight into mechanistic aspects of all GAT subtypes and be utilized to design improved ligands for this highly interesting drug target class.
Asunto(s)
Desarrollo de Medicamentos , Proteínas Transportadoras de GABA en la Membrana Plasmática , Terapia Molecular Dirigida , Enfermedades del Sistema Nervioso , Tiagabina , Ácido gamma-Aminobutírico , Humanos , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Animales , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Tiagabina/farmacología , Trastornos Mentales/tratamiento farmacológico , Trastornos Mentales/fisiopatología , Trastornos Mentales/metabolismoRESUMEN
The taurine transporter (TauT, SLC6A6) is a member of the solute carrier 6 (SLC6) family, which plays multiple physiological roles. The SLC6 family is divided into four subfamilies: GABA (γ-aminobutyric acid), monoamine, glycine and neutral amino acid transporters. Proteins from the GABA group, including the taurine transporter, are primarily considered therapeutic targets for treating central nervous system disorders. However, recent studies have suggested that inhibitors of SLC6A6 could also serve as anticancer agents. Overexpression of TauT has been associated with the progression of colon and gastric cancer. The pool of known ligands of this transporter is limited and the exact spatial structure of taurine transporter remains unsolved. Understanding its structure could aid in the development of novel inhibitors. Therefore, we utilized homology modelling techniques to create models of TauT. Docking studies and molecular dynamics simulations were conducted to describe protein-ligand interactions. We compared the obtained information for TauT with literature data on other members of the GABA transporter group. Our in silico analysis allowed us to characterize the transporter structure and point out amino acids crucial for ligand binding: Glu406, Gly62 and Tyr138. The significance of selected residues was confirmed through structural studies of mutants. These results will aid in the development of novel taurine transporter inhibitors, which can be explored as anticancer agents.
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática , Proteínas de Transporte de Membrana , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Humanos , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/antagonistas & inhibidores , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Ligandos , Secuencia de Aminoácidos , Unión ProteicaRESUMEN
Impaired expression of GABA transporters (GATs) is closely related to the pathogenesis of among others Parkinson's disease and epilepsy. As such, lipophilic nipecotic acid analogs have been extensively studied as GAT1-addressing drugs and radioligands but suffer from limited brain uptake due to the zwitterionic properties of the nipecotic acid moiety. Bioisosteric replacement of the carboxylic acid group is a promising strategy to improve the brain uptake, though it requires knowledge on the binding of these isosteres to GAT1. To screen nipecotic acid isosteres for their affinity to GAT1 in a time- and cost-effective manner, this research aims to develop a molecular imprinted polymer (MIP) that mimics the natural binding site of GAT1 and can act as an alternative screening tool to the current radiometric and mass spectrometry cellular-based assays. To this end, a nipecotic acid MIP was created using the electropolymerization of ortho-phenylenediamine (oPD) by cyclic voltammetry (CV). The optimization of the generated receptor layer was achieved by varying the scan rate (50-250 mV/s) and number of CV cycles (5-12), yielding an optimized MIP with an average imprinting factor of 2.6, a linear range of 1-1000 nm, and a theoretical LOD of 0.05 nm, as analyzed by electrical impedance spectroscopy (EIS). Selectivity studies facilitated the investigation of major binding interactions between the MIP and the substrate, building an experimental model that compares characteristics of various analogs. Results from this model indicate that the substrate carboxylic acid group plays a more important role in binding than an amine group, after comparing the binding of cyclohexanecarboxylic acid (average IF of 1.7) and piperidine (average IF of 0.46). The research culminates in a discussion regarding the feasibility of the in vitro model, comparing the synthetic system against the biological performance of GAT1. Thus, evaluating if it is possible to generate a synthetic GAT1 mimic, and if so, provide directions for follow-up research.
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Polímeros Impresos Molecularmente , Ácidos Nipecóticos , Polímeros Impresos Molecularmente/química , Ácidos Nipecóticos/química , Ácidos Nipecóticos/metabolismo , Humanos , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Impresión MolecularRESUMEN
Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling.In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Here we demonstrate that betaine at low concentration modulates the GABA uptake by GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. This modulation occurs through the temporal inhibition of the transporter, wherein prolonged occupancy by betaine impedes the swift transition of the transporter to the inward conformation. Importantly, the modulatory effect of betaine on GAT1 is reversible, as the blocking of GAT1 disappears with increased extracellular GABA. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine has a dual role in GAT1: at mM concentration acts as a slow substrate, and at µM as a temporal blocker of GABA, when it is below its K0.5. Given this unique modulatory characteristic and lack of any harmful side effects, betaine emerges as a promising neuromodulator of the inhibitory pathways improving GABA homeostasis via GAT1, thereby conferring neuroprotection against excitotoxicity.
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Betaína , Proteínas Transportadoras de GABA en la Membrana Plasmática , Homeostasis , Ácido gamma-Aminobutírico , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Betaína/farmacología , Betaína/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Homeostasis/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Simulación de Dinámica Molecular , Humanos , Ratas , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/metabolismo , Células HEK293RESUMEN
Clinical surgery can lead to severe neuroinflammation and cognitive dysfunctions. It has been reported that astrocytes mediate memory formation and postoperative cognitive dysfunction (POCD), however, the thalamic mechanism of astrocytes in mediating POCD remains unknown. Here, we report that reactive astrocytes in zona incerta (ZI) mediate surgery-induced recognition memory impairment in male mice. Immunostaining results showed that astrocytes are activated with GABA transporter-3 (GAT-3) being down-expressed, and neurons were suppressed in the ZI. Besides, our work revealed that reactive astrocytes caused increased tonic current in ZI neurons. Up-regulating the expression of GAT-3 in astrocytes ameliorates surgery-induced recognition memory impairment. Together, our work demonstrates that the reactive astrocytes in the ZI play a crucial role in surgery-induced memory impairment, which provides a new target for the treatment of surgery-induced neural dysfunctions.
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Astrocitos , Proteínas Transportadoras de GABA en la Membrana Plasmática , Trastornos de la Memoria , Regulación hacia Arriba , Zona Incerta , Animales , Masculino , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Trastornos de la Memoria/metabolismo , Ratones , Regulación hacia Arriba/efectos de los fármacos , Astrocitos/metabolismo , Zona Incerta/metabolismo , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Complicaciones Cognitivas Postoperatorias/metabolismo , Complicaciones Cognitivas Postoperatorias/prevención & control , Reconocimiento en Psicología/efectos de los fármacos , Reconocimiento en Psicología/fisiologíaRESUMEN
1,2-Dichloroethane (1,2-DCE) is a powerfully toxic neurotoxin, which is a common environmental pollutant. Studies have indicated that 1,2-DCE long-term exposure can result in adverse effects. Nevertheless, the precise mechanism remains unknown. In this study, behavioral results revealed that 1,2-DCE long-term exposure could cause anxiety and learning and memory ability impairment in mice. The contents of γ-aminobutyric acid (GABA) and glutamine (Gln) in mice's prefrontal cortex decreased, whereas that of glutamate (Glu) increased. With the increase in dose, the activities of glutamate decarboxylase (GAD) decreased and those of GABA transaminase (GABA-T) increased. The protein and mRNA expressions of GABA transporter-3 (GAT-3), vesicular GABA transporter (VGAT), GABA A receptor α2 (GABAARα2), GABAARγ2, K-Cl cotransporter isoform 2 (KCC2), GABA B receptor 1 (GABABR1), GABABR2, protein kinase A (PKA), cAMP-response element binding protein (CREB), p-CREB, brain-derived neurotrophic factor (BDNF), c-fos, c-Jun and the protein of glutamate dehydrogenase (GDH) and PKA-C were decreased, while the expression levels of GABA transporter-1 (GAT-1) and Na-K-2Cl cotransporter isoform 1 (NKCC1) were increased. However, there was no significant change in the protein content of succinic semialdehyde dehydrogenase (SSADH). The expressions of adenylate cyclase (AC) and cyclic adenosine monophosphate (cAMP) contents were also reduced. In conclusion, the results of this study show that exposure to 1,2-DCE could lead to anxiety and cognitive impairment in mice, which may be related to the disturbance of GABA metabolism and its receptors along with the cAMP-PKA-CREB pathway.
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Ansiedad , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico , Dicloruros de Etileno , Transducción de Señal , Ácido gamma-Aminobutírico , Animales , Ratones , Ácido gamma-Aminobutírico/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dicloruros de Etileno/toxicidad , Masculino , Ansiedad/inducido químicamente , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/metabolismo , AMP Cíclico/metabolismo , Contaminantes Ambientales/toxicidad , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Glutamato Descarboxilasa/metabolismoRESUMEN
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The termination of GABA transmission is through the action of GABA transporters (GATs). mGAT4 (encoded by Slc6a11) is another GAT besides GAT1 (encoded by Slc6a1) that functions in GABA reuptake in CNS. Research on the function of mGAT4 is still in its infancy. We developed an mGat4 knockout mouse model (mGat4-/- mice) and performed a series of behavioral analyses for the first time to study the effect of mGat4 on biological processes in CNS. Our results indicated that homozygous mGat4-/- mice had less depression, anxiety-like behavior and more social activities than their wild-type littermate controls. However, they had weight loss and showed motor incoordination and imbalance. Meanwhile, mGat4-/- mice showed increased pain threshold and hypoalgesia behavior in nociceptive stimulus and learning and memory impairments. The expression of multiple components of the GABAergic system including GAD67, GABAA and KCC2 was altered. There is little or no compensatory change in mGat1. In a word, mGat4 may play a key role in normal motor coordination, sensation, emotion, learning and memory and could be the potential target of neurological disorders.
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Proteínas Transportadoras de GABA en la Membrana Plasmática , Ratones Noqueados , Animales , Masculino , Ratones , Ansiedad/genética , Ansiedad/metabolismo , Conducta Animal , Depresión/genética , Depresión/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Glutamato Descarboxilasa/metabolismo , Glutamato Descarboxilasa/genética , Cotransportadores de K Cl , Ratones Endogámicos C57BL , Simportadores/genética , Simportadores/metabolismoRESUMEN
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.
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Enfermedad de Alzheimer , Butirilcolinesterasa , Ratones , Animales , Butirilcolinesterasa/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Inhibidores de la Colinesterasa/metabolismo , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Diseño de Fármacos , Ácido Aspártico Endopeptidasas/metabolismo , Acetilcolinesterasa/metabolismoRESUMEN
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/).
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática , Estudios de Asociación Genética , Mutación Missense , Humanos , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , FenotipoRESUMEN
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.
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Lipopolisacáridos , Microglía , Microglía/metabolismo , Lipopolisacáridos/farmacología , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Bestrofinas/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
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.
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Epilepsia Tipo Ausencia , Etosuximida , Humanos , Ratones , Animales , Niño , Etosuximida/uso terapéutico , Haploinsuficiencia/genética , Ácidos Nipecóticos/uso terapéutico , Epilepsia Tipo Ausencia/tratamiento farmacológico , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismoRESUMEN
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.
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Cloruros , Ácido gamma-Aminobutírico , Ratas , Animales , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Microscopía por Crioelectrón , Cloruros/metabolismo , Cloruros/farmacología , Ácido gamma-Aminobutírico/metabolismo , Sitios de UniónRESUMEN
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.
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Ácido gamma-Aminobutírico , Humanos , Tiagabina , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Conformación MolecularRESUMEN
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.
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Relojes Circadianos , Ritmo Circadiano , Animales , Ritmo Circadiano/genética , Relojes Circadianos/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Núcleo Supraquiasmático/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Mamíferos/metabolismoRESUMEN
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.
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Proteínas Transportadoras de GABA en la Membrana Plasmática , Proteínas del Tejido Nervioso , Sinteninas , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Sinteninas/genética , Sinteninas/metabolismo , Tirosina/metabolismo , Animales , Ratones , Línea CelularRESUMEN
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.
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Artritis Reumatoide , Ácido gamma-Aminobutírico , Humanos , Artritis Reumatoide/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Inflamación , Receptores de GABA/metabolismoRESUMEN
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.
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Epilepsias Mioclónicas , Proteínas Transportadoras de GABA en la Membrana Plasmática , Membrana Dobles de Lípidos , Humanos , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Epilepsias Mioclónicas/metabolismo , Epilepsias Mioclónicas/patologíaRESUMEN
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.
Asunto(s)
Anticonvulsivantes , Simulación de Dinámica Molecular , Humanos , Tiagabina , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Microscopía por Crioelectrón , Anticonvulsivantes/farmacologíaRESUMEN
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.
Asunto(s)
Receptores de GABA-A , Ácido gamma-Aminobutírico , Animales , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Ratones , Terminales Presinápticos/metabolismo , Receptores de GABA-A/metabolismo , Sinapsis/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
OBJECTIVE: Mutations in γ-aminobutyric acid (GABA) transporter 1 (GAT-1)-encoding SLC6A1 have been associated with myoclonic atonic epilepsy and other phenotypes. We determined the patho-mechanisms of the mutant GAT-1, in order to identify treatment targets. METHODS: We conducted whole-exome sequencing of patients with myoclonic atonic epilepsy (MAE) and characterized the seizure phenotypes and EEG patterns. We studied the protein stability and structural changes with homology modeling and machine learning tools. We characterized the function and trafficking of the mutant GAT-1 with 3H radioactive GABA uptake assay and confocal microscopy. We utilized different models including a knockin mouse and human astrocytes derived from induced pluripotent stem cells (iPSCs). We focused on astrocytes because of their direct impact of astrocytic GAT-1 in seizures. RESULTS: We identified four novel SLC6A1 variants associated with MAE and 2 to 4 Hz spike-wave discharges as a common EEG feature. Machine learning tools predicted that the variant proteins are destabilized. The variant protein had reduced expression and reduced GABA uptake due to endoplasmic reticular retention. The consistent observation was made in cortical and thalamic astrocytes from variant-knockin mice and human iPSC-derived astrocytes. The Slc6a+/A288V mouse, representative of MAE, had increased 5-7 Hz spike-wave discharges and absence seizures. INTERPRETATION: SLC6A1 variants in various locations of the protein peptides can cause MAE with similar seizure phenotypes and EEG features. Reduced GABA uptake is due to decreased functional GAT-1, which, in thalamic astrocytes, could result in increased extracellular GABA accumulation and enhanced tonic inhibition, leading to seizures and abnormal EEGs.