Γ-aminobutyric Acid Transporter Research Articles

MdSINA2-MdNAC104 Module Regulates Apple Alkaline Resistance by Affecting γ-Aminobutyric Acid Synthesis and Transport.

Soil alkalization is an adverse factor limiting plant growth and yield. As a signaling molecule and secondary metabolite, γ-aminobutyric acid (GABA) responds rapidly to alkaline stress and enhances the alkaline resistance of plants. However, the molecular mechanisms by which the GABA pathway adapts to alkaline stress remain unclear. In this study, a transcription factor, MdNAC104 is identified, from the transcriptome of the alkaline-stressed roots of apple, which effectively reduces GABA levels and negatively regulates alkaline resistance. Nevertheless, applying exogenous GABA compensates the negative regulatory mechanism of overexpressed MdNAC104 on alkaline resistance. Further research confirms that MdNAC104 repressed the GABA biosynthetic gene MdGAD1/3 and the GABA transporter gene MdALMT13 by binding to their promoters. Here, MdGAD1/3 actively regulates alkaline resistance by increasing GABA synthesis, while MdALMT13 promotes GABA accumulation and efflux in roots, resulting in an improved resistance to alkaline stress. This subsequent assays reveal that MdSINA2 interacts with MdNAC104 and positively regulates root GABA content and alkaline resistance by ubiquitinating and degrading MdNAC104 via the 26S proteasome pathway. Thus, the study reveals the regulation of alkaline resistance and GABA homeostasis via the MdSINA2-MdNAC104-MdGAD1/3/MdALMT13 module in apple. These findings provide novel insight into the molecular mechanisms of alkaline resistance in plants.

Evaluation of quality consistency between dispensing granules and traditional decoction of Bombyx batryticatus based on peptidomics and in silico simulations.

The application of traditional Chinese medicine dispensing granules is becoming increasingly prevalent. However, the consistency of dispensing granules with traditional decoction remains controversial. In this study, the consistency of peptide composition and pharmacodynamics between dispensing granules and traditional decoction of Bombyx batryticatus (BB) were assessed. A peptidomics method based on LC-tandem mass spectrometry technology was used to evaluate peptide composition similarity between BB traditional decoction and dispensing granules. The results revealed notable differences in peptide sequences between the two dosage forms, with only 8.55% of peptides shared between them. To evaluate the potential pharmacodynamic effects of the two dosage forms on epilepsy, virtual screening was used to identify potential active peptides, including blood-brain barrier permeability, toxicity prediction, and molecular docking. BB traditional decoction demonstrated a higher number and greater abundance of potential active peptides than BB dispensing granules, suggesting that BB traditional decoction may have a more favorable effect in treating epilepsy compared with BB dispensing granules. Moreover, molecular docking and molecular dynamics simulation studies confirmed the mechanism of action of active peptides to γ-aminobutyric acid transporter 1 (GAT-1). This study provides a scientific basis for the evaluation of quality consistency between BB traditional decoction and dispensing granules.

Pharmacological evaluation of E2730, a novel selective uncompetitive GAT1 inhibitor, on epileptiform activities in resected brain tissues from human focal cortical dysplasia ex vivo

Focal cortical dysplasia (FCD) is an important etiology of focal epilepsy in children and adults. However, only a few preclinical models sufficiently reproduce the characteristic histopathologic features of FCD. To improve the success rate of clinical trials for antiseizure medications (ASMs) in patients with FCD, more human-relevant preclinical models are needed, and epileptic foci resected from patients are a powerful tool for this purpose. Here, we conducted ex vivo studies using epileptic foci resected from patients with FCD type II to evaluate the pharmacologic effects of the ASM candidate E2730, a selective uncompetitive inhibitor of γ-aminobutyric acid transporter 1. We used the same ex vivo assay system to assess carbamazepine (CBZ), an ASM often prescribed for focal epilepsy, as a reference. At the higher dose tested (200 µM), both E2730 and CBZ suppressed spontaneous epileptiform activities almost completely. At the lower dose (100 µM), CBZ reduced the area of brain tissue showing epileptiform activity, whereas E2730 significantly decreased the number of epileptiforms. These findings suggest that E2730—both as a single agent and in combination with CBZ—merits evaluation in clinical trials involving patients with FCD.

γ-Aminobutyric acid transporter and GABAA receptor mechanisms in Slc6a1+/A288V and Slc6a1+/S295L mice associated with developmental and epileptic encephalopathies.

We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 (SLC6A1) in vitro and concluded that a partial or complete loss of γ-aminobutyric acid uptake due to impaired protein trafficking is the primary aetiology. Impairment of γ-aminobutyric acid transporter 1 function could cause compensatory changes in the expression of γ-aminobutyric acid receptors, which, in turn, modify disease pathophysiology and phenotype. Here we used different approaches including radioactive 3H γ-aminobutyric acid uptake in cells and synaptosomes, immunohistochemistry and confocal microscopy as well as brain slice surface protein biotinylation to characterize Slc6a1+/A288V and Slc6a1+/S295L mice, representative of a partial or a complete loss of function of SLC6A1 mutations, respectively. We employed the γ-aminobutyric acid transporter 1-specific inhibitor [3H]tiagabine binding and GABAA receptor subunit-specific radioligand binding to profile the γ-aminobutyric acid transporter 1 and GABAA receptor expression in major brain regions such as cortex, cerebellum, hippocampus and thalamus. We also determined the total and surface expression of γ-aminobutyric acid transporter 1, γ-aminobutyric acid transporter 3 and expression of GABAA receptor in the major brain regions in the knockin mice. We found that γ-aminobutyric acid transporter 1 protein was markedly reduced in cortex, hippocampus, thalamus and cerebellum in both mutant mouse lines. Consistent with the findings of reduced γ-aminobutyric acid uptake for both γ-aminobutyric acid transporter 1(A288V) and γ-aminobutyric acid transporter 1(S295L), both the total and the γ-aminobutyric acid transporter 1-mediated 3H γ-aminobutyric acid reuptake was reduced. We found that γ-aminobutyric acid transporter 3 is only abundantly expressed in the thalamus and there was no compensatory increase of γ-aminobutyric acid transporter 3 in either of the mutant mouse lines. γ-Aminobutyric acid transporter 1 was reduced in both somatic regions and nonsomatic regions in both mouse models, in which a ring-like structure was identified only in the Slc6a1+/A288V mouse, suggesting more γ-aminobutyric acid transporter 1 retention inside endoplasmic reticulum in the Slc6a1+/A288V mouse. The [3H]tiagabine binding was similar in both mouse models despite the difference in γ-aminobutyric acid uptake function and γ-aminobutyric acid transporter 1 protein expression for both mutations. There were no differences in GABAA receptor subtype expression, except for a small increase in the expression of α5 subunits of GABAA receptor in the hippocampus of Slc6a1S295L homozygous mice, suggesting a potential interaction between the expression of this GABAA receptor subtype and the mutant γ-aminobutyric acid transporter 1. The study provides the first comprehensive characterization of the SLC6A1 mutations in vivo in two representative mouse models. Because both γ-aminobutyric acid transporter 1 and GABAA receptors are targets for anti-seizure medications, the findings from this study can help guide tailored treatment options based on the expression and function of γ-aminobutyric acid transporter 1 and GABAA receptor in SLC6A1 mutation-mediated neurodevelopmental and epileptic encephalopathies.

Transmembrane protein modulates seizure in epilepsy: evidence from temporal lobe epilepsy patients and mouse models.

Transmembrane protein (TMEM230) is located in secretory/recycling vesicles, including synaptic vesicles in neurons. However, the functional relationship between TMEM230 and epilepsy is still a mystery. The aims of this study were to investigate the expression of TMEM230 in patients with temporal lobe epilepsy (TLE) and two different mice models of chronic epilepsy, and to determine the probable roles of TMEM230 in epilepsy. Our results showed that TMEM230 expression was increased in the temporal neocortex of epileptic patients and the hippocampus and cortex of epileptic mice compared with that in the control tissues. Moreover, TMEM230 was mainly expressed in the neurons in both humans and mice epileptic brain. TMEM230 co-localized with glutamate vesicular transporter 1 (VGLUT-1), but not with vesicular γ-aminobutyric acid (GABA) transporter (VGAT). Mechanistically, coimmunoprecipitation confirmed that TMEM230 interacted with VGLUT-1, but not with VGAT in the hippocampus of epileptic mice. Lentivirus mediated overexpression of TMEM230 increased mice susceptibility to epilepsy and behavioural phenotypes of epileptic seizures during the kainite (KA)-induced chronic phase of epileptic seizures and the pentylenetetrazole (PTZ) kindling process, whereas lentivirus-mediated TMEM230 downregulation had the opposite effect. These results shed light on the functions of TMEM230 in neurons, suggesting that TMEM230 may play a critical role in the regulation of epileptic activity via influencing excitatory neurotransmission.

The Co-Expression Pattern of Calcium-Binding Proteins with γ-Aminobutyric Acid and Glutamate Transporters in the Amygdala of the Guinea Pig: Evidence for Glutamatergic Subpopulations.

The amygdala has large populations of neurons utilizing specific calcium-binding proteins such as parvalbumin (PV), calbindin (CB), or calretinin (CR). They are considered specialized subsets of γ-aminobutyric acid (GABA) interneurons; however, many of these cells are devoid of GABA or glutamate decarboxylase. The neurotransmitters used by GABA-immunonegative cells are still unknown, but it is suggested that a part may use glutamate. Thus, this study investigates in the amygdala of the guinea pig relationships between PV, CB, or CR-containing cells and GABA transporter (VGAT) or glutamate transporter type 2 (VGLUT2), markers of GABAergic and glutamatergic neurons, respectively. The results show that although most neurons using PV, CB, and CR co-expressed VGAT, each of these populations also had a fraction of VGLUT2 co-expressing cells. For almost all neurons using PV (~90%) co-expressed VGAT, while ~1.5% of them had VGLUT2. The proportion of neurons using CB and VGAT was smaller than that for PV (~80%), while the percentage of cells with VGLUT2 was larger (~4.5%). Finally, only half of the neurons using CR (~53%) co-expressed VGAT, while ~3.5% of them had VGLUT2. In conclusion, the populations of neurons co-expressing PV, CB, and CR are in the amygdala, primarily GABAergic. However, at least a fraction of neurons in each of them co-express VGLUT2, suggesting that these cells may use glutamate. Moreover, the number of PV-, CB-, and CR-containing neurons that may use glutamate is probably larger as they can utilize VGLUT1 or VGLUT3, which are also present in the amygdala.

E2730, an uncompetitive γ-aminobutyric acid transporter-1 inhibitor, suppresses epileptic seizures in a rat model of chronic mesial temporal lobe epilepsy.

More than one third of mesial temporal lobe epilepsy (MTLE) patients are resistant to current antiseizure medications (ASMs), and half experience mild-to-moderate adverse effects of ASMs. There is therefore a strong need to develop and test novel ASMs. The objective of this work is to evaluate the pharmacokinetics and neurological toxicity of E2730, a novel uncompetitive inhibitor of γ-aminobutyric acid transporter-1, and to test its seizure suppression effects in a rat model of chronic MTLE. We first examined plasma levels and adverse neurological effects of E2730 in healthy Wistar rats. Adult male rats were implanted with osmotic pumps delivering either 10, 20, or 100 mg/kg/day of E2730 subcutaneously for 1 week. Blood sampling and behavioral assessments were performed at several timepoints. We next examined whether E2730 suppressed seizures in rats with chronic MTLE. These rats were exposed to kainic acid-induced status epilepticus, and 9 weeks later, when chronic epilepsy was established, were assigned to receive one of the three doses of E2730 or vehicle for 1 week in a randomized crossover design. Continuous video-electroencephalographic monitoring was acquired during the treatment period to evaluate epileptic seizures. Plasma levels following continuous infusion of E2730 showed a clear dose-related increase in concentration. The drug was well tolerated at all doses, and any sedation or neuromotor impairment was mild and transient, resolving within 48 h of treatment initiation. Remarkably, E2730 treatment in chronically epileptic rats led to seizure suppression in a dose-dependent manner, with 65% of rats becoming seizure-free at the highest dose tested. Mean seizure class did not differ between the treatment groups. This study shows that continuous subcutaneous infusion of E2730 over 7 days results in a marked, dose-dependent suppression of spontaneous recurrent seizures, with minimal adverse neurological effects, in a rat model of chronic MTLE. E2730 shows strong promise as an effective new ASM to be translated into clinical trials.

Patient-derived SLC6A1 variant S295L results in an epileptic phenotype similar to haploinsufficient mice.

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.

Astrocyte activities in the external globus pallidus regulate action-selection strategies in reward-seeking behaviors.

An imbalance in goal-directed and habitual behavioral control is a hallmark of decision-making-related disorders, including addiction. Although external globus pallidus (GPe) is critical for action selection, which harbors enriched astrocytes, the role of GPe astrocytes involved in action-selection strategies remained unknown. Using in vivo calcium signaling with fiber photometry, we found substantially attenuated GPe astrocytic activity during habitual learning compared to goal-directed learning. The support vector machine analysis predicted the behavioral outcomes. Chemogenetic activation of the astrocytes or inhibition of GPe pan-neuronal activities facilitates the transition from habit to goal-directed reward-seeking behavior. Next, we found increased astrocyte-specific GABA (γ-aminobutyric acid) transporter type 3 (GAT3) messenger RNA expression during habit learning. Notably, the pharmacological inhibition of GAT3 occluded astrocyte activation-induced transition from habitual to goal-directed behavior. On the other hand, attentional stimuli shifted the habit to goal-directed behaviors. Our findings suggest that the GPe astrocytes regulate the action selection strategy and behavioral flexibility.

Discovery of E2730, a novel selective uncompetitive GAT1 inhibitor, as a candidate for anti-seizure medication.

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. 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. 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. 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.

The Role of Pharmacogenetics in Personalizing the Antidepressant and Anxiolytic Therapy

Pharmacotherapy for neuropsychiatric disorders, such as anxiety and depression, has been characterized by significant inter-individual variability in drug response and the development of side effects. Pharmacogenetics, as a key part of personalized medicine, aims to optimize therapy according to a patient's individual genetic signature by targeting genetic variations involved in pharmacokinetic or pharmacodynamic processes. Pharmacokinetic variability refers to variations in a drug's absorption, distribution, metabolism, and elimination, whereas pharmacodynamic variability results from variable interactions of an active drug with its target molecules. Pharmacogenetic research on depression and anxiety has focused on genetic polymorphisms affecting metabolizing cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes, P-glycoprotein ATP-binding cassette (ABC) transporters, and monoamine and γ-aminobutyric acid (GABA) metabolic enzymes, transporters, and receptors. Recent pharmacogenetic studies have revealed that more efficient and safer treatments with antidepressants and anxiolytics could be achieved through genotype-guided decisions. However, because pharmacogenetics cannot explain all observed heritable variations in drug response, an emerging field of pharmacoepigenetics investigates how epigenetic mechanisms, which modify gene expression without altering the genetic code, might influence individual responses to drugs. By understanding the epi(genetic) variability of a patient's response to pharmacotherapy, clinicians could select more effective drugs while minimizing the likelihood of adverse reactions and therefore improve the quality of treatment.

Structural basis of GABA reuptake inhibition.

γ-Aminobutyric acid (GABA) transporter 1 (GAT1), a member of the neurotransmitter sodium symporter (NSS) family, regulates neuronal excitation by clearing the synaptic cleft of the main inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating GABA levels in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy. Here we present the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor, the anticonvulsant tiagabine. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine. Tiagabine's pharmacodynamic profile, confirmed by our experimental data, suggests initial binding to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step induced-fit mechanism of inhibition. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators.

Drosophila melanogaster as a model for unraveling unique molecular features of epilepsy elicited by human GABA transporter 1 variants.

Mutations in the human γ-aminobutyric acid (GABA) transporter 1 (hGAT-1) can instigate myoclonic-atonic and other generalized epilepsies in the afflicted individuals. We systematically examined fifteen hGAT-1 disease variants, all of which dramatically reduced or completely abolished GABA uptake activity. Many of these loss-of-function variants were absent from their regular site of action at the cell surface, due to protein misfolding and/or impaired trafficking machinery (as verified by confocal microscopy and de-glycosylation experiments). A modest fraction of the mutants displayed correct targeting to the plasma membrane, but nonetheless rendered the mutated proteins devoid of GABA transport, possibly due to structural alterations in the GABA binding site/translocation pathway. We here focused on a folding-deficient A288V variant. In flies, A288V reiterated its impeded expression pattern, closely mimicking the ER-retention demonstrated in transfected HEK293 cells. Functionally, A288V presented a temperature-sensitive seizure phenotype in fruit flies. We employed diverse small molecules to restore the expression and activity of folding-deficient hGAT-1 epilepsy variants, in vitro (in HEK293 cells) and in vivo (in flies). We identified three compounds (chemical and pharmacological chaperones) conferring moderate rescue capacity for several variants. Our data grant crucial new insights into: (i) the molecular basis of epilepsy in patients harboring hGAT-1 mutations, and (ii) a proof-of-principle that protein folding deficits in disease-associated hGAT-1 variants can be corrected using the pharmacochaperoning approach. Such innovative pharmaco-therapeutic prospects inspire the rational design of novel drugs for alleviating the clinical symptoms triggered by the numerous emerging pathogenic mutations in hGAT-1.

Reduced inhibitory and excitatory input onto parvalbumin interneurons mediated by perineuronal net might contribute to cognitive impairments in a mouse model of sepsis-associated encephalopathy

Sepsis-associated encephalopathy (SAE) is commonly defined as diffuse brain dysfunction and can manifest as delirium to coma. Accumulating evidence has suggested that perineuronal net (PNN) plays an important role in the modulation of the synaptic plasticity of central nervous system. We here investigated the role of PNN in SAE induced by lipopolysaccharide (LPS) injection. Behavioral tests were performed by open field, Y-maze, and fear conditioning tests at the indicated time points. The densities of vesicular γ-aminobutyric acid transporter, vesicular glutamate transporter 1, PNN, and parvalbumin (PV) in the hippocampus were evaluated by immunofluorescence. Matrix metalloproteinases-9 (MMP-9) expression and its activity were detected by Western blot and gel zymography, respectively. Local field potential was recorded by in vivo electrophysiology. LPS-treated mice displayed significant cognitive impairments, coincided with activated MMP-9, decreased PNN and PV densities, reduced inhibitory and excitatory input onto PV interneurons enwrapped by PNN, and decreased gamma oscillations in hippocampal CA1. Notably, MMP-9 inhibitor SB-3CT treatment rescued most of these abnormalities. Taken together, our study demonstrates that active MMP-9 mediated PNN remodeling, leading to reduced inhibitory and excitatory input onto PV interneurons and abnormal gamma oscillations in hippocampal CA1, which consequently contributed to cognitive impairments after LPS injection.

Effects of estradiol on dopaminergic synapse formation in the mouse olfactory bulb.

Olfactory glomeruli are the sites of initial synaptic integration in olfactory information processing. They are surrounded by juxtaglomerular (JG) cells, which include periglomerular, superficial short axon, and external tufted cells. A subpopulation of JG cells expresses the dopamine synthetic enzymes, tyrosine hydroxylase (TH), and aromatic l-amino acid decarboxylase (AADC). TH cells corelease γ-aminobutyric acid (GABA) and their processes extend to multiple glomeruli forming intra- and interglomerular circuits. It is well established that 17β-estradiol (E2) exerts wide ranging effects in the central nervous system. However, participation of E2 in the modulation of neurotransmission and synaptic plasticity of TH cells in olfactory glomeruli is unknown. To address this, we subcutaneously implanted a 60-day release pellet of E2 or placebo into intact male mice and compared glomerular TH, AADC, and vesicular γ-aminobutyric acid transporter (VGAT) immunoreactivity between them. High-voltage electron microscopy (HVEM) and ultra-HVEM using immunogold revealed significantly increased immunoreactive intensity at the cellular level for TH and AADC after E2 treatment and for VGAT in TH cells. These results indicate that E2 may affect the interplay between dopaminergic and GABAergic systems. Moreover, random-section electron microscopy analysis showed a significant increase in the number of symmetrical synapses from TH cell to mitral/tufted cell dendrites after E2 treatment. This result was supported by quantitative immunofluorescence staining with synapse markers. Together, these data indicate that E2 may regulate inhibition between TH cells and olfactory bulb neurons within the glomerulus via interaction between dopaminergic and GABAergic systems, thereby contributing to neuromodulation of odor information processing.

Rhizobia Contribute to Salinity Tolerance in Common Beans (Phaseolus vulgaris L.).

Rhizobia are soil bacteria that induce nodule formation on leguminous plants. In the nodules, they reduce dinitrogen to ammonium that can be utilized by plants. Besides nitrogen fixation, rhizobia have other symbiotic functions in plants including phosphorus and iron mobilization and protection of the plants against various abiotic stresses including salinity. Worldwide, about 20% of cultivable and 33% of irrigation land is saline, and it is estimated that around 50% of the arable land will be saline by 2050. Salinity inhibits plant growth and development, results in senescence, and ultimately plant death. The purpose of this study was to investigate how rhizobia, isolated from Kenyan soils, relieve common beans from salinity stress. The yield loss of common bean plants, which were either not inoculated or inoculated with the commercial R. tropici rhizobia CIAT899 was reduced by 73% when the plants were exposed to 300 mM NaCl, while only 60% yield loss was observed after inoculation with a novel indigenous isolate from Kenyan soil, named S3. Expression profiles showed that genes involved in the transport of mineral ions (such as K+, Ca2+, Fe3+, PO43-, and NO3-) to the host plant, and for the synthesis and transport of osmotolerance molecules (soluble carbohydrates, amino acids, and nucleotides) are highly expressed in S3 bacteroids during salt stress than in the controls. Furthermore, genes for the synthesis and transport of glutathione and γ-aminobutyric acid were upregulated in salt-stressed and S3-inocculated common bean plants. We conclude that microbial osmolytes, mineral ions, and antioxidant molecules from rhizobia enhance salt tolerance in common beans.

Analysis of Different Binding Modes for Tiagabine within the GAT-1 Transporter.

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.

GABA transport goes structural

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.

Astrocytic GABA transporter 1 deficit in novel SLC6A1 variants mediated epilepsy: Connected from protein destabilization to seizures in mice and humans

ObjectiveMutations 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. MethodsWe 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. ResultsWe 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. InterpretationSLC6A1 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.

Structural basis of GABA reuptake inhibition.

γ-Aminobutyric acid (GABA) transporter 1 (GAT1)1 regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy2. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine3, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition4. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins.