GABA Transporter Research Articles

In silico characterization, structural modeling, and molecular docking of GabP in citrus and its potential role in GABA uptake

GABA shunt is an important metabolic hub that plays a key role in plant growth, development, and response. Although a functional connection between GABA shunt and the TCA cycle via a mitochondrial GABA permease (gabP) gene was reported previously from model plants, this connection in non-model plants, such as citrus, is poorly studied. Herein, we used bioinformatics and multi-omics for in-silico characterization, structural modeling, and ligand-receptor binding profile of GABA transport carrier (CsgabP) from the non-model plant, Valencia sweet orange (Citrus sinensis) and to explore their potential role(s) in GABA uptake and citrus response to Huanglongbing. Genome-wide analysis showed that the citrus genome encodes for two putative CsgabP proteins (CsgabP-1 [521 aa] and CsgabP-2 [419 aa]) that were relatively highly homologous to each other and homologous to amino acid permease BAT1 proteins from other plant species. Primary structures of both CsgabPs showed high similarity and conserved sequences between them. CsgabPs are highly hydrophobic integral transmembrane transporter proteins with internal hydrophilic N- and C-terminal ends and depend on 9–12 transmembrane segments. The crystallographic three-dimensional (3D) structures of both CsgabPs were predicted and confirmed using AlphaFold as a monomer with 100% coverage, but not experimentally determined crystallographic structures. Targeted metabolomics showed that CsgabP was upregulated upon GABA supplementation in healthy and ‘Candidatus Liberibacter asiaticus’-infected citrus plants. Likewise, CsgabP was involved in citrus response(s) to different biotic stress including infection with ‘Ca. L. asiaticus’ or infestation with its vector, Diaphorina citri. These findings probably clarify the molecular and functional connection between the GABA shunt and the TCA cycle in non-model plants such as citrus.

Sideroflexins enable mitochondrial transport of polar neutral amino acids.

Mitochondria contribute to compartmentalized metabolism in eukaryotic cells, supporting key enzymatic reactions for cell function and energy homeostasis. However, this compartmentalization necessitates regulated metabolite transport across mitochondrial membranes. Although many transport proteins have been identified, several mitochondrial amino acid transporters remain largely uncharacterized. Using CRISPR-Cas9-mediated candidate transporter knockouts coupled with assessment of metabolite transport via a mitochondrial swelling assay, we identify SFXN1, previously characterized for its role in mitochondrial serine transport, as a protein that mediates mitochondrial transport of a range of other polar neutral amino acids including proline, glycine, threonine, taurine, hypotaurine, β-alanine, and γ-aminobutyric acid (GABA). Furthermore, the SFXN1 paralogues SFXN2 and SFXN3 partially complement loss of SFXN1 to enable glycine transport, while SFXN2 and SFXN5 partially complement loss of SFXN1 to enable GABA transport. Altogether, these data suggest that sideroflexins facilitate the transport of polar neutral amino acids across the inner mitochondrial membrane.

Rescue of Epilepsy‐Associated Mutations of the Highly Conserved Glycine Residue 443 in the Human GABA Transporter 1

ABSTRACTThe human γ‐aminobutyric acid (GABA) transporter 1 (hGAT‐1) plays a pivotal role in synaptic neurotransmission by facilitating the clearance of GABA from the synaptic cleft. Pathogenic mutations in the SLC6A1 gene encoding hGAT‐1 have been implicated in a spectrum of neurodevelopmental disorders, including epilepsy, autism spectrum disorder, intellectual disability, and developmental delay. Here, we elucidate the molecular and functional consequences of disease‐associated mutations affecting the highly conserved glycine residue at position 443 (G443) in hGAT‐1. Through a combination of in vitro biochemical analyses, ion flux assays, and pharmacological profiling in HEK293 cells, alongside in vivo studies in Drosophila melanogaster, we demonstrate that substitutions of G443 to aspartate (G443D) or valine (G443V) result in complete abolishment of GABA transport. This severe impairment stems from distinct disruptions in protein folding and trafficking. In particular, G443V is fully retained in the endoplasmic reticulum (ER), as substantiated by de‐glycosylation assays indicating exclusively core‐glycosylated protein bands and confocal co‐localization with the ER chaperone calnexin. The G443D variant, on the other hand, exhibits partial trafficking to the plasma membrane, confirmed by the presence of maturely glycosylated bands, albeit at significantly reduced expression levels relative to the wild type transporter. Treatment with glycerol and 4‐phenylbutyrate (4‐PBA) successfully restored both surface expression and GABA uptake activity of the G443 mutants. Our findings highlight the potential of small‐molecule chaperones as interventions for ameliorating protein misfolding and functional deficits in hGAT‐1‐associated pathologies.

VGluT3 BNST neurons transmit GABA and restrict sucrose consumption

VGluT3 BNST neurons transmit GABA and restrict sucrose consumption

Patch-Clamp- and 15N Isotope Tracer-Based Techniques Reveal the Transport Characteristics of SlGAT1 and the Metabolic Flow of Exogenous GABA Under Salt Stress.

γ-Aminobutyric acid (GABA) plays an important role in plant growth, development, and stress resistance. Exogenous GABA works by increasing the content of endogenous GABA; therefore, GABA transport from the apoplast to the cytosol is critical. In this study, two GABA transporter genes, SlGAT1 and SlGAT2 were identified in tomato. It was verified that the transporter efficiency of SlGAT1 is greater than that of SlGAT2 via a two-electrode voltage clamp (TEVC) and quantitative real-time PCR (qRT-PCR). In combination with its subcellular and tissue expression, SlGAT1 is localised on the plasma membrane and is widely distributed in plants in response to salt stress. In addition, 15N isotope tracing confirmed that exogenous GABA can be absorbed by plant roots and transported to stems and leaves. After transmembrane transport to the cytosol, GABA is converted into glutamate, which is then catabolized to arginine and three branched-chain amino acids (BCAAs), valine, leucine, and isoleucine through four metabolic pathways. Among them, the metabolic processes of glutamate-aspartate-threonine-glycine-serine-BCAAs are crucial. Our findings provide the first evidence that SlGAT1 is a high affinity GABA transporter located on the tomato plasma membrane, that regulates plant cell metabolic pathways, promotes plant growth and enhances plant salt tolerance through GABA transport.

Decoding the role of novel long noncoding RNAs lnc-SLC6A12-1:3 and lnc-SLC6A12-7:5 in regulating the expression of GAD1 and SLC6A12 in cholangiocarcinoma.

Decoding the role of novel long noncoding RNAs lnc-SLC6A12-1:3 and lnc-SLC6A12-7:5 in regulating the expression of GAD1 and SLC6A12 in cholangiocarcinoma.

Evidence for low affinity of GABA at the vesicular monoamine transporter VMAT2 - Implications for transmitter co-release from dopamine neurons.

Midbrain dopamine (DA) neurons comprise a heterogeneous population of cells. For instance, some DA neurons express the vesicular glutamate transporter VGLUT2 allowing these cells to co-release DA and glutamate. Additionally, GABA may be co-released from DA neurons. However, most cells do not express the canonical machinery to synthesize GABA or the vesicular GABA transporter VGAT. Instead, GABA seems to be taken up into DA neurons by a plasmalemmal GABA transporter (GAT1) and stored in synaptic vesicles via the vesicular monoamine transporter VMAT2. Yet, it remains unclear whether GABA indeed interacts with VMAT2. Here, we used radiotracer flux measurements in VMAT2 expressing HEK-293cells and synaptic vesicles from male and female mice to determine whether GABA qualifies as substrate at VMAT2. We found that GABA reduced uptake of VMAT2 substrates in mouse synaptic vesicle preparations from striatum and cerebellum at millimolar concentrations but had no effect in VMAT2-expressing HEK-293cells. Interestingly, while the closely related amino acid glycine did not affect substrate uptake at VMAT2 in mouse synaptic vesicles, the amino sulfonic acid taurine reduced uptake similar to GABA. Lastly, we discovered that the majority of mouse and human midbrain DA neurons in the substantia nigra of either sex expressed VMAT2 and GAT1 suggesting that most of them could be capable of co-releasing DA and GABA. Together, our findings suggest that GABA is a low-affinity substrate at VMAT2 with potential implications for basal ganglia physiology and disease.

Characterizing the phenotype of pre-Bötzinger Complex neurons in rats.

Characterizing the phenotype of pre-Bötzinger Complex neurons in rats.

Dopamine D3 receptor in the nucleus accumbens modulates opioid taking and seeking in mice.

Dopamine D3 receptor in the nucleus accumbens modulates opioid taking and seeking in mice.

Regulation of perisomatic synapses from cholecystokinin basket interneurons through NrCAM and Ankyrin B.

Regulation of perisomatic synapses from cholecystokinin basket interneurons through NrCAM and Ankyrin B.

Voluntary wheel running exercise improves sleep disorder, circadian rhythm disturbance, and neuropathology in an animal model of Alzheimer's disease

INTRODUCTIONThe sleep–wake cycle and circadian rhythm disturbances are common in Alzheimer's disease (AD). However, it is not known if exercise has any benefit for the sleep disorders in AD.METHODSWe conducted a 2‐month voluntary wheel running (VWR) exercise (Ex) in an animal model of AD (APPSWE/PS1dE9 mice). We assessed behavioral circadian rhythm, sleep structure, circadian clock genes, cognitive function, and neurodegeneration in the suprachiasmatic nucleus (SCN), the hippocampus, and the cortex.RESULTSAfter VWR exercise in the AD mice, the rapid eye movement sleep was increased by 89%. The levels of circadian clock genes were significantly changed (brain and muscle arnt‐like protein 1 [BMAL1] and retinoic acid receptor‐related orphan receptorsα [RORα] reduced by 45.7% and 36.4%, reverse erythroblastosis virusα (REV‐ERBα) increased by 119%) in the SCN by immunofluorescence staining, with the mRNA levels were markedly altered (Bmal1 and Rorα decreased by 57% and 68%, Rev‐erbα elevated by 79%) in the hypothalamus at Zeitgeber Time 1; phospho‐tau 231 (p‐tau231) was reduced by 35%, whereas vesicular GABA transporter (VGAT) was elevated by 38.7% in the SCN. In addition, ionized calcium binding adapter molecude 1 (Iba1), glial fibrillary acidic protein (GFAP), amyloid β (Aβ), and p‐tau231 were significantly reduced in the hippocampus and cortex.DISCUSSIONOur results demonstrate that VWR exercise improves sleep disorders, cognitive deficits, and neuropathology in AD mice.HighlightsVoluntary wheel running (VWR) exercise improves the behavioral circadian rhythm disorder and sleep structure disturbance in Alzheimer's disease (AD) mice.After VWR exercise, there is a significant change in the expression levels of circadian clock genes, and a remarkable reduction of tau phosphorylation and axonal damage in the γ‐aminobutyric acid (GABA)ergic neurons in the suprachiasmatic nucleus (SCN).The levels of beta‐site amyloid precurson protein cleaving enzyme 1 (BACE1) and glycogen synthase kinase‐3β (GSK3β) are reduced in the hypothalamus after VWR exercise in AD mice.Furthermore, VWR exercise attenuates cognitive deficits, neuroinflammation, amyloid beta (Aβ), and phospho‐tau protein accumulation in the hippocampus and cortex.

Uncovering the role of Gpr45 in obesity regulation

Uncovering the role of Gpr45 in obesity regulation

Nanoemulsions of Cannabidiol, Δ9-Tetrahydrocannabinol, and Their Combination Similarly Exerted Anticonvulsant and Antioxidant Effects in Mice Treated with Pentyelenetetrazole

Background/Objectives: The main biologically active molecules of Cannabis sativa L. are cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC). Both exert anticonvulsant effects when evaluated as single drugs, but their possible interaction as components of C. sativa extracts has been scarcely studied. For this reason, we evaluated CBD and THC, combined or not, in two seizure models in mice, using an improved vehicle formula. Methods: Firstly, acute seizures were induced by intraperitoneal (i.p.) pentylenetetrazole (PTZ, 80 mg/kg), and mice received CBD or THC at 1, 3, 6, and 10 mg/kg, or a CBD/THC 1:1 combination at 1.5, 3, and 6 mg/kg, per os (p.o.), one hour before PTZ administration. Secondly, mice received p.o. CBD (10 mg/kg), CBD/THC (1.5, 3, and 6 mg/kg), valproic acid (50 mg/kg), or vehicle (nanoemulsions without CBD or THC), one hour before PTZ (30 mg/kg, i.p.) every other day for 21 days. Behavioral, biochemical, and immunohistochemical analyses were performed to assess the response to PTZ, oxidative stress, and astroglial activation. Results: In the acute model, CBD and THC at 3–10 mg/kg, and their combinations, significantly increased latency to generalized seizures and death, and improved survival rates. In the chronic model, similarly to valproic acid, CBD 10 mg/kg and CBD/THC at 1.5 and 3 mg/kg delayed kindling acquisition, while CBD/THC 6 mg/kg had no effect. CBD and CBD/THC treatments reduced oxidative and nitrosative stress and attenuated astrogliosis, as indicated by decreased glial fibrillary acidic protein and GABA transporter 1 expression and increased inwardly rectifying potassium channel 4.1 expression in hippocampal regions. However, no cannabinoid treatment prevented the impairment in novel object recognition and Y maze tests. Conclusions: These findings support the potential role of cannabinoids in counteracting seizures, possibly by reducing oxidative stress and astrogliosis. The study also highlights the importance of nanoemulsions as a delivery vehicle to enhance cannabinoid effectiveness while considering the risks associated with direct cannabinoid receptor activation.

Neurotransmitter-informed connectivity maps and their application for outcome inference after stroke.

Neuroscience has evolved by framing numerous neuropsychiatric conditions as network diseases. Alterations within neurotransmitter (NT) systems are central to the development of these diseases. Recently, normative data on whole-brain NT fingerprints derived from PET tracer data has become accessible; limited data related such information to sequelae after stroke. This work aims to explore (1) the integration of NT data into whole-brain structural connectivity analyses and (2) its potential contribution to understanding outcome variability following stroke. Normative maps of NT receptor and transporter densities were integrated with a normative structural connectome to generate NT-specific connectivity maps for serotonin, dopamine, GABA, glutamate, and acetylcholine receptors and transporters. Stroke lesion data from two independent, matched cohorts comprising a total of 126 severely impaired acute stroke patients were used to assess NT-related network damage on a patient-specific basis across each receptor and transporter distribution. Multivariable logistic regression models were applied to evaluate the relationship between NT-informed network disconnections and functional outcomes three to six months post-stroke, operationalised by the modified Rankin scale (mRS). Analyses were adjusted for lesion-induced global network damage, age, sex, lesion volume, and baseline neurological symptom burden. We present an innovative method to incorporate PET tracer data of various NT systems into normative structural connectome data. The resulting NT-informed connectivity maps revealed distinct spatial distributions consistent with the established literature. In both cohorts of severely impaired stroke patients, incorporating lesion-induced disruptions within specific NT systems provided more insights into variability in stroke outcomes than structural disconnection alone. Notably, greater damage to networks with high dopamine transporter (DAT) density was associated with poorer functional recovery. Based on NT-informed structural connectivity maps with distinct topographical features for individual receptors and transporters, we show that lesion-induced disruptions in large-scale dopaminergic brain networks, beyond global structural network damage, may play a key role in stroke recovery. These insights hold significant translational potential for advancing personalised medicine in stroke care, such as those achieved by targeted pharmacologic interventions.

Update on inherited disorders of GABA metabolism.

Update on inherited disorders of GABA metabolism.

Modulation of GABAergic System in a Chicken Retinal Ischemic Model: The Role of Chloride Cotransporters.

Retinal ischemia is a significant pathological condition that contributes to visual impairment and neuronal cell death in various retinopathies. Evidence suggests that GABA release during ischemic events may exhibit neuroprotective properties, but conflicting findings highlight a potential shift in its effects due to altered chloride ion homeostasis. This study aimed to investigate the role of the GABAergic system in retinal ischemia, focusing on the temporal dynamics of GABA release and its impact on retinal damage. We hypothesized that ischemia-induced changes in GABA transport and chloride ion equilibrium contribute to neuronal damage, which can be mitigated by modulating GABAergic activity. Using an exvivo chick retina model subjected to oxygen and glucose deprivation (OGD), during different times, we assessed morphological changes, cell death, GABA levels, transporter activity, and the levels of chloride cotransporters NKCC1 and KCC2. Pharmacological interventions, including picrotoxin and bumetanide, were used to evaluate neuroprotective effects. Our results revealed that OGD-induced significant morphological changes and cell death in the retina. GABA levels were reduced in a GAT-1-dependent manner, while picrotoxin and bumetanide demonstrated neuroprotective effects by mitigating retinal swelling and modulating the GABAergic system. Notably, OGD increased NKCC1 content, but not KCC2 levels, indicating a disruption in chloride homeostasis. These findings suggest that ischemia-induced alterations in GABAergic activity and chloride transport contribute to retinal damage. Targeting these pathways with pharmacological agents, such as bumetanide, may offer therapeutic strategies for mitigating ischemic retinal injury. Further research is recommended to explore the clinical applicability of these findings in the ischemic retina.

Forebrain microglia-associated synaptic alterations during obesity-related MASLD development

Metabolic dysfunction-associated steatotic liver disease (MASLD), considered as the hepatic manifestation of metabolic syndrome, is linked to cardiometabolic disease development. In addition to peripheral mechanisms, accumulating evidence points to changes in the brain as a factor in MASLD. In particular, we have demonstrated that excitatory signaling from the subfornical organ (SFO), a circumventricular nucleus located outside of the blood-brain-barrier, contributes to hepatic steatosis. While the mechanisms remain unclear, recent evidence indicates that microglia, the immune cell population within the brain, contribute to a number of diseases through alterations in synaptic contacts with neuron populations. Based on this, we hypothesized that SFO microglia contribute to MASLD development during obesity, and further, that this is related to changes in microglia-neuron synaptic interactions. To test this, male C57BL/6J mice were fed a high fat diet (HFD, 60% kcal fat) to induce hepatic steatosis or normal chow for 8 weeks. A chemogenetic strategy was then utilized to selectively inhibit microglial through SFO microinjection of a designer receptors exclusively activated by designer drugs inhibitory viral construct (AAV-CD68-hM4Gi-mCherry). Following surgical recovery, the pharmacological ligand clozapine-N-oxide (CNO; 3 mg/kg i.p., saline served as a control, n=7-11/group) was administered once daily over 3 days to inhibit SFO microglia. Selective inhibition of SFO microglia reduced liver lipid accumulation in mice fed a HFD (e.g. liver triglycerides: 2.7±0.3 vs 1.8±0.2 mM, HFD saline vs CNO, p<0.05) without altering body weight or food intake. Building upon this, we explored whether SFO microglia-neuronal synaptic alterations occurred during MASLD development. Male C57BL/6J mice fed a HFD or normal chow for 8 weeks were microinjected with a vector encoding green fluorescent protein driven under the glutamatergic neuron promoter calcium/calmodulin dependent kinase II (AAV-CAMKII-GFP) to label SFO excitatory neurons, which were then evaluated 3 weeks later using immunohistochemistry [microglia: ionized calcium-binding adapter molecule 1; CAMKII: GFP; inhibitory neurons: vesicular gamma-aminobutyric acid transporter (VGAT)]. Imaris software was used for analysis of synaptic interactions between cell types. 3D analysis throughout the SFO indicated that the number and surface area of SFO CAMKII neurons did not differ between groups (e.g. # CAMKII neurons: 135±16 vs 145±19, normal chow vs HFD, p>0.05). However, the total area of SFO microglia, reflecting microglial activation, was increased in HFD fed mice (75093±14206 vs 167712±29863 µm2, normal chow vs HFD, p<0.05). Interestingly, HFD fed mice demonstrated more than double the number of SFO CAMKII neurons in synaptic contact with microglia compared to control animals (41±4 vs 112±19, normal chow vs HFD, p<0.05). Moreover, the area of microglial synaptic coverage of SFO CAMKII neurons was also increased in HFD fed mice (64±7 vs 126±16 µm2, normal chow vs HFD, p<0.05). Surprisingly, increased microglial contact with SFO excitatory neurons was associated with reduced inhibitory synaptic innervation onto SFO CAMKII neurons (e.g. VGAT contact area: 160±15 vs 106±17 µm2; VGAT contact area/CAMKII neuron: 22±3 vs 15±2%, normal chow vs HFD, p<0.05). Collectively, these findings indicate that: 1) SFO microglia activation contributes to MASLD development during obesity, and; 2) SFO microglia may alter excitatory/inhibitory neuronal balance by disrupting physical interactions between neuron populations. R01DK117007, R01HL141393, AHA932522 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Angiotensin-(1-7) Activates POMC Neurons in the Arcuate Nucleus of the Hypothalamus Through a M as Receptor-Mediated Mechanism

Angiotensin-(1-7) is a protective hormone of the renin-angiotensin system that is well recognized to lower blood pressure and improve metabolic function in animal models of cardiovascular-related diseases; however, the precise mechanisms involved remain incompletely understood. Recent data from our laboratory suggests that the blood pressure lowering effects of angiotensin-(1-7) could occur through a central mechanism of action by inhibiting sympathetic activity to cardiovascular end organs. We have shown that angiotensin-(1-7) mas receptors highly colocalize to proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC), making these neurons a potential target for angiotensin-(1-7) cardiometabolic actions. In this study, we hypothesized that angiotensin-(1-7) activates POMC neurons in the ARC in a ma s receptor-dependent manner. To test this, we used male and female POMC-GFP mice to selectively target POMC neurons in the ARC. We assessed action potential firing of ARC POMC neurons following bath application of 100 nM angiotensin-(1-7) using cell attached patch clamp electrophysiology (n=7-10 cells per group). Angiotensin-(1-7) increased action potential firing of POMC neurons from baseline with no difference in angiotensin-(1-7) responsiveness between sexes (174±26% change in males vs. 133±12% change in females; one-sample t-test p=0.028 males vs. p=0.024 females; unpaired t-test p=0.127). To assess whether this increase in action potential firing occurred through a mas receptor mediated mechanism, we pretreated ARC slices with the selective mas receptor antagonist A779 (1 uM) for at least 30 minutes. Pretreatment with A779 prevented the increase in action potential firing following 100nM angiotensin-(1-7) bath application (106±13% change in males vs. 99±9% change in females; one-sample t-test p=0.549 males vs. p=0.884 females; unpaired t-test p=0.534). We also explored the neurotransmitter identity of ARC POMC neurons that express mas receptor using RNAscope in situ hybridization. We found that nearly 75% of POMC neurons in the ARC co-express mas receptor and mRNA for the vesicular GABA transporter (Vgat). Based on these data, we propose that angiotensin-(1-7) activates GABAergic subpopulations of POMC neurons in the ARC through a mas receptor-mediated mechanism, which may lead to a downstream inhibition of cardiovascular sympathetic activity. Additional studies are ongoing to further test this mechanism of action as well as to examine the impact of angiotensin-(1-7) on activity of ARC POMC neurons in the context of diet-induced obesity. Overall, these data provide new insight into central circuits engaged by angiotensin-(1-7). Funding: NIH F31 HL170693, NIH R01 HL156986 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Neuronal STING-GAT1 signaling maintains paclitaxel-induced neuropathic pain in the spinal cord.

Stimulator of interferon genes (STING), a pivotal immune regulator, has emerged as a contributor to nociception, yet its role in chronic pains remains still unknown. Here, we demonstrate that STING plays a dual role in normal and neuropathic pain in mature male rodents. Stimulator of interferon genes maintains type I interferon (IFN-I) level restraining pain sensitivity in normal and sham control, while activated STING/interferon regulatory factor 3 (IRF3) signaling increases the expression of gamma-aminobutyric acid (GABA) transporter 1 (GAT1) in the spinal cord (SC), thus, generating paclitaxel (PTX)-induced peripheral neuropathy. Genetic interference of STING (STING-/- mice) attenuated PTX-induced mechanical hypersensitivity with attenuated PTX-induced GAT1 increase, preventing PTX-induced increase in tonic GABAA inhibition of the spinal dorsal horn neurons. Stimulator of interferon genes regulates GAT expression through a TANK-binding kinase 1 (TBK1)-IRF3 signaling pathway, with IRF3 as a crucial transcription factor. Silencing neuronal STING, as opposed to its astrocytic counterpart, effectively restrained the PTX-induced mechanical hypersensitivity and GAT1 increase in the SC. Pharmacological inhibition of STING (H-151) efficiently diminished the TBK1/IRF3/GAT1 signaling pathway to alleviate PTX-induced mechanical hypersensitivity. Our findings show that STING-IRF3 serves a dual role: suppressing physiological nociception through IFN-I and acting as a transcriptional regulator of GAT1, contributing to chemotherapy-induced neuropathic pain.

Molecular basis of human GABA transporter 3 inhibition

γ-Aminobutyric acid (GABA) transporters (GATs) are sodium- and chloride-dependent transporters that mediate the reuptake of the inhibitory neurotransmitter GABA after its release from synaptic vesicles. GAT3 transports GABA from the synaptic cleft into astrocytes and modulates synaptic signaling. GAT3 has been implicated in various neurological disorders and neurodegenerative diseases, rendering it a therapeutically important drug target. To understand the mechanism of transport and inhibition, here we determine cryo-electron microscopy structures of human GAT3 in its apo form and in complex with the selective inhibitor SNAP-5114. Unexpectedly, we have discovered that SNAP-5114 acts as a noncompetitive inhibitor at GAT3. SNAP-5114 binds at the orthosteric substrate binding pocket of GAT3 in its inward-open conformation, in agreement with its noncompetitive inhibition of GABA transport. In the apo state, GAT3 also adopts an inward-open conformation with the orthosteric substrate binding pocket exposed to cytoplasm, while an extensive network of interactions closes the extracellular gate. The structures, complemented with mutagenesis and radioligand uptake assays, show that the increased orthosteric substrate binding pocket volume and bulky moieties of SNAP-5114, drive the selective inhibition of GAT3 over GAT1. Our structural and functional studies reveal the mechanism of selective inhibition of GAT3 and provide a framework for GAT3-targeted rational drug design.