{"gene":"SLC1A6","run_date":"2026-06-10T07:46:32","timeline":{"discoveries":[{"year":1996,"finding":"EAAT4 is a postsynaptic glutamate transporter specifically localized to dendritic spines of cerebellar Purkinje cells, as determined by immunoblotting and electron microscopy with a peptide antibody against the N-terminal domain.","method":"Immunohistochemistry, immunoblotting, electron microscopy","journal":"Neuroreport","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct subcellular localization by electron microscopy, replicated across multiple subsequent studies","pmids":["8905715"],"is_preprint":false},{"year":1997,"finding":"EAAT4 is predominantly localized in dendritic spines and distal dendrites of Purkinje cells and forms parasagittal compartments in the rat cerebellum, consistent with its role as a glutamate-gated chloride channel at parallel fiber-Purkinje cell synapses.","method":"Immunohistochemistry, Western blot with site-directed antisera","journal":"Neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct subcellular localization, independently replicated across labs","pmids":["9174061"],"is_preprint":false},{"year":1997,"finding":"EAAT4 is selectively targeted to the extra-junctional membrane of excitatory Purkinje cell synapses (not at the junctional membrane), suggesting it transports glutamate from outside the synaptic cleft and facilitates glutamate diffusion away from the cleft.","method":"Silver-enhanced immunogold electron microscopy","journal":"Neuroreport","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution immunogold EM establishing precise subcellular localization, single lab but rigorous method","pmids":["9261809"],"is_preprint":false},{"year":1998,"finding":"EAAT4 functions as a high-affinity Na+-dependent glutamate transporter with properties of a ligand-gated chloride channel; in Xenopus oocytes expressing rat EAAT4, L-glutamate and other substrates elicited currents predominantly carried by chloride ions.","method":"Xenopus oocyte heterologous expression, electrophysiology (voltage clamp), radiolabeled glutamate uptake","journal":"Brain research. Molecular brain research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro functional reconstitution in oocytes with electrophysiology, consistent with human EAAT4 data","pmids":["9838098"],"is_preprint":false},{"year":1998,"finding":"EAAT4 is enriched in parasagittal zones of Purkinje cell spines and thin dendrites, concentrated at perisynaptic membranes facing astroglia rather than at the synapse proper; cross-linking revealed EAAT4 forms dimers (not trimers as seen for GLAST and GLT).","method":"Immunohistochemistry, immunoblotting with chemical cross-linking (bis-sulfosuccinimidyl suberate)","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative immunogold EM with cross-linking biochemistry, replicated across independent labs","pmids":["9570792"],"is_preprint":false},{"year":2001,"finding":"Two proteins, GTRAP41 and GTRAP48, specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity, identified by yeast two-hybrid screening and confirmed by biochemical characterization.","method":"Yeast two-hybrid screen, Co-IP/pulldown, functional transport assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid plus functional modulation data, published in Nature with characterization of both interactors","pmids":["11242047"],"is_preprint":false},{"year":2004,"finding":"SGK1 (serum and glucocorticoid inducible kinase 1) and its isoforms SGK2, SGK3, and PKB stimulate EAAT4-mediated glutamate-induced current in Xenopus oocytes; the ubiquitin ligase Nedd4-2 decreases EAAT4 membrane abundance, an effect partially reversed by SGK isoforms. SGK1 enhances EAAT4 protein abundance in the cell membrane.","method":"Xenopus oocyte electrophysiology, immunohistochemistry, chemiluminescence membrane abundance assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional electrophysiology in oocytes combined with membrane abundance quantification, single lab","pmids":["15504348"],"is_preprint":false},{"year":2006,"finding":"PKC activation by PMA specifically enhances the substrate-gated Cl- currents of EAAT4 (via non-conventional PKC isoforms) without affecting glutamate transport activity, demonstrating that the ion channel function and transport function of EAAT4 can be independently regulated.","method":"Xenopus oocyte electrophysiology, radiolabeled glutamate uptake, pharmacological inhibitors of PKC","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with orthogonal measurements of transport vs. channel activity, single lab","pmids":["16601148"],"is_preprint":false},{"year":2006,"finding":"EAAT4 knockout mice show selective loss of Purkinje cells with low EAAT4 expression after cardiac arrest, demonstrating that EAAT4 protects Purkinje cells from excitotoxic damage after ischemia and acts in concert with the glial transporter GLAST.","method":"Genetic knockout mouse model, cardiac arrest ischemia model, histological Purkinje cell counting","journal":"Neuroscience research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with defined cellular phenotype and genetic epistasis showing GLAST/EAAT4 interaction, in vivo model","pmids":["16647773"],"is_preprint":false},{"year":2007,"finding":"SGK1 modulates EAAT4 function and cell surface expression via phosphorylation at Thr40 (SGK1 consensus site at the N-terminus); disruption of Thr40 (T40A mutation) or both sites (T40A/T504A) abrogated SGK1-dependent enhancement of glutamate uptake and membrane abundance. SGK1 acts partly by inhibiting the ubiquitin ligase Nedd4-2, which itself reduces EAAT4 activity.","method":"Xenopus oocyte expression, site-directed mutagenesis, radiolabeled glutamate uptake, chemiluminescence membrane abundance, RNA interference","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of phosphorylation sites combined with functional assay and membrane abundance, single lab with multiple orthogonal methods","pmids":["17442044"],"is_preprint":false},{"year":2010,"finding":"PIKfyve (PIP5K3) participates in SGK1-dependent regulation of EAAT4; co-expression of PIKfyve and SGK1 synergistically enhances EAAT4-mediated glutamate-induced current, and an SGK1-phosphorylation site mutant of PIKfyve (S318A) abolishes this effect.","method":"Xenopus oocyte electrophysiology, dominant-negative and phosphorylation-site mutants","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional oocyte assay with mutants establishing pathway, single lab","pmids":["20110679"],"is_preprint":false},{"year":2010,"finding":"AMPK (AMP-activated protein kinase) down-regulates EAAT4 transport activity by reducing membrane abundance of the transporter, without affecting substrate affinity (Km unchanged); constitutively active AMPK decreased maximal glutamate-induced current by ~49%.","method":"Xenopus oocyte electrophysiology, confocal microscopy, chemiluminescence membrane abundance","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional assay with constitutively active and inactive AMPK constructs, single lab","pmids":["20218975"],"is_preprint":false},{"year":2010,"finding":"A conserved aspartate residue (Asp117) in EAAT4 determines pore properties including unitary conductance, anion permeability selectivity, and voltage- and substrate-dependent gating of the associated anion channel; D117A abolishes glutamate- and anion-dependent gating of EAAT4 anion currents.","method":"Heterologous expression in mammalian cells, whole-cell patch clamp, noise analysis, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structure-function mutagenesis with electrophysiology and noise analysis in mammalian cells, single lab with multiple rigorous methods","pmids":["20519505"],"is_preprint":false},{"year":2010,"finding":"EAAT4 in Purkinje cells controls intersynaptic diffusion of climbing fiber transmitter (glutamate); pharmacological blockade of EAAT4 enhanced climbing fiber-induced inhibition of GABA release at basket cell-Purkinje cell synapses; tetanic CF stimulation induced long-term potentiation of glutamate transporter activity that attenuated this spillover. EAAT4 expression level inversely correlates with the degree of CF-mediated inhibition of GABAergic transmission across cerebellar lobules.","method":"Electrophysiology in cerebellar slices, pharmacological EAAT4 blockade, immunohistochemistry","journal":"The European journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — slice electrophysiology with pharmacological dissection plus immunohistochemistry, correlation across regions, use-dependent plasticity demonstrated","pmids":["21070388"],"is_preprint":false},{"year":2013,"finding":"Klotho (as a β-glucuronidase) upregulates EAAT4 transport activity and membrane protein abundance in Xenopus oocytes; the effect is mimicked by recombinant β-Klotho protein and abolished by the β-glucuronidase inhibitor DSAL.","method":"Xenopus oocyte electrophysiology, confocal microscopy, chemiluminescence, recombinant protein treatment","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional oocyte assay with recombinant protein and inhibitor, single lab","pmids":["23923038"],"is_preprint":false},{"year":2016,"finding":"GSK3β up-regulates EAAT4 transport activity in Xenopus oocytes; coexpression of wild-type but not kinase-dead (K85A) GSK3β significantly increases maximal EAAT4-mediated glutamate-induced current without affecting substrate affinity. This effect is blocked by lithium.","method":"Xenopus oocyte electrophysiology, kinase-dead mutant, lithium treatment","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional oocyte assay with kinase-dead control and pharmacological inhibition, single lab","pmids":["27978527"],"is_preprint":false},{"year":2015,"finding":"Caveolin-1 negatively regulates EAAT4 transport activity by decreasing the maximal transport rate without accelerating transporter retrieval from the cell membrane, as shown in Xenopus oocytes.","method":"Xenopus oocyte electrophysiology, brefeldin A treatment","journal":"The Journal of membrane biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — functional oocyte assay, single method, single lab","pmids":["26690923"],"is_preprint":false},{"year":2016,"finding":"Conditional knockout of Rheb1 (Ras homolog enriched in brain) in cerebellar Purkinje cells leads to downregulation of EAAT4 expression and reduced EAAT4-mediated currents, placing Rheb1/mTOR signaling upstream of EAAT4 expression in Purkinje cells.","method":"Conditional knockout mice, immunohistochemistry, electrophysiology (EAAT4 and AMPA receptor currents)","journal":"Cerebellum","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with functional current measurements and immunohistochemistry, single lab","pmids":["26194056"],"is_preprint":false},{"year":2018,"finding":"EAAT4 limits mGluR1 signaling in zebrin-positive (high EAAT4) Purkinje cells to constrain heterogeneous spontaneous firing; mGluR1 antagonists restore regular spontaneous firing and motor behavior in EAAT4 knockout mice. In contrast, loss of GLAST/EAAT1 disrupts zebrin-negative (low EAAT4) Purkinje cells via NMDA receptor overactivation. Genetic epistasis places EAAT4 upstream of mGluR1 in regulating Purkinje cell firing.","method":"EAAT4 knockout mice, electrophysiology (spontaneous firing), pharmacological rescue with mGluR1 and NMDA receptor antagonists, behavioral assays","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with pharmacological epistasis, electrophysiology, and behavioral rescue, multiple orthogonal methods","pmids":["29741614"],"is_preprint":false},{"year":2021,"finding":"Patterned EAAT4 expression in Purkinje cells regulates glutamate spillover from climbing fibers to molecular layer interneurons; regions with high EAAT4 show smaller spillover EPSCs than regions with low EAAT4. Inhibiting glutamate transport normalizes the regional difference, demonstrating EAAT4 as a primary determinant of differential spillover.","method":"Electrophysiology in cerebellar slices from Aldolase C-Venus knock-in mice, pharmacological transporter blockade, postsynaptic receptor analysis","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — slice electrophysiology with genetic visualization of microzones plus pharmacological rescue, multiple controls","pmids":["34400517"],"is_preprint":false},{"year":2026,"finding":"Ethanol at pharmacologically relevant concentrations (25–100 mM) increases glutamate uptake via EAAT4 in Purkinje cells, operating in concert with Na,K-ATPase, thereby restricting glutamate diffusion from climbing fiber synaptic clefts and suppressing intersynaptic modulation of GABAergic interneurons.","method":"Electrophysiology in rat cerebellar slices, pharmacological manipulation of EAAT4","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — slice electrophysiology with pharmacological dissection, single lab, single paper","pmids":["41946908"],"is_preprint":false}],"current_model":"EAAT4 (SLC1A6) is a high-affinity Na+-dependent glutamate transporter expressed predominantly in cerebellar Purkinje cells, where it localizes to extra-junctional perisynaptic membranes of dendritic spines; it functions both as an electrogenic glutamate transporter and a substrate-gated chloride channel (with Asp117 as a key pore-forming residue), limits mGluR1 signaling to regulate spontaneous firing in zebrin-positive Purkinje cells, controls glutamate spillover to molecular layer interneurons in a regionally patterned manner, and is regulated by multiple kinases (SGK1 via Thr40 phosphorylation and Nedd4-2 ubiquitination, AMPK, PKC, PIKfyve, GSK3β, Klotho, and caveolin-1) that primarily alter its membrane abundance; its carboxy-terminal domain interacts with GTRAP41 and GTRAP48 to modulate activity, and its loss causes excitotoxic Purkinje cell death and cerebellar ataxia."},"narrative":{"mechanistic_narrative":"SLC1A6 (EAAT4) is a high-affinity, Na+-dependent glutamate transporter expressed predominantly in cerebellar Purkinje cells, where it shapes synaptic glutamate signaling and protects neurons from excitotoxicity [PMID:8905715, PMID:16647773]. It localizes to extra-junctional, perisynaptic membranes of dendritic spines and distal dendrites facing astroglia, positioning it to clear glutamate spilling out of the synaptic cleft, and it is distributed in parasagittal microzones across the cerebellum [PMID:9261809, PMID:9570792]. Functionally, EAAT4 couples electrogenic glutamate transport to a substrate-gated chloride channel, with a conserved aspartate (Asp117) governing the anion pore's conductance, selectivity, and substrate- and voltage-dependent gating [PMID:9838098, PMID:20519505]; transport and channel functions are separable, as PKC activation selectively enhances the chloride current without altering uptake [PMID:16601148]. Through this perisynaptic uptake, EAAT4 controls intersynaptic diffusion of climbing-fiber glutamate to molecular layer interneurons and limits mGluR1 signaling in zebrin/aldolase-C-positive Purkinje cells, thereby constraining spontaneous firing in a regionally patterned manner [PMID:21070388, PMID:29741614, PMID:34400517]. Its surface abundance and activity are set by a network of regulators acting largely on membrane trafficking: SGK1 phosphorylates Thr40 to oppose Nedd4-2-mediated ubiquitination and downregulation [PMID:15504348, PMID:17442044], while its carboxy-terminal domain binds GTRAP41 and GTRAP48 to modulate transport [PMID:11242047]. Loss of EAAT4 causes excitotoxic Purkinje cell death and motor deficits, the latter reversible by mGluR1 antagonism [PMID:16647773, PMID:29741614].","teleology":[{"year":1996,"claim":"Establishing where EAAT4 acts was the first step: it was identified as a postsynaptic glutamate transporter concentrated in Purkinje cell dendritic spines, distinguishing it from glial transporters.","evidence":"Peptide-antibody immunohistochemistry, immunoblotting, and electron microscopy in cerebellum","pmids":["8905715"],"confidence":"High","gaps":["Did not define transport kinetics or ion coupling","No data on synaptic vs extra-synaptic position"]},{"year":1997,"claim":"Refining localization showed EAAT4 sits at extra-junctional/perisynaptic membranes in parasagittal compartments, implying it handles glutamate diffusing out of the cleft rather than direct synaptic clearance.","evidence":"Site-directed antisera immunohistochemistry and silver-enhanced immunogold EM","pmids":["9174061","9261809"],"confidence":"High","gaps":["Functional consequence of perisynaptic placement not yet measured electrophysiologically","Oligomeric state and partners unknown"]},{"year":1998,"claim":"Heterologous reconstitution established the dual nature of EAAT4 as a Na+-dependent transporter whose currents are dominated by a ligand-gated chloride conductance, and biochemistry showed it forms dimers unlike trimeric glial transporters.","evidence":"Xenopus oocyte voltage clamp, radiolabeled glutamate uptake, and chemical cross-linking with quantitative immunogold EM","pmids":["9838098","9570792"],"confidence":"High","gaps":["Pore residues controlling the anion channel not yet mapped","Physiological role of the chloride current in Purkinje cells untested"]},{"year":2001,"claim":"Identifying GTRAP41 and GTRAP48 as C-terminal interactors gave the first protein partners that tune EAAT4 transport activity.","evidence":"Yeast two-hybrid screen, co-IP/pulldown, and functional transport assays","pmids":["11242047"],"confidence":"High","gaps":["Structural basis of C-terminal binding not resolved","In vivo relevance in Purkinje cells not demonstrated"]},{"year":2004,"claim":"The first kinase/ubiquitin axis was defined, showing EAAT4 surface abundance is set by Nedd4-2-mediated downregulation that SGK isoforms and PKB reverse.","evidence":"Xenopus oocyte electrophysiology with chemiluminescence membrane abundance assays","pmids":["15504348"],"confidence":"Medium","gaps":["Phosphorylation site mediating SGK1 action not yet identified","Performed in oocytes, not Purkinje cells"]},{"year":2006,"claim":"Genetic loss-of-function in mice tied EAAT4 to neuroprotection, showing it shields Purkinje cells from ischemic excitotoxicity in concert with glial GLAST.","evidence":"EAAT4 knockout mice subjected to cardiac-arrest ischemia with histological Purkinje cell counting","pmids":["16647773"],"confidence":"High","gaps":["Did not address basal firing or synaptic signaling roles","Mechanism of GLAST cooperation not molecularly defined"]},{"year":2006,"claim":"Transport and channel functions were shown to be independently regulable, as PKC selectively boosts the chloride current without changing uptake.","evidence":"Oocyte electrophysiology and radiolabeled uptake with PKC inhibitors/activators","pmids":["16601148"],"confidence":"Medium","gaps":["PKC phosphorylation site on EAAT4 not mapped","Physiological setting of channel-specific modulation unknown"]},{"year":2007,"claim":"The SGK1 mechanism was pinned to Thr40 phosphorylation that opposes Nedd4-2, converting the earlier observation into a defined regulatory site.","evidence":"Oocyte expression with T40A/T504A site-directed mutagenesis, uptake, membrane abundance, and RNAi","pmids":["17442044"],"confidence":"High","gaps":["In vivo phosphorylation in Purkinje cells not confirmed","Upstream signals activating SGK1 toward EAAT4 unclear"]},{"year":2010,"claim":"The structural determinant of the anion channel was localized to Asp117, explaining how a single residue sets conductance, anion selectivity, and gating.","evidence":"Mammalian-cell whole-cell patch clamp with noise analysis and D117A mutagenesis","pmids":["20519505"],"confidence":"High","gaps":["No atomic structure of the EAAT4 pore","Relationship of Asp117 to the glutamate transport pathway not resolved"]},{"year":2010,"claim":"Additional trafficking regulators were placed in the network — PIKfyve cooperates with SGK1 to enhance EAAT4, while AMPK downregulates it by reducing surface abundance without altering Km.","evidence":"Oocyte electrophysiology with phospho-site and constitutively active/inactive kinase mutants and confocal microscopy","pmids":["20110679","20218975"],"confidence":"Medium","gaps":["Effects shown only in oocytes","Endogenous relevance to Purkinje cell physiology untested"]},{"year":2010,"claim":"Slice physiology connected EAAT4 to circuit function, showing it limits climbing-fiber glutamate spillover onto basket-cell synapses and undergoes use-dependent potentiation, with expression inversely tracking spillover-mediated GABA inhibition across lobules.","evidence":"Cerebellar slice electrophysiology with pharmacological EAAT4 blockade and immunohistochemistry","pmids":["21070388"],"confidence":"High","gaps":["Molecular basis of transporter potentiation not defined","Did not test downstream behavior"]},{"year":2013,"claim":"Klotho's β-glucuronidase activity was shown to upregulate EAAT4 surface abundance, extending the regulatory repertoire to an extracellular enzyme.","evidence":"Oocyte electrophysiology with recombinant β-Klotho and the β-glucuronidase inhibitor DSAL","pmids":["23923038"],"confidence":"Medium","gaps":["Mechanism linking glucuronidase activity to EAAT4 trafficking unknown","Oocyte-only system"]},{"year":2016,"claim":"Two further modulators were defined: GSK3β increases maximal transport (lithium-blockable) and caveolin-1 decreases maximal transport rate without accelerating retrieval, and Rheb1/mTOR was placed upstream of EAAT4 expression in vivo.","evidence":"Oocyte electrophysiology with kinase-dead and pharmacological controls; conditional Rheb1 knockout mice with immunohistochemistry and current recordings","pmids":["27978527","26690923","26194056"],"confidence":"Medium","gaps":["Caveolin-1 and GSK3β actions shown only in oocytes","How Rheb1/mTOR controls EAAT4 transcription/translation not detailed"]},{"year":2018,"claim":"Genetic epistasis revealed the circuit logic of EAAT4: it limits mGluR1 signaling in zebrin-positive Purkinje cells to maintain regular firing and motor behavior, with mGluR1 antagonism rescuing knockout deficits.","evidence":"EAAT4 knockout mice with spontaneous-firing electrophysiology, pharmacological mGluR1/NMDA rescue, and behavioral assays","pmids":["29741614"],"confidence":"High","gaps":["How perisynaptic uptake mechanistically gates mGluR1 not fully resolved","Human disease link not directly established"]},{"year":2021,"claim":"Patterned EAAT4 expression was shown to be the primary determinant of regional differences in climbing-fiber glutamate spillover to molecular layer interneurons.","evidence":"Cerebellar slice electrophysiology in aldolase-C-Venus knock-in mice with pharmacological transporter blockade","pmids":["34400517"],"confidence":"High","gaps":["Functional consequence of differential spillover for cerebellar output not defined","Did not test behavioral readouts of microzonal spillover"]},{"year":2026,"claim":"A modulatory role for ethanol was added, showing it enhances EAAT4-mediated uptake in concert with Na,K-ATPase to restrict climbing-fiber glutamate diffusion and suppress GABAergic interneuron modulation.","evidence":"Rat cerebellar slice electrophysiology with pharmacological EAAT4 manipulation","pmids":["41946908"],"confidence":"Medium","gaps":["Molecular mechanism of ethanol action on EAAT4 unknown","Single lab, single study"]},{"year":null,"claim":"How the many oocyte-defined regulators integrate in vivo to set EAAT4 surface levels, and whether SLC1A6 variants cause human cerebellar ataxia, remain open.","evidence":"","pmids":[],"confidence":"Low","gaps":["No atomic structure of human EAAT4","Most kinase/trafficking regulation shown only in heterologous systems","No timeline evidence of a causative human Mendelian variant"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[3,9,11]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,3,12]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[3,12]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,2,4]}],"pathway":[{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[13,18,19]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[3,9]}],"complexes":[],"partners":["GTRAP41","GTRAP48","NEDD4L","SGK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P48664","full_name":"Excitatory amino acid transporter 4","aliases":["Sodium-dependent glutamate/aspartate transporter","Solute carrier family 1 member 6"],"length_aa":564,"mass_kda":61.6,"function":"Sodium-dependent, high-affinity amino acid transporter that mediates the uptake of L-glutamate and also L-aspartate and D-aspartate (PubMed:26690923, PubMed:7791878). Functions as a symporter that transports one amino acid molecule together with two or three Na(+) ions and one proton, in parallel with the counter-transport of one K(+) ion. Mediates Cl(-) flux that is not coupled to amino acid transport; this avoids the accumulation of negative charges due to aspartate and Na(+) symport (PubMed:7791878). Plays a redundant role in the rapid removal of released glutamate from the synaptic cleft, which is essential for terminating the postsynaptic action of glutamate (Probable)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/P48664/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SLC1A6","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SLC1A6","total_profiled":1310},"omim":[{"mim_id":"604471","title":"SOLUTE CARRIER FAMILY 1 (GLUTAMATE TRANSPORTER), MEMBER 7; SLC1A7","url":"https://www.omim.org/entry/604471"},{"mim_id":"600637","title":"SOLUTE CARRIER FAMILY 1 (HIGH AFFINITY ASPARTATE/GLUTAMATE TRANSPORTER), MEMBER 6; SLC1A6","url":"https://www.omim.org/entry/600637"},{"mim_id":"600300","title":"SOLUTE CARRIER FAMILY 1 (GLIAL HIGH AFFINITY GLUTAMATE TRANSPORTER), MEMBER 2; SLC1A2","url":"https://www.omim.org/entry/600300"},{"mim_id":"131340","title":"PRODYNORPHIN; PDYN","url":"https://www.omim.org/entry/131340"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Intermediate filaments","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":105.2}],"url":"https://www.proteinatlas.org/search/SLC1A6"},"hgnc":{"alias_symbol":["EAAT4"],"prev_symbol":[]},"alphafold":{"accession":"P48664","domains":[{"cath_id":"1.10.3860.10","chopping":"65-202_262-536","consensus_level":"medium","plddt":89.0789,"start":65,"end":536}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P48664","model_url":"https://alphafold.ebi.ac.uk/files/AF-P48664-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P48664-F1-predicted_aligned_error_v6.png","plddt_mean":80.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SLC1A6","jax_strain_url":"https://www.jax.org/strain/search?query=SLC1A6"},"sequence":{"accession":"P48664","fasta_url":"https://rest.uniprot.org/uniprotkb/P48664.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P48664/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P48664"}},"corpus_meta":[{"pmid":"9570792","id":"PMC_9570792","title":"The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia.","date":"1998","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/9570792","citation_count":268,"is_preprint":false},{"pmid":"11968459","id":"PMC_11968459","title":"Why do Purkinje cells die so easily after global brain ischemia? Aldolase C, EAAT4, and the cerebellar contribution to posthypoxic myoclonus.","date":"2002","source":"Advances in neurology","url":"https://pubmed.ncbi.nlm.nih.gov/11968459","citation_count":211,"is_preprint":false},{"pmid":"11242047","id":"PMC_11242047","title":"Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins.","date":"2001","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/11242047","citation_count":198,"is_preprint":false},{"pmid":"10812201","id":"PMC_10812201","title":"The high-affinity glutamate transporters GLT1, GLAST, and EAAT4 are regulated via different signalling mechanisms.","date":"2000","source":"Neurochemistry international","url":"https://pubmed.ncbi.nlm.nih.gov/10812201","citation_count":175,"is_preprint":false},{"pmid":"19747495","id":"PMC_19747495","title":"Reduced expression of glutamate transporters vGluT1, EAAT2 and EAAT4 in learned helpless rats, an animal model of depression.","date":"2009","source":"Neuropharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/19747495","citation_count":142,"is_preprint":false},{"pmid":"8905715","id":"PMC_8905715","title":"EAAT4 is a post-synaptic glutamate transporter at Purkinje cell synapses.","date":"1996","source":"Neuroreport","url":"https://pubmed.ncbi.nlm.nih.gov/8905715","citation_count":129,"is_preprint":false},{"pmid":"9174061","id":"PMC_9174061","title":"EAAT4, a glutamate transporter with properties of a chloride channel, is predominantly localized in Purkinje cell dendrites, and forms parasagittal compartments in rat cerebellum.","date":"1997","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/9174061","citation_count":91,"is_preprint":false},{"pmid":"9261809","id":"PMC_9261809","title":"Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses.","date":"1997","source":"Neuroreport","url":"https://pubmed.ncbi.nlm.nih.gov/9261809","citation_count":52,"is_preprint":false},{"pmid":"9838098","id":"PMC_9838098","title":"Molecular cloning and expression of the rat EAAT4 glutamate transporter subtype.","date":"1998","source":"Brain research. Molecular brain research","url":"https://pubmed.ncbi.nlm.nih.gov/9838098","citation_count":50,"is_preprint":false},{"pmid":"9630235","id":"PMC_9630235","title":"Expression of two glutamate transporters, GLAST and EAAT4, in the human cerebellum: their correlation in development and neonatal hypoxic-ischemic damage.","date":"1998","source":"Journal of neuropathology and experimental neurology","url":"https://pubmed.ncbi.nlm.nih.gov/9630235","citation_count":49,"is_preprint":false},{"pmid":"16647773","id":"PMC_16647773","title":"Glutamate transporters GLAST and EAAT4 regulate postischemic Purkinje cell death: an in vivo study using a cardiac arrest model in mice lacking GLAST or EAAT4.","date":"2006","source":"Neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/16647773","citation_count":47,"is_preprint":false},{"pmid":"18770868","id":"PMC_18770868","title":"High-affinity Na+/K+-dependent glutamate transporter EAAT4 is expressed throughout the rat fore- and midbrain.","date":"2008","source":"The Journal of comparative neurology","url":"https://pubmed.ncbi.nlm.nih.gov/18770868","citation_count":47,"is_preprint":false},{"pmid":"12951653","id":"PMC_12951653","title":"Neuronal glutamate transporter EAAT4 is expressed in astrocytes.","date":"2003","source":"Glia","url":"https://pubmed.ncbi.nlm.nih.gov/12951653","citation_count":46,"is_preprint":false},{"pmid":"17221839","id":"PMC_17221839","title":"Association study of polymorphisms in the glutamate transporter genes SLC1A1, SLC1A3, and SLC1A6 with schizophrenia.","date":"2007","source":"American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/17221839","citation_count":42,"is_preprint":false},{"pmid":"20110679","id":"PMC_20110679","title":"Regulation of the glutamate transporter EAAT4 by PIKfyve.","date":"2010","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/20110679","citation_count":38,"is_preprint":false},{"pmid":"15504348","id":"PMC_15504348","title":"Stimulation of the EAAT4 glutamate transporter by SGK protein kinase isoforms and PKB.","date":"2004","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/15504348","citation_count":37,"is_preprint":false},{"pmid":"23049999","id":"PMC_23049999","title":"Glutamate transporters EAAT4 and EAAT5 are expressed in vestibular hair cells and calyx endings.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23049999","citation_count":35,"is_preprint":false},{"pmid":"20519505","id":"PMC_20519505","title":"A conserved aspartate determines pore properties of anion channels associated with excitatory amino acid transporter 4 (EAAT4).","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20519505","citation_count":35,"is_preprint":false},{"pmid":"17022974","id":"PMC_17022974","title":"Analysis of cerebellar Purkinje cells using EAAT4 glutamate transporter promoter reporter in mice generated via bacterial artificial chromosome-mediated transgenesis.","date":"2006","source":"Experimental neurology","url":"https://pubmed.ncbi.nlm.nih.gov/17022974","citation_count":34,"is_preprint":false},{"pmid":"29741614","id":"PMC_29741614","title":"Loss of cerebellar glutamate transporters EAAT4 and GLAST differentially affects the spontaneous firing pattern and survival of Purkinje cells.","date":"2018","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29741614","citation_count":33,"is_preprint":false},{"pmid":"20218975","id":"PMC_20218975","title":"Down-regulation of Na+-coupled glutamate transporter EAAT3 and EAAT4 by AMP-activated protein kinase.","date":"2010","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20218975","citation_count":29,"is_preprint":false},{"pmid":"9129177","id":"PMC_9129177","title":"Changes in expression and distribution of the glutamate transporter EAAT4 in developing mouse Purkinje cells.","date":"1997","source":"Neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/9129177","citation_count":25,"is_preprint":false},{"pmid":"11209956","id":"PMC_11209956","title":"Expression of the high-affinity glutamate transporter EAAT4 in mammalian cerebral cortex.","date":"2001","source":"Neuroreport","url":"https://pubmed.ncbi.nlm.nih.gov/11209956","citation_count":25,"is_preprint":false},{"pmid":"9367257","id":"PMC_9367257","title":"Expression of a glutamate transporter subtype, EAAT4, in the developing human cerebellum.","date":"1997","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/9367257","citation_count":23,"is_preprint":false},{"pmid":"17442044","id":"PMC_17442044","title":"EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase.","date":"2007","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17442044","citation_count":21,"is_preprint":false},{"pmid":"23923038","id":"PMC_23923038","title":"Klotho sensitivity of the neuronal excitatory amino acid transporters EAAT3 and EAAT4.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23923038","citation_count":19,"is_preprint":false},{"pmid":"20011524","id":"PMC_20011524","title":"Patterned neuroprotection in the Inpp4a(wbl) mutant mouse cerebellum correlates with the expression of Eaat4.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/20011524","citation_count":19,"is_preprint":false},{"pmid":"16601148","id":"PMC_16601148","title":"Enhancement of substrate-gated Cl- currents via rat glutamate transporter EAAT4 by PMA.","date":"2006","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/16601148","citation_count":14,"is_preprint":false},{"pmid":"17490622","id":"PMC_17490622","title":"Glutamate transporter EAAT4 is increased in hippocampal astrocytes following lateral fluid-percussion injury in the rat.","date":"2007","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/17490622","citation_count":14,"is_preprint":false},{"pmid":"34400517","id":"PMC_34400517","title":"Climbing Fiber-Mediated Spillover Transmission to Interneurons Is Regulated by EAAT4.","date":"2021","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/34400517","citation_count":13,"is_preprint":false},{"pmid":"27978527","id":"PMC_27978527","title":"Up-Regulation of Excitatory Amino Acid Transporters EAAT3 and EAAT4 by Lithium Sensitive Glycogen Synthase Kinase GSK3ß.","date":"2016","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/27978527","citation_count":13,"is_preprint":false},{"pmid":"20211693","id":"PMC_20211693","title":"Quantitative analysis of EAAT4 promoter activity in neurons and astrocytes of mouse somatic sensory cortex.","date":"2010","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/20211693","citation_count":13,"is_preprint":false},{"pmid":"33927617","id":"PMC_33927617","title":"Radioresistant Nasopharyngeal Carcinoma Cells Exhibited Decreased Cisplatin Sensitivity by Inducing SLC1A6 Expression.","date":"2021","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/33927617","citation_count":11,"is_preprint":false},{"pmid":"21070388","id":"PMC_21070388","title":"Glutamate transporter EAAT4 in Purkinje cells controls intersynaptic diffusion of climbing fiber transmitter mediating inhibition of GABA release from interneurons.","date":"2010","source":"The European journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/21070388","citation_count":11,"is_preprint":false},{"pmid":"26690923","id":"PMC_26690923","title":"Caveolin-1 Sensitivity of Excitatory Amino Acid Transporters EAAT1, EAAT2, EAAT3, and EAAT4.","date":"2015","source":"The Journal of membrane biology","url":"https://pubmed.ncbi.nlm.nih.gov/26690923","citation_count":6,"is_preprint":false},{"pmid":"26194056","id":"PMC_26194056","title":"Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells.","date":"2016","source":"Cerebellum (London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/26194056","citation_count":5,"is_preprint":false},{"pmid":"14625012","id":"PMC_14625012","title":"EAAT4 mRNA expression is preserved in the cerebellum of prion protein-deficient mice.","date":"2003","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/14625012","citation_count":2,"is_preprint":false},{"pmid":"40481876","id":"PMC_40481876","title":"Glutamate transporter SLC1A6 promotes resistance to immunotherapy in cancer.","date":"2025","source":"Cancer immunology, immunotherapy : CII","url":"https://pubmed.ncbi.nlm.nih.gov/40481876","citation_count":0,"is_preprint":false},{"pmid":"41946908","id":"PMC_41946908","title":"Ethanol disrupts cerebellar synaptic modulation by enhancing EAAT4-mediated glutamate uptake.","date":"2026","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/41946908","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":21962,"output_tokens":4765,"usd":0.068681,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12977,"output_tokens":4641,"usd":0.090455,"stage2_stop_reason":"end_turn"},"total_usd":0.159136,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"EAAT4 is a postsynaptic glutamate transporter specifically localized to dendritic spines of cerebellar Purkinje cells, as determined by immunoblotting and electron microscopy with a peptide antibody against the N-terminal domain.\",\n      \"method\": \"Immunohistochemistry, immunoblotting, electron microscopy\",\n      \"journal\": \"Neuroreport\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct subcellular localization by electron microscopy, replicated across multiple subsequent studies\",\n      \"pmids\": [\"8905715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"EAAT4 is predominantly localized in dendritic spines and distal dendrites of Purkinje cells and forms parasagittal compartments in the rat cerebellum, consistent with its role as a glutamate-gated chloride channel at parallel fiber-Purkinje cell synapses.\",\n      \"method\": \"Immunohistochemistry, Western blot with site-directed antisera\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct subcellular localization, independently replicated across labs\",\n      \"pmids\": [\"9174061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"EAAT4 is selectively targeted to the extra-junctional membrane of excitatory Purkinje cell synapses (not at the junctional membrane), suggesting it transports glutamate from outside the synaptic cleft and facilitates glutamate diffusion away from the cleft.\",\n      \"method\": \"Silver-enhanced immunogold electron microscopy\",\n      \"journal\": \"Neuroreport\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution immunogold EM establishing precise subcellular localization, single lab but rigorous method\",\n      \"pmids\": [\"9261809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"EAAT4 functions as a high-affinity Na+-dependent glutamate transporter with properties of a ligand-gated chloride channel; in Xenopus oocytes expressing rat EAAT4, L-glutamate and other substrates elicited currents predominantly carried by chloride ions.\",\n      \"method\": \"Xenopus oocyte heterologous expression, electrophysiology (voltage clamp), radiolabeled glutamate uptake\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro functional reconstitution in oocytes with electrophysiology, consistent with human EAAT4 data\",\n      \"pmids\": [\"9838098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"EAAT4 is enriched in parasagittal zones of Purkinje cell spines and thin dendrites, concentrated at perisynaptic membranes facing astroglia rather than at the synapse proper; cross-linking revealed EAAT4 forms dimers (not trimers as seen for GLAST and GLT).\",\n      \"method\": \"Immunohistochemistry, immunoblotting with chemical cross-linking (bis-sulfosuccinimidyl suberate)\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative immunogold EM with cross-linking biochemistry, replicated across independent labs\",\n      \"pmids\": [\"9570792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Two proteins, GTRAP41 and GTRAP48, specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity, identified by yeast two-hybrid screening and confirmed by biochemical characterization.\",\n      \"method\": \"Yeast two-hybrid screen, Co-IP/pulldown, functional transport assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid plus functional modulation data, published in Nature with characterization of both interactors\",\n      \"pmids\": [\"11242047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SGK1 (serum and glucocorticoid inducible kinase 1) and its isoforms SGK2, SGK3, and PKB stimulate EAAT4-mediated glutamate-induced current in Xenopus oocytes; the ubiquitin ligase Nedd4-2 decreases EAAT4 membrane abundance, an effect partially reversed by SGK isoforms. SGK1 enhances EAAT4 protein abundance in the cell membrane.\",\n      \"method\": \"Xenopus oocyte electrophysiology, immunohistochemistry, chemiluminescence membrane abundance assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional electrophysiology in oocytes combined with membrane abundance quantification, single lab\",\n      \"pmids\": [\"15504348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PKC activation by PMA specifically enhances the substrate-gated Cl- currents of EAAT4 (via non-conventional PKC isoforms) without affecting glutamate transport activity, demonstrating that the ion channel function and transport function of EAAT4 can be independently regulated.\",\n      \"method\": \"Xenopus oocyte electrophysiology, radiolabeled glutamate uptake, pharmacological inhibitors of PKC\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with orthogonal measurements of transport vs. channel activity, single lab\",\n      \"pmids\": [\"16601148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EAAT4 knockout mice show selective loss of Purkinje cells with low EAAT4 expression after cardiac arrest, demonstrating that EAAT4 protects Purkinje cells from excitotoxic damage after ischemia and acts in concert with the glial transporter GLAST.\",\n      \"method\": \"Genetic knockout mouse model, cardiac arrest ischemia model, histological Purkinje cell counting\",\n      \"journal\": \"Neuroscience research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined cellular phenotype and genetic epistasis showing GLAST/EAAT4 interaction, in vivo model\",\n      \"pmids\": [\"16647773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SGK1 modulates EAAT4 function and cell surface expression via phosphorylation at Thr40 (SGK1 consensus site at the N-terminus); disruption of Thr40 (T40A mutation) or both sites (T40A/T504A) abrogated SGK1-dependent enhancement of glutamate uptake and membrane abundance. SGK1 acts partly by inhibiting the ubiquitin ligase Nedd4-2, which itself reduces EAAT4 activity.\",\n      \"method\": \"Xenopus oocyte expression, site-directed mutagenesis, radiolabeled glutamate uptake, chemiluminescence membrane abundance, RNA interference\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of phosphorylation sites combined with functional assay and membrane abundance, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"17442044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PIKfyve (PIP5K3) participates in SGK1-dependent regulation of EAAT4; co-expression of PIKfyve and SGK1 synergistically enhances EAAT4-mediated glutamate-induced current, and an SGK1-phosphorylation site mutant of PIKfyve (S318A) abolishes this effect.\",\n      \"method\": \"Xenopus oocyte electrophysiology, dominant-negative and phosphorylation-site mutants\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional oocyte assay with mutants establishing pathway, single lab\",\n      \"pmids\": [\"20110679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"AMPK (AMP-activated protein kinase) down-regulates EAAT4 transport activity by reducing membrane abundance of the transporter, without affecting substrate affinity (Km unchanged); constitutively active AMPK decreased maximal glutamate-induced current by ~49%.\",\n      \"method\": \"Xenopus oocyte electrophysiology, confocal microscopy, chemiluminescence membrane abundance\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional assay with constitutively active and inactive AMPK constructs, single lab\",\n      \"pmids\": [\"20218975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"A conserved aspartate residue (Asp117) in EAAT4 determines pore properties including unitary conductance, anion permeability selectivity, and voltage- and substrate-dependent gating of the associated anion channel; D117A abolishes glutamate- and anion-dependent gating of EAAT4 anion currents.\",\n      \"method\": \"Heterologous expression in mammalian cells, whole-cell patch clamp, noise analysis, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structure-function mutagenesis with electrophysiology and noise analysis in mammalian cells, single lab with multiple rigorous methods\",\n      \"pmids\": [\"20519505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EAAT4 in Purkinje cells controls intersynaptic diffusion of climbing fiber transmitter (glutamate); pharmacological blockade of EAAT4 enhanced climbing fiber-induced inhibition of GABA release at basket cell-Purkinje cell synapses; tetanic CF stimulation induced long-term potentiation of glutamate transporter activity that attenuated this spillover. EAAT4 expression level inversely correlates with the degree of CF-mediated inhibition of GABAergic transmission across cerebellar lobules.\",\n      \"method\": \"Electrophysiology in cerebellar slices, pharmacological EAAT4 blockade, immunohistochemistry\",\n      \"journal\": \"The European journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — slice electrophysiology with pharmacological dissection plus immunohistochemistry, correlation across regions, use-dependent plasticity demonstrated\",\n      \"pmids\": [\"21070388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Klotho (as a β-glucuronidase) upregulates EAAT4 transport activity and membrane protein abundance in Xenopus oocytes; the effect is mimicked by recombinant β-Klotho protein and abolished by the β-glucuronidase inhibitor DSAL.\",\n      \"method\": \"Xenopus oocyte electrophysiology, confocal microscopy, chemiluminescence, recombinant protein treatment\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional oocyte assay with recombinant protein and inhibitor, single lab\",\n      \"pmids\": [\"23923038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"GSK3β up-regulates EAAT4 transport activity in Xenopus oocytes; coexpression of wild-type but not kinase-dead (K85A) GSK3β significantly increases maximal EAAT4-mediated glutamate-induced current without affecting substrate affinity. This effect is blocked by lithium.\",\n      \"method\": \"Xenopus oocyte electrophysiology, kinase-dead mutant, lithium treatment\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional oocyte assay with kinase-dead control and pharmacological inhibition, single lab\",\n      \"pmids\": [\"27978527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Caveolin-1 negatively regulates EAAT4 transport activity by decreasing the maximal transport rate without accelerating transporter retrieval from the cell membrane, as shown in Xenopus oocytes.\",\n      \"method\": \"Xenopus oocyte electrophysiology, brefeldin A treatment\",\n      \"journal\": \"The Journal of membrane biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — functional oocyte assay, single method, single lab\",\n      \"pmids\": [\"26690923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Conditional knockout of Rheb1 (Ras homolog enriched in brain) in cerebellar Purkinje cells leads to downregulation of EAAT4 expression and reduced EAAT4-mediated currents, placing Rheb1/mTOR signaling upstream of EAAT4 expression in Purkinje cells.\",\n      \"method\": \"Conditional knockout mice, immunohistochemistry, electrophysiology (EAAT4 and AMPA receptor currents)\",\n      \"journal\": \"Cerebellum\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with functional current measurements and immunohistochemistry, single lab\",\n      \"pmids\": [\"26194056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EAAT4 limits mGluR1 signaling in zebrin-positive (high EAAT4) Purkinje cells to constrain heterogeneous spontaneous firing; mGluR1 antagonists restore regular spontaneous firing and motor behavior in EAAT4 knockout mice. In contrast, loss of GLAST/EAAT1 disrupts zebrin-negative (low EAAT4) Purkinje cells via NMDA receptor overactivation. Genetic epistasis places EAAT4 upstream of mGluR1 in regulating Purkinje cell firing.\",\n      \"method\": \"EAAT4 knockout mice, electrophysiology (spontaneous firing), pharmacological rescue with mGluR1 and NMDA receptor antagonists, behavioral assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with pharmacological epistasis, electrophysiology, and behavioral rescue, multiple orthogonal methods\",\n      \"pmids\": [\"29741614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Patterned EAAT4 expression in Purkinje cells regulates glutamate spillover from climbing fibers to molecular layer interneurons; regions with high EAAT4 show smaller spillover EPSCs than regions with low EAAT4. Inhibiting glutamate transport normalizes the regional difference, demonstrating EAAT4 as a primary determinant of differential spillover.\",\n      \"method\": \"Electrophysiology in cerebellar slices from Aldolase C-Venus knock-in mice, pharmacological transporter blockade, postsynaptic receptor analysis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — slice electrophysiology with genetic visualization of microzones plus pharmacological rescue, multiple controls\",\n      \"pmids\": [\"34400517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Ethanol at pharmacologically relevant concentrations (25–100 mM) increases glutamate uptake via EAAT4 in Purkinje cells, operating in concert with Na,K-ATPase, thereby restricting glutamate diffusion from climbing fiber synaptic clefts and suppressing intersynaptic modulation of GABAergic interneurons.\",\n      \"method\": \"Electrophysiology in rat cerebellar slices, pharmacological manipulation of EAAT4\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — slice electrophysiology with pharmacological dissection, single lab, single paper\",\n      \"pmids\": [\"41946908\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EAAT4 (SLC1A6) is a high-affinity Na+-dependent glutamate transporter expressed predominantly in cerebellar Purkinje cells, where it localizes to extra-junctional perisynaptic membranes of dendritic spines; it functions both as an electrogenic glutamate transporter and a substrate-gated chloride channel (with Asp117 as a key pore-forming residue), limits mGluR1 signaling to regulate spontaneous firing in zebrin-positive Purkinje cells, controls glutamate spillover to molecular layer interneurons in a regionally patterned manner, and is regulated by multiple kinases (SGK1 via Thr40 phosphorylation and Nedd4-2 ubiquitination, AMPK, PKC, PIKfyve, GSK3β, Klotho, and caveolin-1) that primarily alter its membrane abundance; its carboxy-terminal domain interacts with GTRAP41 and GTRAP48 to modulate activity, and its loss causes excitotoxic Purkinje cell death and cerebellar ataxia.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SLC1A6 (EAAT4) is a high-affinity, Na+-dependent glutamate transporter expressed predominantly in cerebellar Purkinje cells, where it shapes synaptic glutamate signaling and protects neurons from excitotoxicity [#0, #8]. It localizes to extra-junctional, perisynaptic membranes of dendritic spines and distal dendrites facing astroglia, positioning it to clear glutamate spilling out of the synaptic cleft, and it is distributed in parasagittal microzones across the cerebellum [#2, #4]. Functionally, EAAT4 couples electrogenic glutamate transport to a substrate-gated chloride channel, with a conserved aspartate (Asp117) governing the anion pore's conductance, selectivity, and substrate- and voltage-dependent gating [#3, #12]; transport and channel functions are separable, as PKC activation selectively enhances the chloride current without altering uptake [#7]. Through this perisynaptic uptake, EAAT4 controls intersynaptic diffusion of climbing-fiber glutamate to molecular layer interneurons and limits mGluR1 signaling in zebrin/aldolase-C-positive Purkinje cells, thereby constraining spontaneous firing in a regionally patterned manner [#13, #18, #19]. Its surface abundance and activity are set by a network of regulators acting largely on membrane trafficking: SGK1 phosphorylates Thr40 to oppose Nedd4-2-mediated ubiquitination and downregulation [#6, #9], while its carboxy-terminal domain binds GTRAP41 and GTRAP48 to modulate transport [#5]. Loss of EAAT4 causes excitotoxic Purkinje cell death and motor deficits, the latter reversible by mGluR1 antagonism [#8, #18].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Establishing where EAAT4 acts was the first step: it was identified as a postsynaptic glutamate transporter concentrated in Purkinje cell dendritic spines, distinguishing it from glial transporters.\",\n      \"evidence\": \"Peptide-antibody immunohistochemistry, immunoblotting, and electron microscopy in cerebellum\",\n      \"pmids\": [\"8905715\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define transport kinetics or ion coupling\", \"No data on synaptic vs extra-synaptic position\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Refining localization showed EAAT4 sits at extra-junctional/perisynaptic membranes in parasagittal compartments, implying it handles glutamate diffusing out of the cleft rather than direct synaptic clearance.\",\n      \"evidence\": \"Site-directed antisera immunohistochemistry and silver-enhanced immunogold EM\",\n      \"pmids\": [\"9174061\", \"9261809\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of perisynaptic placement not yet measured electrophysiologically\", \"Oligomeric state and partners unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Heterologous reconstitution established the dual nature of EAAT4 as a Na+-dependent transporter whose currents are dominated by a ligand-gated chloride conductance, and biochemistry showed it forms dimers unlike trimeric glial transporters.\",\n      \"evidence\": \"Xenopus oocyte voltage clamp, radiolabeled glutamate uptake, and chemical cross-linking with quantitative immunogold EM\",\n      \"pmids\": [\"9838098\", \"9570792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Pore residues controlling the anion channel not yet mapped\", \"Physiological role of the chloride current in Purkinje cells untested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identifying GTRAP41 and GTRAP48 as C-terminal interactors gave the first protein partners that tune EAAT4 transport activity.\",\n      \"evidence\": \"Yeast two-hybrid screen, co-IP/pulldown, and functional transport assays\",\n      \"pmids\": [\"11242047\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of C-terminal binding not resolved\", \"In vivo relevance in Purkinje cells not demonstrated\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The first kinase/ubiquitin axis was defined, showing EAAT4 surface abundance is set by Nedd4-2-mediated downregulation that SGK isoforms and PKB reverse.\",\n      \"evidence\": \"Xenopus oocyte electrophysiology with chemiluminescence membrane abundance assays\",\n      \"pmids\": [\"15504348\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphorylation site mediating SGK1 action not yet identified\", \"Performed in oocytes, not Purkinje cells\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Genetic loss-of-function in mice tied EAAT4 to neuroprotection, showing it shields Purkinje cells from ischemic excitotoxicity in concert with glial GLAST.\",\n      \"evidence\": \"EAAT4 knockout mice subjected to cardiac-arrest ischemia with histological Purkinje cell counting\",\n      \"pmids\": [\"16647773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address basal firing or synaptic signaling roles\", \"Mechanism of GLAST cooperation not molecularly defined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Transport and channel functions were shown to be independently regulable, as PKC selectively boosts the chloride current without changing uptake.\",\n      \"evidence\": \"Oocyte electrophysiology and radiolabeled uptake with PKC inhibitors/activators\",\n      \"pmids\": [\"16601148\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PKC phosphorylation site on EAAT4 not mapped\", \"Physiological setting of channel-specific modulation unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"The SGK1 mechanism was pinned to Thr40 phosphorylation that opposes Nedd4-2, converting the earlier observation into a defined regulatory site.\",\n      \"evidence\": \"Oocyte expression with T40A/T504A site-directed mutagenesis, uptake, membrane abundance, and RNAi\",\n      \"pmids\": [\"17442044\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo phosphorylation in Purkinje cells not confirmed\", \"Upstream signals activating SGK1 toward EAAT4 unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The structural determinant of the anion channel was localized to Asp117, explaining how a single residue sets conductance, anion selectivity, and gating.\",\n      \"evidence\": \"Mammalian-cell whole-cell patch clamp with noise analysis and D117A mutagenesis\",\n      \"pmids\": [\"20519505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No atomic structure of the EAAT4 pore\", \"Relationship of Asp117 to the glutamate transport pathway not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Additional trafficking regulators were placed in the network — PIKfyve cooperates with SGK1 to enhance EAAT4, while AMPK downregulates it by reducing surface abundance without altering Km.\",\n      \"evidence\": \"Oocyte electrophysiology with phospho-site and constitutively active/inactive kinase mutants and confocal microscopy\",\n      \"pmids\": [\"20110679\", \"20218975\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effects shown only in oocytes\", \"Endogenous relevance to Purkinje cell physiology untested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Slice physiology connected EAAT4 to circuit function, showing it limits climbing-fiber glutamate spillover onto basket-cell synapses and undergoes use-dependent potentiation, with expression inversely tracking spillover-mediated GABA inhibition across lobules.\",\n      \"evidence\": \"Cerebellar slice electrophysiology with pharmacological EAAT4 blockade and immunohistochemistry\",\n      \"pmids\": [\"21070388\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of transporter potentiation not defined\", \"Did not test downstream behavior\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Klotho's β-glucuronidase activity was shown to upregulate EAAT4 surface abundance, extending the regulatory repertoire to an extracellular enzyme.\",\n      \"evidence\": \"Oocyte electrophysiology with recombinant β-Klotho and the β-glucuronidase inhibitor DSAL\",\n      \"pmids\": [\"23923038\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking glucuronidase activity to EAAT4 trafficking unknown\", \"Oocyte-only system\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Two further modulators were defined: GSK3β increases maximal transport (lithium-blockable) and caveolin-1 decreases maximal transport rate without accelerating retrieval, and Rheb1/mTOR was placed upstream of EAAT4 expression in vivo.\",\n      \"evidence\": \"Oocyte electrophysiology with kinase-dead and pharmacological controls; conditional Rheb1 knockout mice with immunohistochemistry and current recordings\",\n      \"pmids\": [\"27978527\", \"26690923\", \"26194056\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Caveolin-1 and GSK3β actions shown only in oocytes\", \"How Rheb1/mTOR controls EAAT4 transcription/translation not detailed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Genetic epistasis revealed the circuit logic of EAAT4: it limits mGluR1 signaling in zebrin-positive Purkinje cells to maintain regular firing and motor behavior, with mGluR1 antagonism rescuing knockout deficits.\",\n      \"evidence\": \"EAAT4 knockout mice with spontaneous-firing electrophysiology, pharmacological mGluR1/NMDA rescue, and behavioral assays\",\n      \"pmids\": [\"29741614\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How perisynaptic uptake mechanistically gates mGluR1 not fully resolved\", \"Human disease link not directly established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Patterned EAAT4 expression was shown to be the primary determinant of regional differences in climbing-fiber glutamate spillover to molecular layer interneurons.\",\n      \"evidence\": \"Cerebellar slice electrophysiology in aldolase-C-Venus knock-in mice with pharmacological transporter blockade\",\n      \"pmids\": [\"34400517\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of differential spillover for cerebellar output not defined\", \"Did not test behavioral readouts of microzonal spillover\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"A modulatory role for ethanol was added, showing it enhances EAAT4-mediated uptake in concert with Na,K-ATPase to restrict climbing-fiber glutamate diffusion and suppress GABAergic interneuron modulation.\",\n      \"evidence\": \"Rat cerebellar slice electrophysiology with pharmacological EAAT4 manipulation\",\n      \"pmids\": [\"41946908\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of ethanol action on EAAT4 unknown\", \"Single lab, single study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many oocyte-defined regulators integrate in vivo to set EAAT4 surface levels, and whether SLC1A6 variants cause human cerebellar ataxia, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No atomic structure of human EAAT4\", \"Most kinase/trafficking regulation shown only in heterologous systems\", \"No timeline evidence of a causative human Mendelian variant\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [3, 9, 11]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 3, 12]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [3, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 2, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [13, 18, 19]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [3, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"GTRAP41\", \"GTRAP48\", \"NEDD4L\", \"SGK1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}