Mutating a Conserved Proline Residue within the Trimerization Domain Modifies Na+ Binding to Excitatory Amino Acid Transporters and Associated Conformational Changes

Hotzy, Jasmin; Schneider, Nicole; Kovermann, Peter; Fahlke, Christoph

doi:10.1074/jbc.m113.489385

Cited by 22 publications

(28 citation statements)

References 29 publications

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“…He carries a heterozygous SLC1A3 missense mutation (RefSeq NM_001166695.1: c.869C>G: p.Pro290Arg) predicting a proline-to-arginine replacement in transmembrane domain 5 (p.P290R) of the human EAAT1 (Jen et al, 2005). EAATs function as both secondary-active glutamate transporters and anion channels (Fahlke, Kortzak, & Machtens, 2016), and we demonstrated that the P290R substitution impairs glutamate transport, but causes gain-offunction of EAAT1 anion channels (Hotzy, Schneider, Kovermann, & Fahlke, 2013;Winter, Kovermann, & Fahlke, 2012). To understand how increased EAAT1 anion currents cause ataxia in EA6 we generated a transgenic mouse model-the Slc1a3 P290R/+ mouse-and demonstrated that excessive chloride efflux in the second postnatal week results in Bergmann glia apoptosis and cerebellar atrophy (Kovermann et al, 2020).…”

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confidence: 75%

Functional consequences of SLC1A3 mutations associated with episodic ataxia 6

et al. 2020

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The episodic ataxias (EA) are a group of inherited neurological diseases characterized by paroxysmal cerebellar incoordination. There exist nine forms of episodic ataxia with distinct neurological symptoms and genetic origins. Episodic ataxia type 6 (EA6) differs from other EA forms in long attack duration, epilepsy and absent myokymia, nystagmus, and tinnitus. It has been described in seven families, and mutations in SLC1A3, the gene encoding the glial glutamate transporter EAAT1, were reported in each family. How these mutations affect EAAT1 expression, subcellular localization, and function, and how such alterations result in the complex neurological phenotype of EA6 is insufficiently understood. We here compare the functional consequences of all currently known mutations by heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch clamp recordings of EAAT1 transport and anion currents. We observed impairments of multiple EAAT1 properties ranging from changes in transport function, impaired trafficking to increased protein expression. Many mutations caused only slight changes illustrating how sensitively the cerebellum reacts on impaired EAAT1 functions.

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Functional consequences of SLC1A3 mutations associated with episodic ataxia 6

et al. 2020

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“…Our clinical and functional data re-inforce the concept that disturbed glutamate homeostasis from genetic variations in SLC1A3 could lead to a broad spectrum of neurological manifestations with overlapping features. The first SLC1A3 mutation (P290R) 8 , arose de novo in a single patient with episodic ataxia, hemiplegia with migraine, and epilepsy, impairs glutamate transport and enhances h EAAT1 anion currents 27 , 32 , likely reducing glial intracellular [Cl − ] 33 , 34 . Another SLC1A3 mutation, found in multiple members of a family with episodic ataxia (C186S), was reported to slightly reduce glutamate uptake levels 9 and to modify intracellular transport of EAAT1 35 , while h EAAT1 anion currents were not studied.…”

Section: Discussionmentioning

confidence: 99%

Impaired K+ binding to glial glutamate transporter EAAT1 in migraine

Kovermann

Hessel

Kortzak

et al. 2017

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SLC1A3 encodes the glial glutamate transporter hEAAT1, which removes glutamate from the synaptic cleft via stoichiometrically coupled Na+-K+-H+-glutamate transport. In a young man with migraine with aura including hemiplegia, we identified a novel SLC1A3 mutation that predicts the substitution of a conserved threonine by proline at position 387 (T387P) in hEAAT1. To evaluate the functional effects of the novel variant, we expressed the wildtype or mutant hEAAT1 in mammalian cells and performed whole-cell patch clamp, fast substrate application, and biochemical analyses. T387P diminishes hEAAT1 glutamate uptake rates and reduces the number of hEAAT1 in the surface membrane. Whereas hEAAT1 anion currents display normal ligand and voltage dependence in cells internally dialyzed with Na+-based solution, no anion currents were observed with internal K+. Fast substrate application demonstrated that T387P abolishes K+-bound retranslocation. Our finding expands the phenotypic spectrum of genetic variation in SLC1A3 and highlights impaired K+ binding to hEAAT1 as a novel mechanism of glutamate transport dysfunction in human disease.

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“…Bipolar cells exhibit a somatodendritic chloride gradient that might serve inversion of GABAergic horizontal cell input (44). We recently reported that a naturally occurring mutation found in a patient with episodic ataxia type 6 modifies the gating of EAAT1 (38,45), resulting in a profound inward-rectification, and we postulated that P290R EAAT1 might affect intracellular anion concentrations in Bergmann glia. The gating of EAAT5 closely resembles gating of P290R EAAT1 anion channels, and it is thus tempting to speculate that EAAT5 might fulfill a similar function in retinal neurons.…”

Section: Discussionmentioning

confidence: 99%