Structure of a glutamate transporter homologue from Pyrococcus horikoshii

Yernool, Dinesh; Boudker, Olga; Jin, Yan; Gouaux, Eric

doi:10.1038/nature03018

Cited by 769 publications

(1,223 citation statements)

References 48 publications

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“…[ Figure (Haugeto et al, 1996;Yernool et al, 2004;Gendreau et al, 2004). When fresh brain tissue is rapidly homogenized directly in sodium dodecyl sulfate (SDS), only monomers are seen on the Western blots regardless of whether reducing agents have been added (Fig.…”

Section: Sample Quality -Proteolysis and True Oligomers Versus In Vitmentioning

confidence: 99%

“…This means that the diameter of a red blood cell is about 9 times larger than the scale bar shown in figure 8. A plasma membrane is about 5 nm thick and the width of the extracellular space (the distances between neighboring cellular extensions) is typically in the range 20-40 nm, while the diameter of a glutamate transporter trimer is believed to be about 8 nm (Yernool et al, 2004). Cellular elements are tightly intermingled (Kirov et al, 1999;Sorra and Harris, 2000;Witcher et al, 2010;Harris and Weinberg, 2012;Mathiisen et al, 2010).…”

Section: Correlation Between Labeling Intensity In Tissue Sections Anmentioning

confidence: 99%

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Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Danbolt

Zhou

Furness

et al. 2016

Glia

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Immunocytochemistry and Western blotting are still major methods for protein localization, but they rely on the specificity of the antibodies. Validation of antibody specificity remains challenging mostly because ideal negative controls are often unavailable. Further, immunochemical labeling patterns are also influenced by a number of other factors such as postmortem changes, fixation procedures and blocking agents as well as the general assay conditions (e.g., buffers, temperature, etc.). Western blotting similarly depends on tissue collection and sample preparation as well as the electrophoretic separation, transfer to blotting membranes and the immunochemical probing of immobilized molecules. Publication of inaccurate information on protein distribution has downstream consequences for other researchers because the interpretation of physiological and pharmacological observations depends on information on where ion channels, receptors, enzymes or transporters are located. Despite numerous reports, some of which are strongly worded, erroneous localization data are being published. Here we describe the extent of the problem and illustrate the nature of the pitfalls with examples from studies of neurotransmitter transporters. We explain the importance of supplementing immunochemical observations with other measurements (e.g., mRNA levels and distribution, protein activity, mass spectrometry, electrophysiological recordings, etc.) and why quantitative considerations are integral parts of the quality control. Further, we propose a practical strategy for researchers who plan to embark on a localization study. We also share our thoughts about guidelines for quality control. GLIA 2016;64:2045–2064

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Section: Sample Quality -Proteolysis and True Oligomers Versus In Vitmentioning

confidence: 99%

Section: Correlation Between Labeling Intensity In Tissue Sections Anmentioning

confidence: 99%

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Danbolt

Zhou

Furness

et al. 2016

Glia

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“…Structural information is available for the glutamate transporter homolog Glt Ph from the archaeon Pyrococcus horikoshii, which is selective for aspartate rather than glutamate and which couples the uptake of substrate to the symport of three sodium ions [3][4][5][6][7][8] . The protein is a homotrimer, with each protomer consisting of two domains.…”

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confidence: 99%

“…3 Fig. 4a) 4,5 . The two helical hairpins (HP1 and HP2) in the transport domain, which may form intra-and extracellular gates, respectively, occlude the substrate-binding site in both Glt Ph and Glt Tk (Supplementary Fig.…”

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Crystal structure of a substrate-free aspartate transporter

Jensen

Guskov

Rempel

et al. 2013

Nat Struct Mol Biol

104

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Episodic Ataxia Associated With EAAT1 Mutation C186S Affecting Glutamate Reuptake

Vries¹,

Mamsa²,

Stam³

et al. 2009

Arch Neurol

131

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Background: Episodic ataxia (EA) is variably associated with additional neurologic symptoms. At least 4 genes have been implicated. Recently, a mutation in the SLC1A3 gene encoding the glutamate transporter EAAT1 was identified in a patient with severe episodic and progressive ataxia, seizures, alternating hemiplegia, and migraine headache. The mutant EAAT1 showed severely reduced uptake of glutamate. The syndrome was designated EA6 and shares overlapping clinical features with EA2, which is caused by mutations in CACNA1A. Objective: To test the role of the SLC1A3 gene in EA. Design: Genetic and functional studies. We analyzed the coding region of the SLC1A3 gene by direct sequencing. Setting: Academic research. Patients: DNA samples from 20 patients with EA (with or without interictal nystagmus) negative for CACNA1A mutations were analyzed. Main Outcome Measures: We identified 1 novel EAAT1 mutation in a family with EA and studied the functional consequences of this mutation using glutamate uptake assay. Results: We identified a missense C186S mutation that segregated with EA in 3 family members. The mutant EAAT1 showed a modest but significant reduction of glutamate uptake. Conclusions: We broadened the clinical spectrum associated with SLC1A3 mutations to include milder manifestations of EA without seizures or alternating hemiplegia. The severity of EA6 symptoms appears to be correlated with the extent of glutamate transporter dysfunction.

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Structure of a glutamate transporter homologue from Pyrococcus horikoshii

Cited by 769 publications

References 48 publications

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Crystal structure of a substrate-free aspartate transporter

Episodic Ataxia Associated With EAAT1 Mutation C186S Affecting Glutamate Reuptake

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