A Trimeric Quaternary Structure Is Conserved in Bacterial and Human Glutamate Transporters

Gendreau, Sandra; Voswinkel, Stephan; Torres-Salazar, Delany; Lang, Niklas; Heidtmann, H.; Detro‐Dassen, Silvia; Schmalzing, Günther; Hidalgo, Patricia; Fahlke, Christoph

doi:10.1074/jbc.m408038200

Cited by 113 publications

(102 citation statements)

References 47 publications

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“…For example, biochemical studies on human EAAT2 showed that the transporter exists as a trimer (8); cross-linking of pairs of cysteines introduced at the tips of HP1 and HP2 loops, on HP1 tip and TM7, and on HP2 tip and TM7 suggested that these regions are located within close proximity (9,10), in accord with their spatial distances observed in the resolved Glt Ph structure. Additionally, residues in the mammalian EAATs equivalent to the Glt Ph residues Asp-394 and Arg-397, the serine-rich motif (Ser-277-Ser-279), and the conserved NMDGT motif have been proposed to be involved in substrate binding (11)(12)(13)(14), consistent with their interaction with the bound aspartate observed in the high resolution structure of Glt Ph .…”

mentioning

confidence: 51%

Time-resolved Mechanism of Extracellular Gate Opening and Substrate Binding in a Glutamate Transporter

Shrivastava

Jiang

Amara

et al. 2008

Journal of Biological Chemistry

121

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Glutamate transporters, also referred to as excitatory amino acid transporters (EAATs), are membrane proteins that regulate glutamatergic signal transmission by clearing excess glutamate after its release at synapses. A structure-based understanding of their molecular mechanisms of function has been elusive until the recent determination of the x-ray structure of an archaeal transporter, Glt Ph . Glt Ph exists as a trimer, with each subunit containing a core region that mediates substrate translocation. In the present study a series of molecular dynamics simulations have been conducted and analyzed in light of new experimental data on substrate binding properties of EAATs. The simulations provide for the first time a full atomic description of the time-resolved events that drive the recognition and binding of substrate. The core region of each subunit exhibits an intrinsic tendency to open the helical hairpin HP2 loop, the extracellular gate, within tens of nanoseconds exposing conserved polar residues that serve as attractors for substrate binding. The NMDGT motif on the partially unwound part of the transmembrane helix TM7 and the residues Asp-390 and Asp-394 on TM8 are also distinguished by their important role in substrate binding and close interaction with mediating water molecules and/or sodium ions. The simulations reveal a Na ؉ binding site comprised in part of Leu-303 on TM7 and Asp-405 on TM8 and support a role for sodium ions in stabilizing substrate-bound conformers. The functional importance of Leu-303 or its counterpart Leu-391 in human EAAT1 (hEAAT1) is confirmed by site-directed mutagenesis and Na ؉ dependence assays conducted with hEAAT1 mutants L391C and L391A.

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mentioning

confidence: 51%

Time-resolved Mechanism of Extracellular Gate Opening and Substrate Binding in a Glutamate Transporter

Shrivastava

Jiang

Amara

et al. 2008

Journal of Biological Chemistry

121

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“…These data, combined with biochemical evidence, also predict that the functional glutamate transporter has a homotrimeric quaternary structure, which is conserved in bacterial and human transporters (53,156). Glutamate transporters account for the bulk transport of glutamate across the plasma membrane of cells and act quickly to buffer synaptically released glutamate (140).…”

Section: Foran and Trottimentioning

confidence: 95%

Glutamate Transporters and the Excitotoxic Path to Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis

Foran

Trotti²

2009

Antioxidants & Redox Signaling

247

141

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Responsible for the majority of excitatory activity in the central nervous system (CNS), glutamate interacts with a range of specific receptor and transporter systems to establish a functional synapse. Excessive stimulation of glutamate receptors causes excitotoxicity, a phenomenon implicated in both acute and chronic neurodegenerative diseases [e.g., ischemia, Huntington's disease, and amyotrophic lateral sclerosis (ALS)]. In physiology, excitotoxicity is prevented by rapid binding and clearance of synaptic released glutamate by high-affinity, Na þ -dependent glutamate transporters and amplified by defects to the glutamate transporter and receptor systems. ALS pathogenetic mechanisms are not completely understood and characterized, but excitotoxicity has been regarded as one firm mechanism implicated in the disease because of data obtained from ALS patients and animal and cellular models as well as inferred by the documented efficacy of riluzole, a generic antiglutamatergic drug, has in patients. In this article, we critically review the several lines of evidence supporting a role for glutamate-mediated excitotoxicity in the death of motor neurons occurring in ALS, putting a particular emphasis on the impairment of the glutamate-transport system. Antioxid. Redox Signal. 11, 1587-1602. Glutamate in the Central Nervous SystemL -Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS). A nonessential amino acid, glutamate is continuously converted to a-ketoglutarate through deamination by glutamate dehydrogenase or by transamination by one of the transaminases and metabolized through the tricarboxylic acid cycle to succinate, fumarate, and malate, successively. Glutamate is also the product of the deamination of glutamine by phosphateactivated glutaminase, a mitochondrial and possibly neuronspecific enzyme (80). Synaptically released glutamate activates a family of ligand-gated ion channels (ionotropic receptors) and G protein-coupled receptors (metabotropic receptors), and its action is terminated by specific reuptake systems located mainly in astrocytes surrounding the synapse. In astrocytes, glutamate is then converted into glutamine, which does not have neurotransmitter properties and can be released and made available for neurons to convert it back to glutamate through a glutamine-reuptake system. Glutamate is then packed by vesicular glutamate transporters in synaptic vesicles, ready to be released again (35,129) (Fig. 1).

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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%

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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A Trimeric Quaternary Structure Is Conserved in Bacterial and Human Glutamate Transporters

Cited by 113 publications

References 47 publications

Time-resolved Mechanism of Extracellular Gate Opening and Substrate Binding in a Glutamate Transporter

Time-resolved Mechanism of Extracellular Gate Opening and Substrate Binding in a Glutamate Transporter

Glutamate Transporters and the Excitotoxic Path to Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis

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

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