Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters

Silverstein, Nechama; Ewers, David; Forrest, Lucy R.; Fahlke, Christoph; Kanner, Baruch I.

doi:10.1074/jbc.m115.682666

Cited by 9 publications

(14 citation statements)

References 42 publications

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“…3) using the γ-protons (very similar results of 458 μM and 984 μM respectively were obtained if the β-protons were used). These values are higher than those previously observed in detergent micelles (between 120-250 μM) 10,16 , however it must be noted that the sodium ion concentration used here is half of that used in those previous studies. It is also known that due to fast protein-ligand rebinding in solution, K D values obtained from STD NMR are always greater than, or equal to, the true thermodynamic value 38 .…”

Section: Resultscontrasting

confidence: 69%

“…This difficulty extends to include medium to high throughput determination of substrate and inhibitor binding affinities, essential for drug target screening. Until now methods such as fluorescence-based assays 10,15,16 , isothermal titration calorimetry 12,15,17 and uptake experiments 18 have been utilised in the determination of substrate K M and K D values in both detergent-solubilised and membrane-reconstituted protein environments. Such methods have yielded results that vary considerably with Na + concentration, clearly highlighting GltPh's sodium dependency for substrate binding.…”

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confidence: 99%

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Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding

Hall

Sohail

Cabrita

et al. 2020

Sci Rep

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Saturation-transfer difference (STD) NMR spectroscopy is a fast and versatile method which can be applied for drug-screening purposes, allowing the determination of essential ligand binding affinities (KD). Although widely employed to study soluble proteins, its use remains negligible for membrane proteins. Here the use of STD NMR for KD determination is demonstrated for two competing substrates with very different binding affinities (low nanomolar to millimolar) for an integral membrane transport protein in both detergent-solubilised micelles and reconstituted proteoliposomes. GltPh, a homotrimeric aspartate transporter from Pyrococcus horikoshii, is an archaeal homolog of mammalian membrane transport proteins—known as excitatory amino acid transporters (EAATs). They are found within the central nervous system and are responsible for fast uptake of the neurotransmitter glutamate, essential for neuronal function. Differences in both KD’s and cooperativity are observed between detergent micelles and proteoliposomes, the physiological implications of which are discussed.

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Section: Resultscontrasting

confidence: 69%

mentioning

confidence: 99%

Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding

Hall

Sohail

Cabrita

et al. 2020

Sci Rep

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“…The transporters are actually complex molecules as they also function as chloride channels (Fairman et al, 1995;Kavanaugh, 1996a, Wadiche et al, 1995a;Wadiche et al, 1995b;Wadiche and Kavanaugh, 1998;Bergles et al, 2002;Koch et al, 2007;Ryan and Mindell, 2007;Zhou et al, 2014a;Fahlke et al, 2016;LeVine et al, 2016) and even transport water (MacAulay et al, 2001, MacAulay et al, 2004. Although the mammalian transporters have not yet been crystallized, we know quite a lot about their structure (Kanner, 2007;Vandenberg and Ryan, 2013;Gouaux, 2009;Verdon et al, 2014;Silverstein et al, 2015;Fahlke et al, 2016;LeVine et al, 2016).…”

Section: Identification Of Plasma Membrane Glutamate Transportersmentioning

confidence: 99%

Neuronal vs glial glutamate uptake: Resolving the conundrum

Danbolt

Furness

Zhou

2016

Neurochemistry International

186

152

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Neither normal brain function nor the pathological processes involved in neurological diseases can be adequately understood without knowledge of the release, uptake and metabolism of glutamate. The reason for this is that glutamate (a) is the most abundant amino acid in the brain, (b) is at the cross-roads between several metabolic pathways, and (c) serves as the major excitatory neurotransmitter. In fact most brain cells express glutamate receptors and are thereby influenced by extracellular glutamate. In agreement, brain cells have powerful uptake systems that constantly remove glutamate from the extracellular fluid and thereby limit receptor activation. It has been clear since the 1970s that both astrocytes and neurons express glutamate transporters. However, the relative contribution of neuronal and glial transporters to the total glutamate uptake activity, however, as well as their functional importance, has been hotly debated ever since. The present short review provides (a) an overview of what we know about neuronal glutamate uptake as well as an historical description of how we got there, and (b) a hypothesis reconciling apparently contradicting observations thereby possibly resolving the paradox. ABBREVIATIONSCre, Cyclization recombinase (Le and Sauer, 2000); EAAC1, glutamate transporter (EAAT3; slc1a1; Kanai and Hediger, 1992; Bjørås et al., 1996); EAAT, excitatory amino acid transporter (synonym to glutamate transporter); GABA, γ-aminobutyric acid; GLAST, rat glutamate transporter (EAAT1; slc1a3; Storck et al., 1992;Tanaka, 1993a); GLT-1, rat glutamate transporter (EAAT2; slc1a2; Pines et al., 1992); GLUL, glutamine synthetase; TBOA, DL-threo-β-benzyloxyaspartate AbstractNeither normal brain function nor the pathological processes involved in neurological diseases can be adequately understood without knowledge of the release, uptake and metabolism of glutamate. The reason for this is that glutamate (a) is the most abundant amino acid in the brain, (b) is at the cross-roads between several metabolic pathways, and (c) serves as the major excitatory neurotransmitter. In fact most brain cells express glutamate receptors and are thereby influenced by extracellular glutamate. In agreement, brain cells have powerful uptake systems that constantly remove glutamate from the extracellular fluid and thereby limit receptor activation. It has been clear since the 1970s that astrocytes and neurons express glutamate transporters. The relative contribution of neuronal and glial transporters to the total glutamate uptake activity, however, as well as their functional importance, has been hotly debated ever since. The present short review provides (a) an overview of what we know about neuronal glutamate uptake as well as an historical description of how we got there, and (b) an hypothesis reconciling apparently contradicting observations thereby possibly resolving the paradox.

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“…Glt Ph -WT, Glt Ph -G354S, and Glt Ph -G354L were expressed, purified (45), and reconstitution using Sephadex G50 spin columns and radioactive uptake were done as described (7,38).…”

Section: Mutagenesis Expression Purification Reconstitution and Ramentioning

confidence: 99%

Both reentrant loops of the sodium-coupled glutamate transporters contain molecular determinants of cation selectivity

Silverstein

Sliman

Stockner

et al. 2018

Journal of Biological Chemistry

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In the brain, glutamate transporters terminate excitatory neurotransmission by removing this neurotransmitter from the synapse via cotransport with three sodium ions into the surrounding cells. Structural studies have identified the binding sites of the three sodium ions in glutamate transporters. The residue side-chains directly interact with the sodium ions at the Na1 and Na3 sites and are fully conserved from archaeal to eukaryotic glutamate transporters. The Na2 site is formed by three main-chain oxygens on the extracellular reentrant hairpin loop HP2 and one on transmembrane helix 7. A glycine residue on HP2 is located closely to the three main-chain oxygens in all glutamate transporters, except for the astroglial transporter GLT-1, which has a serine residue at that position. Unlike for WT GLT-1, substitution of the serine residue to glycine enables sustained glutamate transport also when sodium is replaced by lithium. Here, using functional and simulation studies, we studied the role of this serine/glycine switch on cation selectivity of substrate transport. Our results indicate that the side-chain oxygen of the serine residues can form a hydrogen bond with a main-chain oxygen on transmembrane helix 7. This leads to an expansion of the Na2 site such that water can participate in sodium coordination at Na2. Furthermore, we found other molecular determinants of cation selectivity on the nearby HP1 loop. We conclude that subtle changes in the composition of the two reentrant hairpin loops determine the cation specificity of acidic amino acid transport by glutamate transporters.

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Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters

Cited by 9 publications

References 42 publications

Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding

Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding

Neuronal vs glial glutamate uptake: Resolving the conundrum

Both reentrant loops of the sodium-coupled glutamate transporters contain molecular determinants of cation selectivity

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