Transporters play a central role in synaptic transmission. They are responsible for removal of neurotransmitters from the synaptic cleft and their storage in synaptic vesicles. In this project we propose to obtain mechanistic information at the molecular level on 2 transporters. These are GLT-1, a plasma membrane (Na+K+)-coupled transporter of the neurotransmitter glutamate, and EmrE-a unique bacterial multidrug transporter that provides an experimental paradigm to study the vesicular H+-coupled neurotransmitter transporters.In recent years, the study of bacterial homologues has provided important information on the structure of ion channels and transporters. The availability of new high-resolution structures of transporters from several families mark a new and stimulating era in defining mechanisms in molecular detail. We will progress towards understanding mechanisms by using the available structural and biochemical information to (i) further explore the residues in the binding pocket and to modify specificities and affinities of various substrates; (ii) study the molecular determinants of ion binding and (iii) to explore the conformational transitions that occur upon ion and substrate binding. In addition to impacting on the central question of the structural basis of ion-coupled transporter function, our studies may provide important clues for the role of these transporters not only under normal physiological conditions, but also in disease.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Biophysics of Synapses, Channels, and Transporters Study Section (BSCT)
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Silberberg, Shai D
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Hebrew University of Jerusalem
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Silverstein, Nechama; Sliman, Alaa; Stockner, Thomas et al. (2018) Both reentrant loops of the sodium-coupled glutamate transporters contain molecular determinants of cation selectivity. J Biol Chem 293:14200-14209
Yaffe, Dana; Forrest, Lucy R; Schuldiner, Shimon (2018) The ins and outs of vesicular monoamine transporters. J Gen Physiol 150:671-682
Yaffe, Dana; Vergara-Jaque, Ariela; Forrest, Lucy R et al. (2016) Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis. Proc Natl Acad Sci U S A 113:E7390-E7398
Silverstein, Nechama; Ewers, David; Forrest, Lucy R et al. (2015) Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters. J Biol Chem 290:28988-96
Schuldiner, Shimon (2014) Competition as a way of life for H(+)-coupled antiporters. J Mol Biol 426:2539-46
Shabaneh, Mustafa; Rosental, Noa; Kanner, Baruch I (2014) Disulfide cross-linking of transport and trimerization domains of a neuronal glutamate transporter restricts the role of the substrate to the gating of the anion conductance. J Biol Chem 289:11175-82
Yaffe, Dana; Vergara-Jaque, Ariela; Shuster, Yonatan et al. (2014) Functionally important carboxyls in a bacterial homologue of the vesicular monoamine transporter (VMAT). J Biol Chem 289:34229-40
Yaffe, Dana; Radestock, Sebastian; Shuster, Yonatan et al. (2013) Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2. Proc Natl Acad Sci U S A 110:E1332-41
Ugolev, Yelena; Segal, Tali; Yaffe, Dana et al. (2013) Identification of conformationally sensitive residues essential for inhibition of vesicular monoamine transport by the noncompetitive inhibitor tetrabenazine. J Biol Chem 288:32160-71
Silverstein, Nechama; Crisman, Thomas J; Forrest, Lucy R et al. (2013) Cysteine scanning mutagenesis of transmembrane helix 3 of a brain glutamate transporter reveals two conformationally sensitive positions. J Biol Chem 288:964-73

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