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 two classes of transporters. These are GLT-1 and EAAC1, the (Na+ + K+)-coupled plasma membrane transporters of the neurotransmitter glutamate, and rVMAT2, a vesicular H+-coupled monoamine transporter. Crystal structures relevant to this project have become available: GltPh and MFS transporters, bacterial homologues of GLT-1 and VMAT, respectively. In turn, now the structures provide important clues to continue our studies towards the understanding of the mechanism of transport. In this process, biochemical information is essential to validate the existing structures and those to come and to understand them in the context of function. A synergistic interaction with computational biologists also will lead to the generation of new models to be tested with our experimental tools. Using biochemical and biophysical analysis of mutants, generated by rational design or by directed evolution, we will progress towards understanding mechanism by using the available structural and biochemical information to (i) further explore the residues in the binding pocket and to modify specificities and affinities for various substrates;(ii) study the molecular determinants of ion binding and the nature of the coupling of ion and substrate fluxes 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.

Public Health Relevance

Neurotransporters located in the plasma membrane and in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Evidence for their central role is that they are the targets of many psychoactive drugs, such as Prozac, Cocaine and Amphetamines, and that their malfunctioning leads to severe pathological conditions. Hence the study of the mode of action of the neurotransporters, as proposed in this research project, is fundamental for understanding the communication between nerve cells in the normal and diseased brain.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Biophysics of Neural Systems Study Section (BPNS)
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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
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
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; 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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