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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS016708-28
Application #
8232538
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Silberberg, Shai D
Project Start
1981-01-01
Project End
2013-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
28
Fiscal Year
2012
Total Cost
$252,238
Indirect Cost
$18,684
Name
Hebrew University of Jerusalem
Department
Type
DUNS #
600044978
City
Jerusalem
State
Country
Israel
Zip Code
91904
Schuldiner, Shimon (2014) Competition as a way of life for H(+)-coupled antiporters. J Mol Biol 426:2539-46
Schuldiner, Shimon (2009) EmrE, a model for studying evolution and mechanism of ion-coupled transporters. Biochim Biophys Acta 1794:748-62
Adam, Yoav; Edwards, Robert H; Schuldiner, Shimon (2008) Expression and function of the rat vesicular monoamine transporter 2. Am J Physiol Cell Physiol 294:C1004-11
Elbaz, Yael; Salomon, Tal; Schuldiner, Shimon (2008) Identification of a glycine motif required for packing in EmrE, a multidrug transporter from Escherichia coli. J Biol Chem 283:12276-83
Steiner-Mordoch, Sonia; Soskine, Misha; Solomon, Dalia et al. (2008) Parallel topology of genetically fused EmrE homodimers. EMBO J 27:17-26
Adam, Yoav; Tayer, Naama; Rotem, Dvir et al. (2007) The fast release of sticky protons: kinetics of substrate binding and proton release in a multidrug transporter. Proc Natl Acad Sci U S A 104:17989-94
Teichman, Shlomit; Kanner, Baruch I (2007) Aspartate-444 is essential for productive substrate interactions in a neuronal glutamate transporter. J Gen Physiol 129:527-39
Schuldiner, Shimon (2007) When biochemistry meets structural biology: the cautionary tale of EmrE. Trends Biochem Sci 32:252-8
Shlaifer, Irina; Kanner, Baruch I (2007) Conformationally sensitive reactivity to permeant sulfhydryl reagents of cysteine residues engineered into helical hairpin 1 of the glutamate transporter GLT-1. Mol Pharmacol 71:1341-8
Rotem, Dvir; Steiner-Mordoch, Sonia; Schuldiner, Shimon (2006) Identification of tyrosine residues critical for the function of an ion-coupled multidrug transporter. J Biol Chem 281:18715-22

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