1985 — 2000 |
Schuldiner, Shimon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanism of Active Neurotransmitter Transport @ Hebrew University of Jerusalem
DESCRIPTION: This project explores the molecular mechanism of two ion-coupled transporters: The (Na+ + K+)-coupled glutamate transporter GLT-1 from brain an the H+-organic cation multidrug antiporter EmrE from E. coli. These two system provide unique experimental paradigms for structure/function relationships. In GLT-1 we have already identified several amino acid residues lining the translocation pathway. Moreover, homologous bacterial transporters provide a powerful tool to apply bacterial genetics to study the system. In the case of EmrE, it is the smallest ion-coupled transporter and displays unique stability and solubility properties. This makes it amenable to structural studies. These investigations will progress towards understanding the molecular mechanism by (a) identification of residues in the translocation pathway, (b) in depth biochemical and electrophysiological analysis of the role of critical residues, (c) identification of new ones upon study of second site revertants of the bacterially expressed proteins, and (d) study of the topology, helix packing and oligomeric structure. In addition to contributing to understanding the structural basis of ion-coupled transporter function, these studies may provide important clues to the role of these transporters not only under normal physiological conditions, but also in disease.
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2001 — 2014 |
Kanner, Baruch I Schuldiner, Shimon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Mechanisms of Active Neurotransmitter Transport @ Hebrew University of Jerusalem
DESCRIPTION (provided by applicant): 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.
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