2004 — 2012 |
Grewer, Christof Theodor |
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. |
Molecular Basis of Glutamate Transport @ University of Miami School of Medicine
[unreadable] DESCRIPTION (provided by applicant): In the mammalian brain the extracellular concentration of the excitatory neurotransmitter glutamate is, among other factors, controlled by transporters, which actively take up glutamate into glial cells and neurons. The broad aim of this proposal is to investigate the molecular mechanisms by which these transporters accomplish this glutamate uptake. Recombinant glutamate transporters will be expressed in HEK293 cells and Xenopus oocytes, and currents that originate from electrogenic glutamate transport will be recorded using the patch clamp technique. Currents will be induced by glutamate concentration jumps generated within 100 mus, allowing the resolution of rapid transporter reaction steps in time. The hypotheses that guide this research are: Both inward and outward glutamate transport occur via a multistep electrogenic mechanism; charged amino acids in the transmembrane segments contribute to the function and electrogenicity of the transporter; translocation of glutamate across the membrane requires molecular movement of the transport protein. Experiments with the following specific aims will test these hypotheses: (1) To determine the reaction steps associated with outward glutamate transport, by investigating the sequence and voltage dependence of intracellular Na+ and glutamate binding. (2) To determine the contributions of conserved charged amino acids to transporter function and electrogenicity, by investigating transporters with specifically charge-neutralized amino acids. (3) To characterize molecular movement of the transporter machinery by determining which parts of the transporter move and at what steps during a complete transport cycle they move. Movement of the transporter will be detected by the movement of charges attached to the transporter at specific sites. Understanding the molecular mechanism of glutamate transport will give insight into the involvement of glutamate transporters in acute and slowly progressing neurodegenerative diseases, such as stroke, amyotrophic lateral sclerosis, and Alzheimer's disease. [unreadable] [unreadable] [unreadable]
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0.958 |
2015 — 2018 |
Grewer, Christof |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Dynamics and Energetics of Secondary-Active Glutamate Transport
The plasma membrane separates the interior of the cell from the extracellular space and provides a barrier to cell entry/exit for many molecules (for example nutrient or signaling molecules) that are essential for cell survival. Glutamate is one such molecule, which is not only an amino acid needed for the synthesis of proteins, but also the most important neurotransmitter in the mammalian brain, responsible for the majority of excitatory neurotransmission. Glutamate is transported across the plasma membrane through active glutamate transporters, which play essential roles in controlling the levels of glutamate in the mammalian brain and other tissue. This project explores the fundamental principles by which these transporters work. Understanding these principles is important because it will contribute to our knowledge not only of the role of glutamate and nutrient movement between cellular compartments in the mammalian brain, as well as other organs of the body, but also glutamate homeostasis, which is important for cellular function in general. Furthermore, the developed methods for analyzing transport will be applicable to potential future studies of other neurotransmitter and/or nutrient transporters and membrane proteins. On the educational level, the project provides an invaluable opportunity for undergraduate students to become involved in cutting-edge biophysical research, providing training in basic physical, quantitative approaches to be applied to a significant biological problem. The investigator is continuing previous efforts of undergraduate involvement (including members of under-represented groups), which has led to many publications with undergraduate students as co-authors, as well as students successfully moving on to careers in research. The investigator is also very active in local and regional activities that promote the sciences at all levels, including demonstrations at schools, science fair involvement, and assuming leadership roles in the local American Chemical Society (ACS) section and the Science Olympiad. Such activities are necessary to ensure a vibrant local science community and to foster excitement of the upcoming generations of young scientists for science and research.
Active glutamate transport is a multi-step process that requires multiple ion/substrate binding steps, as well as conformational changes. Despite recent progress towards understanding the transport process through functional studies, as well as the identification of structural models of a bacterial homologue of mammalian glutamate transporters in several states, basic questions about the mechanism, specificity, and dynamics of interaction of the transporters with cations, in particular K+ and Li+, as well as the energetic contributions to energy barriers that control the transport rate and specific structural changes in the transport cycle remain unresolved. Answers to these questions are obtained by applying a combination of experimental and computational methods, allowing the investigation of the dynamics of glutamate transport in real time, with microsecond time resolution, and to test predictions from all-atom and simplified molecular models. This work has the potential to reveal new aspects of glutamate movement across the cellular membrane, in particular with respect to the cooperation of polar and non-polar forces, which are fine tuned to adjust the energy barriers associated with ion/substrate binding, as well as conformational changes. The novel insight may uncover more general concepts that are employed by membrane transport proteins, in order to provide a permeation pathway for polar molecules across the hydrophobic membrane.
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0.958 |
2019 |
Grewer, Christof Theodor |
R15Activity Code Description: Supports small-scale research projects at educational institutions that provide baccalaureate or advanced degrees for a significant number of the Nation’s research scientists but that have not been major recipients of NIH support. The goals of the program are to (1) support meritorious research, (2) expose students to research, and (3) strengthen the research environment of the institution. Awards provide limited Direct Costs, plus applicable F&A costs, for periods not to exceed 36 months. This activity code uses multi-year funding authority; however, OER approval is NOT needed prior to an IC using this activity code. |
How to Combat Glutamate Release by Reverse Transport: Mechanistic Studies and Development of Selective Efflux Inhibitors @ State University of Ny,Binghamton
Project Summary/Abstract Glutamate transporters play essential roles in controlling the levels of the neurotransmitter glutamate in the mammalian brain. However, when energy supply is impaired, for example in a stroke, glutamate transporters run in reverse, releasing the excitotoxin glutamate into the extracellular space, resulting in neuronal cell death. The long-term goals of this proposal are to explore the fundamental physical principles by which these transporters release glutamate through reverse transport, and to develop methods to prevent this glutamate release. Despite progress towards these goals through functional studies, as well as the identification of structural models of a bacterial homologue of mammalian glutamate transporters in several states, basic questions about the mechanism of reverse transport, its activation by external potassium, and the pharmacology of reverse transport blockers remain unresolved. We propose two specific aims to address these important questions: Aim 1 will develop means to selectively block glutamate release by reverse transport, without blocking glutamate uptake, and will identify new paradigms of inhibition mechanism. The hypothesis will be tested that introduction of a pro-drug blocker into the cytosol will enable the specific inhibition of reverse transport by binding of the blocker to the intracellular substrate binding site. Aim 2 will determine mechanisms by which extracellular potassium activates and regulates glutamate release through reverse transport, and how potassium cooperates with sodium to form previously unknown states of the transporter. Aim 2 will also identify structural elements contributing to interaction with, and reverse translocation of K+. The proposed experiments will test the hypotheses that external K+ has an inhibitory effect on glutamate release at high concentrations, and that it can bind in the presence of Na+ to form a novel K+/Na+ co-binding state. To approach these two aims, a combination of experimental and computational methods will be used, allowing us to rapidly assess glutamate release through reverse transport, and model it using kinetic and all-atom molecular dynamics simulations. Conceptually and methodologically the proposed research is innovative, because we expect to identify novel aspects of reverse transport mechanism, as well as develop novel methods to measure and modulate the rate of reverse transport, based on extensive preliminary data. Understanding the physical principles underlying glutamate release through reverse transport, as well as its modulation by inhibitors, is important because it will contribute to our knowledge of the role of glutamate transport in the normally-functioning brain. In addition, the expected results could be ultimately used to extend existing, or devise new strategies, to reduce the destructive role of glutamate release through glutamate reverse transport in neurodegenerative disease and stroke.
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0.958 |