In the brain, glutamate transporters reside in the plasma membranes of glial cells and neurons at glutamatergic synapses, where they mediate neurotransmitter uptake, allowing multiple rounds of signaling and preventing excitotoxicity. These essential proteins harness the energy of the electrochemical gradients of cations to drive concentrative uptake of the neurotransmitter, and also mediate passive anion fluxes gated by glutamate and sodium binding. Although dysfunction of glutamate transporters is associated with a plethora of diseases and pathological states, strategies for the pharmacological intervention are greatly limited due, at least in part, to the lack of understanding of their kinetc mechanism. To fill this gap, the overarching goal of the proposed research is to advance our understanding of the relationship between the dynamics, structure and function of glutamate transporters. In these studies we rely on the use of a related bacterial homologue, GltPh, a well- established model system, which has proven exceptionally useful in understanding the structure and mechanism of glutamate transporters. Using this system, we propose to establish the nature of the events that limit the rates of substrate uptake and the structures of the key intermediates. The proposed mechanism of glutamate transporters entails trans-membrane movements of the so-called transport domain, which carries the substrate across the membrane, relative to the scaffolding domain, which remains largely static in the membrane. Our key postulate, based on the published preliminary data, is that this process occurs via dynamic intermediates, in which the interface between the transport domain and the scaffold becomes partially hydrated and, therefore, structurally """"""""unlocked"""""""". We propose to substantiate our hypothesis that these """"""""unlocking"""""""" events are rate limiting to the transport cycle, and that mutations that favor the dynamic states accelerate transport. We further seek to test our hypothesis that similar """"""""unlocked"""""""" on- or off-pathway intermediates of the transport cycle mediate anion permeation. Finally, we aim to establish how the membrane environment and the energizing trans-membrane electrochemical gradients affect the dynamics and the thermodynamics of the transporter and how our findings in GltPh relate to the eukaryotic glutamate transporters. In this project, we have established a broad-based collaboration, combining crystallography and functional experiments with single molecule fluorescence imaging methods that allow us to follow domain movements in real time;protein engineering;pulsed dipolar electron spin resonance spectroscopy;and multi-scale computational and modeling approaches. Collectively, these multidisciplinary efforts will establish the feasibility o developing allosteric transporter activators, shed light on the mechanism of physiologically relevant anion permeation, and evaluate the modulatory role of the membrane.

Public Health Relevance

In the brain, glutamate transporters clear the neurotransmitter following synaptic signaling, and thus, play essential roles in learning, memory formation and cognition as well as in the survival of neurons. Using a closely related bacterial homologue, we aim to investigate the dynamic and the mechanical properties of these miniature machines, observing their motions and structures with a broad range of biophysical and structural techniques.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
1R37NS085318-01A1
Application #
8761326
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Silberberg, Shai D
Project Start
2014-07-15
Project End
2018-06-30
Budget Start
2014-07-15
Budget End
2015-06-30
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Physiology
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10065
Oh, SeCheol; Boudker, Olga (2018) Kinetic mechanism of coupled binding in sodium-aspartate symporter GltPh. Elife 7:
Jentsch, Julian-Alexander; Kiburu, Irene; Pandey, Kalpana et al. (2018) Structural basis of sterol binding and transport by a yeast StARkin domain. J Biol Chem 293:5522-5531
Riederer, Erika A; Focke, Paul J; Georgieva, Elka R et al. (2018) A facile approach for the in vitro assembly of multimeric membrane transport proteins. Elife 7:
Ruan, Yi; Miyagi, Atsushi; Wang, Xiaoyu et al. (2017) Direct visualization of glutamate transporter elevator mechanism by high-speed AFM. Proc Natl Acad Sci U S A 114:1584-1588
Akyuz, Nurunisa; Georgieva, Elka R; Zhou, Zhou et al. (2015) Transport domain unlocking sets the uptake rate of an aspartate transporter. Nature 518:68-73
Boudker, Olga; Oh, SeCheol (2015) Isothermal titration calorimetry of ion-coupled membrane transporters. Methods 76:171-182