AMPA receptors mediate fast excitatory responses in the mammalian central nervous system, and ultimately control motor and cognitive functions. In neurons AMPA receptors co-assemble with auxiliary subunits. One member being transmembrane AMPA receptor regulatory proteins (TARPs), which are essential for the normal physiological functioning of these receptors. One of the dramatic effects of TARPs on AMPA receptor gating is a large increase in efficacy such that even some antagonists such as 6-cyano-7-nitroquinoxaline-2,3-dione act as partial agonists. Additionally, TARPs slow deactivation of AMPA receptor when agonist is removed and slow the rate and extent of desensitization when agonist is continually applied. These changes in properties are expected to dictate the kinetics and shape of synaptic transmission. In addition to the rapid modulations, the presence of certain subtypes of TARP's leads to resensitization on longer time scales, from the tens of milliseconds to seconds. The prolonged opening of the receptors in these long resensitization processes may play a role in glutamate mediated excitotoxicity. Here we propose to gain a fundamental understanding of the structure-dynamic changes controlling the increase in efficacy and resensitization in the AMPA receptors in the presence of TARPs using a combination of biophysical and electrophysiological methods. For the proposed study, we will use luminescence resonance energy transfer to study the conformational changes in the protein. For specific labeling of the protein with fluorescent probes we will introduce unnatural amino acids at specific sites on the protein. The conformational changes thus observed will be further verified with functional investigations of mutant proteins with mutations introduced at strategic sites that affect the observed conformational change or the stability of a specific conformational state. Finally, the position of the TARP ecto domain will be mapped to the AMPA receptor extracellular domain using mass spectrometry. To study the interaction sites between the two proteins the unnatural amino acid p-benzoyl phenylalanine will be introduced at various sites and those sites leading to crosslinking will be analyzed using high resolution tandem mass spectrometry. The proposed functional and structural investigations will provide a comprehensive understanding of the mechanism by which TARPs modulates AMPA receptor function.

Public Health Relevance

The AMPA subtype of glutamate receptors are the main mediators of fast excitatory synaptic transmission. These receptors are modulated by auxiliary subunits called transmembrane AMPA receptor regulatory proteins and defects in the auxiliary subunit results in seizures. Here we will study the mechanism of activation in the receptors in complex with the auxiliary proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM113212-03
Application #
9178663
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Dunsmore, Sarah
Project Start
2014-12-05
Project End
2018-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77225
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Coombs, Ian D; MacLean, David M; Jayaraman, Vasanthi et al. (2017) Dual Effects of TARP ?-2 on Glutamate Efficacy Can Account for AMPA Receptor Autoinactivation. Cell Rep 20:1123-1135
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Sirrieh, Rita E; MacLean, David M; Jayaraman, Vasanthi (2015) Subtype-dependent N-methyl-D-aspartate receptor amino-terminal domain conformations and modulation by spermine. J Biol Chem 290:12812-20
Sirrieh, Rita E; MacLean, David M; Jayaraman, Vasanthi (2015) A conserved structural mechanism of NMDA receptor inhibition: A comparison of ifenprodil and zinc. J Gen Physiol 146:173-81