AMPA (?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) subtype ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the central nervous system. Alteration in AMPA receptor function is associated with numerous devastating neurological diseases including Alzheimer's disease, amyotrophic lateral sclerosis, epilepsy and ischemia. Regulation of AMPA receptor activity is therefore an important clinical goal. AMPA receptors function by opening their ion channel for the flow of permeant cations in response to binding of agonist glutamate to the receptor. This process of ion channel opening or activation gating is accompanied by typically slower process of desensitization. Desensitization leads to the closure of the ion channel pore and appears as a 10-500 fold reduction of AMPA receptor- mediated currents in the continuous presence of glutamate. Desensitization represents a natural way of suppressing AMPA receptor activity that protects neurons from the excitotoxic calcium overload. Enhancing or weakening desensitization is therefore an effective way of regulating AMPA receptor activity in pathological conditions. An alternative way to suppress AMPA receptor activity that has therapeutic potential is to use ion channel blockers, molecules that plug the channel pore. At the molecular level, desensitization and ion channel block are expected to have common features that allow them to lock the ion channel in the closed conformation. We plan to study AMPA receptor desensitization and block using a combination of structural and functional approaches and to reveal similarities and differences in these processes. Accordingly, our specific aims are: 1) Obtaining a high-resolution structure of an AMPA receptor in the desensitized state and 2) Building a structural model of ion channel block. AMPA receptor is a challenging target for structure-functional studies because it represents a multimeric integral membrane protein of a large size with typically low expression level. To achieve our goals, we will use a combination of structural and functional approaches including modern crystallographic techniques, Fluorescence-based Size Exclusion Chromatography (FSEC) and electrophysiology. We will use different crystallization methods and temperatures, screen detergents, lipids and ligands to obtain full length AMPA receptor structures in the desensitized and blocker-bound states. We will then combine nascent structural information with electrophysiological recordings and kinetic modeling to figure out mechanisms of AMPA receptor desensitization and block. Achieving our aims will have a significant impact on molecular neuroscience and will result in a new structural/functional model of AMPA receptor that can serve as a dynamic template for theoretical prediction, in silico fitting and chemical synthesis of new compounds that can be tested in different models of neurological diseases and eventually become safe and effective medications.

Public Health Relevance

AMPA subtype ionotropic glutamate receptors mediate majority of excitatory neurotransmission in the central nervous system and their disfunction is associated with devastating neurological diseases, such as Alzheimer's disease, amyotropic lateral sclerosis, epilepsy and ischemia. Our goal is to study AMPA receptors using a combination of biophysical and biochemical techniques and to obtain detailed information about their structure and function that will help to develop new drug design strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS083660-01A1
Application #
8650439
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Silberberg, Shai D
Project Start
2013-09-30
Project End
2018-06-30
Budget Start
2013-09-30
Budget End
2014-06-30
Support Year
1
Fiscal Year
2013
Total Cost
$347,180
Indirect Cost
$128,430
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Yelshanskaya, Maria V; Li, Minfen; Sobolevsky, Alexander I (2014) Structure of an agonist-bound ionotropic glutamate receptor. Science 345:1070-4