3-aminobutyric acid (GABA)-gated chloride channels (GABAA and GABAC receptors) play important roles in fast synaptic inhibition in the mammalian brain. They are also the major targets of many clinically useful neuroactive compounds. Dysfunction of these GABA-gated ion channels can result in epilepsy and other neural disorders. Binding of GABA to its receptor is thought to initiate a conformational wave starting from the binding site(s) and propagating to the gating machinery to open the pore. Gaining insights into the dynamic structural basis of these conformational rearrangements therefore is the key step to understanding the mechanism of GABA receptor activation and antagonism. However, despite intensive structure-function relationship studies in the past, structural dynamics underlying GABA receptor activation is still not fully understood. This project seeks to provide new insights into structural bases for GABAC receptor function by employing techniques successfully adapted in this laboratory.
The first aim i s to use site-specific fluorescence combined with substituted cysteine accessibility analysis and electrophysiological recording to define agonist-induced structural rearrangements in the subunit interface underlying channel activation.
The second aim i s to use fluorescence resonance energy transfer (FRET) to detect conformational changes not located in subunit interface and to deduce their moving direction during receptor activation.
The third aim i s to use site-specific fluorescence and FRET to define agonist-induced movements antagonizable by noncompetitive antagonists.
The fourth aim i s to validate the functional significance of the moving residues identified by previous aims by single channel analysis and explore coupling mechanism by mutant cycle analysis. Collectively, this work will allow us to detect conformational changes in and out of subunit interface underlying GABA receptor activation and antagonism. These studies will test the project's central hypothesis that GABA receptor activation involves an agonist-induced rotation of the N-terminal domain, which then propagates to the gating machinery to open the pore. Insights gained from this work will illuminate mechanisms of receptor activation and antagonism that have potential applicability more generally to the entire class of ligand-gated ion channels. The findings also will provide a structural basis for development of new therapeutics or research tools targeting GABA receptors.

Public Health Relevance

This project seeks to provide new insights into structural basis for the mechanism of GABA receptor function by employing techniques successfully adapted in this laboratory. The findings will be fundamental to understanding the pathological processes of GABA receptor dysfunction and the mechanism of action for those drugs targeting GABA receptors. The findings will also provide a structural basis for development of new therapeutics or research tools targeting GABA receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085237-03
Application #
8214631
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Dunsmore, Sarah
Project Start
2010-04-01
Project End
2015-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
3
Fiscal Year
2012
Total Cost
$240,265
Indirect Cost
$85,825
Name
St. Joseph's Hospital and Medical Center
Department
Type
DUNS #
131606022
City
Phoenix
State
AZ
Country
United States
Zip Code
85013
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