NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications Gamma-aminobutyric acid type A receptors (GABAARs) mediate synaptic inhibition in the brain and are modulated by a variety of clinically important drugs, such as benzodiazepines, barbiturates, steroids, anesthetics and anti-convulsants. GABAARs belong to the pentameric ligand-gated ion channel (pLGIC) gene superfamily that includes nicotinic, 5-HT3 and glycine receptors. Mutations in these receptors are responsible for a number of 'channelopathies', such as congenital myasthenic syndromes, epileptic disorders and hereditary hyperekplexia. Our long-term goal is to understand the function of the GABAAR in terms of its molecular structure. While recent crystallographic advances have provided valuable structural models of the GABAAR, achieving a full understanding of function also requires knowledge of protein dynamics. Little is known about the protein motions that occur when ligand binds and the protein switches between inactive/closed and active/open channel states. In our current NIH funded grant, we are examining how, on a structural level, GABA binding triggers channel gating and how BZD binding is coupled to receptor modulation. The approach combines site-directed mutagenesis, disulfide crosslinking, mutant cycle analysis, substituted cysteine accessibility method, patch-clamping and kinetic analysis. Recently, crystal structures of prokaryotic pLGIC homologs in closed and open channel states have been solved. The identification of these prokaryotic homologs, which can be purified in large amounts, provides us with an unparalleled opportunity to study the dynamics of this family of channels using techniques such as electron paramagnetic resonance. In this supplemental competitive revision, we are proposing experiments to study the structure and dynamics of the prokaryotic pLGIC homolog from Gloeobacter violaceus (GLIC) using site-directed spin-labeling methods and electron paramagnetic resonance spectroscopy. The experiments will be interpreted with the aid of recently elucidated atomic-level structures to gain a deeper understanding of the molecular mechanisms underlying the function of pLGICs. We cannot hope to predict the actions of a drug or ligand or predict the outcome of a disease-causing mutation without dissecting the movements in the protein that mediate its function. We are requesting supplemental funds not to fill a funding gap, but, rather, to allow us to extend our experiments into a new domain that will further advance the field. As bacterial homologs of K+ channels have led to major advances in our understanding of K+ channel structure and function, we are confident that studying prokaryotic pLGICs will lead to significant new insights.
The opening and closing of ligand-gated ion channels, which lie in the membranes of nerve cells, regulate information flow throughout the brain. Defects in these channels lead to a wide variety of diseases, such as myasthenia, hyperekplexia and epilepsy. These channels are also the targets of a number of clinically used drugs, including muscle relaxants, sedative-hypnotics, anti-convulsants, anxiolytics, intravenous and volatile anesthetics, anti-emetics, drugs for nicotine addiction and drugs to treat Alzheimer's disease. We cannot hope to predict the actions of a drug, design safer and more effective drugs, develop better therapeutic strategies or predict the outcome of a disease-causing mutation without knowledge of how these channels work at a molecular level. The research proposed here utilizes a powerful structural approach that will increase our understanding of the molecular motions underlying how a bacterial pentameric ligand-gated ion channel homologue functions. The data will establish testable hypotheses for elucidating how related eukaryotic ligand-gated ion channels function.
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