The overall goal of the proposed work is to increase understanding of the basic mechanisms involved in communication between nerve cells in the brain. The proposal builds on previous findings demonstrating that increased atmospheric pressure (hyperbaric exposure) is a direct, highly selective blocker of a little explored, poorly defined process-allostenc coupling-that modulates the effectiveness of a family of neurotransmitters receptors in the brain, called ligand gated ion channels (LOICs), that play major roles in nerve-nerve communication. In particular, the proposed work focuses on one of these channels. This channel responds to gamma-aminobutyric acid (GABA) and is called the GABAA channel. When GABA binds to its receptor on the GABAA the channel opens a channel and allows chloride ions to enter the nerve cell. The resultant build-up of chloride ions in the nerve cell inhibits the cells action and decreases its excitablity. The GABAA system is the major inhibitory system in the mammalian brain.
The effectiveness of GABA' 5 action in opening chloride ion channels can be modulated by several classes of compounds that act at distinct but interacting sites on the GABAA receptor. These sites include those for the benzodiazepines, barbiturates and neuroactive steroids. When one of these compounds (ligands) bind to their respective sites on the GABAA receptor, it causes a conformation change in the receptor that affects the ability of other ligands to bind or affect the receptor. This process, referred to as allosteric coupling between sites, modulates the effectiveness of the primary agonist-GAB A-as well as the effectiveness of other allosteric modulators.
The molecular structures and functions of the portions of the GAB AA receptor that bind ligands has been extensively studied. In contrast, little attention has been devoted to understanding the elements that underlie allosteric coupling. The elements mediating coupling are difficult to study directly due to a lack of tools. Recent behavioral and biochemical findings in our laboratory suggest that hyperbaric exposure offers a new approach that can help in studying allosteric coupling. Moreover, this work with hyperbaric exposure suggests that there are fundamental, previously unrecognized, differences in the manner in which binding sites on the GABAA receptor are coupled and that hyperbaric exposure can be used to study these differences.
The specific objective of the research to be undertaken is to test two hypotheses: Hypothesis 1: The different pattern of sensitivity to pressure antagonism among allosteric modulators of GABAA receptor function will provide new insights into the structural and functional determinants of coupling. The logic for this hypothesis is based on the assumption that the selectivity of pressure antagonism results from pressure's ability to block common physico-chemical changes underlying coupling and that the sensitivity of these physico-chemical changes to pressure reflect similarities in their underlying molecular structures. The hypothesis will be tested by systematically investigating four predictions regarding the selectivity of pressure antagonism based on known functional distinctions in coupling within and between different sites on the GABAA receptor using biochemical measures of GABAA receptor function in mouse brain cell membranes. Hypothesis 2: Differences in the structural and functional determinants of GABAA receptor coupling identified in ABSTRACT DRAAT 12/4/98
testing Hypothesis 1 reflect differences in the protein subunits that comprise the receptor. This hypothesis will be tested by determining the sensitivity to pressure antagonism of allosterically modulated events using biochemical and molecular biological techniques.
The proposed work will increase knowledge regarding the manner in which the effectiveness of nerve cell transmission is controlled. The proposed work will lay the foundation for future studies that will use hyperbaric exposure in combination with molecular manipulations in recombinant cells to identify molecular components that mediate allosteric coupling. This information in turn will facilitate the development of molecular models of these structures. The proposed work will also lay the foundation for future investigations that will investigate coupling in other allosterically modulated channels (e.g., NMDA, 5HT3...). These studies could support known and/or could reveal previously unrecognized similarities between allosterically modulated ion channels. Finally, future studies will investigate whether differences in coupling mechanisms have currently unrecognized physiological and behavioral significance. Therefore, the proposed and future work should lead to important new insights regarding the role LGICs play in mediating and modulating brain function and behavior.
