General anesthesia plays an indispensable role in modern medicine and affects more than 100,000 patients daily in the United States alone. Despite its clinical importance, the lack of a mechanistic understanding of general anesthetic action has restricted our ability to control drug side effects and anesthesia-related morbidity. The goal of this competing renewal is to advance the understanding of the molecular mechanisms underlying the action of general anesthetics. We have recently made several breakthroughs in the structural and dynamic characterization of anesthetic action on pentameric ligand-gated ion channels (pLGICs), which are targets for general anesthetics and other therapeutic drugs. We have used NMR to analyze anesthetic interactions with the transmembrane domains of neuronal nAChRs. In addition, we have successfully integrated X-ray crystallography and electrophysiology into the characterization of anesthetic binding and modulations on prokaryotic pLGICs. With this new momentum, we are ready to test the central hypothesis that a receptor's sensitivity to anesthetics is controlled by redistribution of the equilibrium populations among preexisting conformations of the receptor. Only those sites that can significantly shift the conformational equilibrium upon anesthetic binding will generate a functional impact. The three new specific aims are: (1) to reveal the structural and dynamic basis of anesthetic inhibition of ELIC, a pLGIC from the Erwinia chrysanthemi. (2) To engineer chimeras of ELIC and human GABAARs such that the chimeras mimic the pharmacological profiles of the parent human GABAARs, but are more amenable to structural investigation. (3) To determine structures of the ELIC-GABAAR chimeras and the underlying mechanisms of anesthetic action in these channels. The innovation of the proposed research is reflected in the integrated approaches and strategies that will generate novel experimental structures for GABAARs and provide an unprecedented opportunity for gaining new insights into actions of general anesthetics. The significance of the proposed studies lies in establishing a framework that directly links channel structure and dynamics to anesthetic action, as well as revealing and understanding structures and dynamics of allosteric sites where anesthetics and other modulators act on pLGICs. The results from the proposed investigations will have a strong impact on the rational design of novel anesthetics with reduced side effects and in the development of other therapeutics.
General anesthesia is a fundamental component of modern medicine. The lack of a mechanistic understanding on how general anesthetics work has hampered the clinical management of anesthetic side effects and anesthesia-related morbidity. The pentameric ligand-gated ion channels are targets of general anesthetics; but little high-resolution structural information of the human receptor channels is currently available. The proposed research will take a multifaceted and integrated approach to establish a framework that directly links channel dynamics to anesthetic action. It will also determine the atomic resolution channel structures and mechanisms of channel gating that are crucial for the rational design of novel anesthetics with reduced side effects and for the development of other therapeutics.
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