Using chemical genetics to investigate mechanisms of anesthetic action

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DESCRIPTION (provided by applicant): Since the discovery of anesthesia over a century ago, the use of these agents has revolutionized the fields of surgery and perioperative care. Despite their widespread use, however, relatively little is known about the mechanisms by which anesthetic agents exert their effects on the central nervous system. For example, recent work has established that kinases are involved in many aspects of anesthetic action, but mechanistic studies of these pathways have been limited by the lack of well defined methods for studying kinase activity. This proposal describes a chemical genetic approach for identifying direct substrates of the tyrosine kinase c-Src in the presence and absence of common anesthetic agents. C-Src is critical in several neuronal signalling pathways, including those believed to modulate pain perception, memory and neuron survival. This approach utilizes an unnatural ATP analog (N6-benzyl-ATP) that is not recognized by wild-type c-Src yet is a specific substrate of a mutant form of the enzyme, c-Src-as1. The analog kinase delivers a chemically unique phosphate mimic, phosphorothioate, to substrates of c-Src-as1. This unique functional group can be modified for recognition by an antibody and will enable purification of direct c-Src-as1 substrates in a model neuron cell line. We will utilize this methodology to examine how c-Src-as1 activity varies in the presence or absence of several key classes of anesthetic agents. The work described here will provide valuable information on how anesthetic agents exert their effects on neuronal signalling pathways. The long term goal of this research in public health is to utilize this information to identify groups that are likely to benefit from a particular anesthetic as well as groups that are at high risk for complications of anesthesia, such as awareness during surgery. This work may also provide information for the development of new anesthetics directed at a particular neural pathway.