Photolabeling of Anesthetic Steroid Binding Sites

Washington University, Saint Louis MO

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Abstract

Certain endogenous steroids and their synthetic analogues (neuroactive steroids) produce profound and rapid effects on the central nervous system ranging from general anesthesia to seizures. While these effects are thought to result from steroid interactions with specific binding sites on the GABA-A receptor, molecular biological and biochemical studies have failed to identify candidate regions or residues that might contribute to a binding site. The overall objective of this project is to identify and characterize the binding sites for neuroactive steroids using photo-affinity labeling techniques. To achieve this goal ZCM-43, a recently developed neuroactive steroid photo-affinity labeling reagent, will be used to photo-label a 35-kDa protein in brain. There are four specific aims of this project: (1) Identify and sequence the 35-kDa protein in brain. There are four specific aims of this project: (1) Identify and sequence the 35-kDa protein using two-dimensional electrophoresis and mass spectrometry. The structural specificity of steroid interaction with the 35-kDa protein will also be examined. (2) Determine the relationship between neuroactive steroid binding to 35-kDa protein and GABA-A receptor modulation using immunoprecipitation, radioligand binding and gene "knockout" techniques. (3) Determine if GABA-A receptors or other proteins in brain also have binding sites for neuroactive steroids. These studies will be performed using novel photo-labeling reagents in which the photo-reactive group is placed in various positions on the steroid backbone. (4) Determine if neuroactive steroids compete with cholesterol for binding sites at the lipid-protein interface These studies will be performed by manipulating membrane cholesterol and examining the effects on GABA-A receptor function as well as by using a cholesterol photo-labeling reagent. The information gained from this project will provide the background knowledge and pharmacological tools to: 1) approach the question of how endogenous neurosteroids modulate CNS function in health and disease and; 2) develop new pharmaceutical agents including potent steroidal anesthetics with minimal side effects, novel anti-convulsants and neuroactive steroid antagonists.