PROJECT 2 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. These effects are thought to result from steroid interactions with specific binding sites on the GABA-A receptor. The overall goal of this project is to identify and characterize the binding sites on the GABA-A receptor with which neuroactive steroids interact to produce their inhibitory (anesthetic) effects. Two novel neurosteroid analogue photoaffinity labeling reagents, CW12(a Sa-reduced steroid) and CW14 (a 5p- reduced steroid) have been developed to achieve this goal. Both CW12 and CW14 are potent and efficacious modulators of GABA-A receptor function. When these reagents are exposed to UV light at 350 nM, they form a reactive group (a carbene) that allows them to covalently attach to (photolabel) their binding site. CW12 photolabels GABA-A receptor subunits both in rat brain and in cultured cells. Assays of GABA-A receptor function demonstrate that CW12 and CW14 selectively photolabel the neuroactive steroid binding site(s) responsible for modulation of GABA-A receptor function. The aim of this project is to identify the GABA-A receptor subunits that are photolabeled by CW12and/or CW14 and to identify the specific amino acid residues that are modified by the photolabeling reagents. Photolabeling studies will be performed in cultured mammalian cells transfected with various combinations of epitope-tagged GABA-A receptors and in rat brain tissue. Photolabeled GABA-A receptors will be isolated using immunoprecipitation and digested with proteases to generate steroid- modified and unmodified peptides. The steroid-modified peptides will then be analyzed using capillary high-performance liquid chromatography-linear quadrupole ion trap-Fourier transform ion cyclotron mass spectrometry to determine their sequence and to identify the modified amino acids. The information gained from this project will provide the background knowledge and tools to: (1) determine 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 anticonvulsants and anxiolytics and neuroactive steroid antagonists.