The primary objective of the research proposed in this fellowship application is to further the understanding of neuroactive steroid and anesthetic action at the GABA/A receptor by photoincorporating novel radiolabeled neuroactive steroid and anesthetic ligands into purified recombinant GABA/A receptor of known subunit composition. The data collected during this investigation will provide valuable information for understanding the molecular targets of general anesthetics and for modeling the allosteric effects of both neuroactive steroids and general anesthetics at the GABA/A receptor. Specifically, a novel neuroactive steroid anesthetic photolabel (HBPP) will be used to label amino acids within or near the binding site for neuroactive steroids on recombinant alpha1beta2gamma2 GABA/A receptor purified from Sf9 cells. Following photoincorporation, the subunits of the receptor are separated using SDS- PAGE and the labeled subunit(s) is fragmented using V8 protease. The fragments generated from protease digestion are identified using photoaffinity labels and antibodies that bind to known domains of GABA/A receptor subunits and are then sequenced. Following the identification of the amino acid residue(s) bound by HBPP, chimeras and site-directed mutants at or near the area bound by HBPP will be generated to verify the contribution of the labeled amino acid to the binding and/or action of neuroactive steroids. This mutational analysis has the potential to identify amino acids not labeled by HBPP that contribute to neuroactive steroid binding and/or action. The general anesthetics promegestone, azi- octanol, and etomidate will also be used as photolables to label amino acid residues within or near the anesthetic binding site at recombinant alpha1beta2gamma2 GABA/A receptor. The identification of amino acid residues bound by these anesthetics will proceed as described above.
| Sawyer, Gregory W; Chiara, David C; Olsen, Richard W et al. (2002) Identification of the bovine gamma-aminobutyric acid type A receptor alpha subunit residues photolabeled by the imidazobenzodiazepine [3H]Ro15-4513. J Biol Chem 277:50036-45 |
