. The overall hypothesis of Project 2 is that general anesthetics bind to several distinct sites on the GABAAR and that each distinct site has its own pharmacology. ,We aim to devise agents that bind selectively to each distinct site. Furthermore, the affinity for each site depends on: (1) the receptor's subunit composition.
We aim to develop anesthetics that target specific subpopulations of GABAARs, such as ?-subunit containing extrasynaptic vs. ?-subunit containing synaptic receptors;(2) its conformational state. The best general anesthetics will bind with higher affinity to the open than to the resting state, the ratio of affinities defining the anesthetic's efficacy. In extreme cases, affinity for the resting state may be so low that a binding site may only be detectable in the open state of a GABAAR. The project is supported by: (1) the Synthetic Chemistry Core (Core B), with whom we will devise new anesthetics and anesthetic photolabels to support all aims;(2) the Protein Chemistry Core (Core C) who provide HPLC assays required in Aim 1 and the sequencing required in Aims 2 &3, and (3) the Protein Production Core (Core D) who provide the large amounts of GABAARs of various subunit compositions required in all aims.
Aim 1 : The PPG has discovered that [3H]R-azi-etomidate and a barbiturate, [3H]R-mTFD-MPAB, bind at different sites between subunits in the transmembrane domain. These sites are homologous in secondary structure, but differ subtly in sequence. These two agents provide us with tools to study the structure activity relationships that govern selective binding to their separate sites. In addition, the PPG has determined that there is a separate nonhomologous site in the extracellular domain that a convulsant barbiturate interacts with. Because general anesthetics often cause excitation and close analogs are often convulsant or pro-convulsant, it is important to discover the structural rules governing binding to this convulsant site so that undesirable excitatory side effects can be eliminated when designing new general anesthetics, thus lowering toxicity and improving patient care.
Aim 2 seeks new sites on the GABAAR. (1) Inhalational anesthetics can fit into smaller binding pockets than those the PPG has detected to date for intravenous anesthetics, so we will devise smaller photolabels to test this hypothesis. (2) What are the structure activity relationships of the transmembrane domain sites neighboring ?-subunits in GABAARs representative of extrasynaptic receptors? Aim 3 uses time resolved photolabeling to test the hypotheses that: (1) There are intrasubunit sites within the bundle of four helices in the transmembrane domain that only become occupied upon activation, and (2) the prediction of the allosteric model that the relative affinity of an anesthetic for the open vs. the resting state is proportional to efficacy for enhancing GABA-induced currents. This interdisciplinary project aims to improve patient safety by providing new information and concepts that will guide the development of more selective general anesthetics.
|Forman, Stuart A; Chiara, David C; Miller, Keith W (2015) Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors. Neuropharmacology 96:169-77|
|Feng, H-J; Jounaidi, Y; Haburcak, M et al. (2014) Etomidate produces similar allosteric modulation in *1*3ýý and *1*3ýý2L GABA(A) receptors. Br J Pharmacol 171:789-98|
|Jayakar, Selwyn S; Zhou, Xiaojuan; Chiara, David C et al. (2014) Multiple propofol-binding sites in a ?-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog. J Biol Chem 289:27456-68|
|Dostalova, Zuzana; Zhou, Xiaojuan; Liu, Aiping et al. (2014) Human ?1?3?2L gamma-aminobutyric acid type A receptors: High-level production and purification in a functional state. Protein Sci 23:157-66|
|Olsen, Richard W; Li, Guo-Dong; Wallner, Martin et al. (2014) Structural models of ligand-gated ion channels: sites of action for anesthetics and ethanol. Alcohol Clin Exp Res 38:595-603|
|Hamouda, Ayman K; Stewart, Deirdre S; Chiara, David C et al. (2014) Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate. Mol Pharmacol 85:735-46|
|Chiara, David C; Gill, Jonathan F; Chen, Qiang et al. (2014) Photoaffinity labeling the propofol binding site in GLIC. Biochemistry 53:135-42|
|Jayakar, Selwyn S; Dailey, William P; Eckenhoff, Roderic G et al. (2013) Identification of propofol binding sites in a nicotinic acetylcholine receptor with a photoreactive propofol analog. J Biol Chem 288:6178-89|
|Stewart, Deirdre S; Hotta, Mayo; Desai, Rooma et al. (2013) State-dependent etomidate occupancy of its allosteric agonist sites measured in a cysteine-substituted GABAA receptor. Mol Pharmacol 83:1200-8|
|Forman, Stuart A (2013) A paradigm shift from biophysical to neurobiological: the fading influence of Claude Bernard's ideas about general anesthesia. Anesthesiology 118:984-5|
Showing the most recent 10 out of 85 publications