- Project 1 (Locating General Anesthetic Binding Sites in GABA-A and Glycine Receptors) Our broad goal is to determine the locations in human GABAA receptors (GABAAR) of the binding sites for general anesthetics of diverse chemical structures and the affinities of anesthetics for the different classes of sites. In the previous grant period, we used photoreactive etomidate and barbiturate analogs to establish for the first time that there are at least two classes of intersubunit general anesthetic binding sites in the transmembrane domain of a human ??? GABAAR. One class is located at the interfaces between subunits containing the transmitter binding sites in the extracellular domain and the second class at interfaces not containing the transmitter binding sites. Etomidate binds at anesthetic concentrations with >100-fold selectivity to the first class of sites, and certain barbiturates bind with high selectivity to the second class. We also determined that propofol binds non-selectively to both sites, but only at concentrations greater than necessary to produce anesthesia or potentiate GABA responses, and anesthetic steroids do not bind to either class of sites.
Our first aim for this renewal is to use novel photoreactive analogs of anesthetic steroids, barbiturates, and propofol to identify in expressed, human ?1?3?2 GABAAR additional anesthetic binding sites important for GABAAR potentiation or inhibition. It is our hypothesis that anesthetic steroids bind to intrasubunit sites at the interfaces between transmembrane helices that are also in contact with lipid. A photoreactive analog of a convulsant barbiturate will be used to identify novel binding sites important for GABAAR inhibition. Photoreactive propofol analogs will be used to determine whether propofol binds in a GABAAR to intrasubunit sites equivalent to those identified previously in nicotinic acetylcholine receptors and a prokaryote GABAAR homolog. We will determine in Aims 2 and 3 whether anesthetics bind to equivalent sites in a GABAAR subtype that is expressed extrasynaptically (?4?3d) and in glycine receptors, the receptor most similar in structure to GABAARs and the major inhibitory receptor in the spinal cord. The proposed experiments make use of the Synthetic Chemistry, Protein Chemistry, and Protein Production Cores. Our studies, in conjunction with the development and characterization of novel, potent anesthetics and time-resolved photolabeling studies of Project 2 (Miller) and the functional studies of Project 3 (Forman), should provide an unprecedented view of the number and diversity of general anesthetic binding sites in GABAARs and provide a starting point for the development of anesthetics selective for GABAAR subtypes.
|Ziemba, Alexis M; Szabo, Andrea; Pierce, David W et al. (2018) Alphaxalone Binds in Inner Transmembrane ?+-?- Interfaces of ?1?3?2 ?-Aminobutyric Acid Type A Receptors. Anesthesiology 128:338-351|
|Forman, Stuart A (2018) Combining Mutations and Electrophysiology to Map Anesthetic Sites on Ligand-Gated Ion Channels. Methods Enzymol 602:369-389|
|Woll, Kellie A; Zhou, Xiaojuan; Bhanu, Natarajan V et al. (2018) Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors. FASEB J 32:4172-4189|
|McGrath, Megan; Yu, Zhiyi; Jayakar, Selwyn S et al. (2018) Etomidate and Etomidate Analog Binding and Positive Modulation of ?-Aminobutyric Acid Type A Receptors: Evidence for a State-dependent Cutoff Effect. Anesthesiology 129:959-969|
|Feng, Hua-Jun; Forman, Stuart A (2018) Comparison of ??? and ??? GABAA receptors: Allosteric modulation and identification of subunit arrangement by site-selective general anesthetics. Pharmacol Res 133:289-300|
|McGrath, Megan; Ma, Celena; Raines, Douglas E (2018) Dimethoxy-etomidate: A Nonhypnotic Etomidate Analog that Potently Inhibits Steroidogenesis. J Pharmacol Exp Ther 364:229-237|
|Zhou, Xiaojuan; Desai, Rooma; Zhang, Yinghui et al. (2018) High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing ?4?3? subunits. PLoS One 13:e0191583|
|Jounaidi, Youssef; Cotten, Joseph F; Miller, Keith W et al. (2017) Tethering IL2 to Its Receptor IL2R? Enhances Antitumor Activity and Expansion of Natural Killer NK92 Cells. Cancer Res 77:5938-5951|
|Yu, Zhiyi; Cohen, Jonathan B (2017) Enantiomeric barbiturates bind distinct inter- and intrasubunit binding sites in a nicotinic acetylcholine receptor (nAChR). J Biol Chem 292:17258-17271|
|Jayakar, Selwyn S; Ang, Gordon; Chiara, David C et al. (2017) Photoaffinity Labeling of Pentameric Ligand-Gated Ion Channels: A Proteomic Approach to Identify Allosteric Modulator Binding Sites. Methods Mol Biol 1598:157-197|
Showing the most recent 10 out of 116 publications