LOCATING GENERAL ANESTHETIC BINDING SITES IN GABAA AND ACETYLCHOLINE RECEPTORS Our goal is to determine the number and location of the binding sites for general anesthetics in GABAARS, a major site of action of a structurally diverse group of general anesthetics used clinically, and to compare them with their binding sites in nicotinic acetylcholine receptors (AcChoRs), receptors that are both members of the """"""""Cys-loop"""""""" superfamily of neurotransmitter-gated ion channels. Most anesthetics potentiate the action of GABA at the inhibitory GABAA receptors, while they inhibit the excitatory AcChoRs in brain and skeletal muscle. Within a single receptor multiple potential anesthetic binding sites are present in the pockets that exist within receptor subunits and at subunit interfaces, including the ion channel. It is our hypothesis that each anesthetic, depending on structural class, will interact preferentially with different receptor sites. An improved understanding of the diversity of general anesthetic binding sites within neurotransmitter-gated ion channels will allow for the design of safer anesthetic agents. We propose to use photoaffinity labeling and protein chemistry techniques to identify the binding sites for photoreactive aliphatic alcohols and analogs of etomidate, propofol, and barbiturates in GABAARS isolated from detergent extracts of bovine brain and cultured cells, in neuronal a4p2 AcChoRs isolated from cultured cells, and in muscle-type AcChoRs in nicotinic postsynaptic membranes isolated from Torpedo electric organ. We will determine whether a single anesthetic binding site in a GABAAR (or AcChoR) can be occupied by drugs that, depending on structure, act as potentiators or inhibitors. These photoaffinity labeling studies make use of the Synthetic Chemistry, Protein Chemistry and Protein Production Cores. Our structural studies are complementary to the time- resolved photolabeling studies in (Miller/Raines Project) and to the mutational analyses (Forman Project) designed to determine whether the sites we identify are sites of functional potentiation or inhibition.

Public Health Relevance

GABAARS in the brain are a major site of action of many clinically used anesthetics of diverse chemical structure, but the number and location of anesthetic binding sites within GABAARS or in the structurally related nicotinic acetylcholine receptors remains unknown. Identification of these sites will contribute to the development of anesthetics with greater selectivity and fewer side-effects.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM058448-14
Application #
8381229
Study Section
Special Emphasis Panel (ZGM1-PPBC-0)
Project Start
Project End
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
14
Fiscal Year
2012
Total Cost
$368,422
Indirect Cost
$70,409
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Ma, Celena; Pejo, Ervin; McGrath, Megan et al. (2017) Competitive Antagonism of Anesthetic Action at the ?-Aminobutyric Acid Type A Receptor by a Novel Etomidate Analog with Low Intrinsic Efficacy. Anesthesiology 127:824-837
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
Savechenkov, Pavel Y; Chiara, David C; Desai, Rooma et al. (2017) Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands. Eur J Med Chem 136:334-347
Feng, Hua-Jun; Forman, Stuart A (2017) Comparison of ??? and ??? GABAA receptors: Allosteric modulation and identification of subunit arrangement by site-selective general anesthetics. Pharmacol Res :
Nourmahnad, Anahita; Stern, Alex T; Hotta, Mayo et al. (2016) Tryptophan and Cysteine Mutations in M1 Helices of ?1?3?2L ?-Aminobutyric Acid Type A Receptors Indicate Distinct Intersubunit Sites for Four Intravenous Anesthetics and One Orphan Site. Anesthesiology 125:1144-1158
Forman, Stuart A; Miller, Keith W (2016) Mapping General Anesthetic Sites in Heteromeric ?-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes. Anesth Analg 123:1263-1273
Ziemba, Alexis M; Forman, Stuart A (2016) Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of ?1?3?2L GABAA Receptors. PLoS One 11:e0154031
Zhang, Xi (2016) Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias. Mol Cell Proteomics 15:3665-3684
Amlong, Corey A; Perkins, Mark G; Houle, Timothy T et al. (2016) Contrasting Effects of the ?-Aminobutyric Acid Type A Receptor ?3 Subunit N265M Mutation on Loss of Righting Reflexes Induced by Etomidate and the Novel Anesthetic Barbiturate R-mTFD-MPAB. Anesth Analg 123:1241-1246
Chiara, David C; Jounaidi, Youssef; Zhou, Xiaojuan et al. (2016) General Anesthetic Binding Sites in Human ?4?3? ?-Aminobutyric Acid Type A Receptors (GABAARs). J Biol Chem 291:26529-26539

Showing the most recent 10 out of 108 publications