General anesthetics are widely used during surgery and other clinical procedures, yet they are extremely dangerous and their mechanisms remain poorly understood. Understanding their actions at the molecular level will enable development of new drugs with more therapeutic specificity. The overall aim of this project is to define the mechanisms of potent general anesthetics at known therapeutic targets: synaptic and extrasynaptic GABAA receptors. Photolabeling, structural homology modeling, mutant studies, and mechanistic analysis have established structural and functional models for etomidate actions at synaptic aiP2Y2L receptors, providing a new paradigm for other potent anesthetics such as propofol and barbiturates, and extra-synaptic GABAARS containing different subunits. Our working hypothesis is that at synaptic cci(32/372 and extrasynaptic OAPSS GABAA receptors, different potent anesthetics bind to distinct sub-regions of intra-membrane pockets formed at a-p subunit interfaces, with each subunit contributing a channel-lining transmembrane M2 domain and one other: a-M1 and P-M3. Anesthetic binding facilitates rearrangement of these pockets, stabilizing open channel states, thereby reducing neuronal activity. We propose to electrophysiologically study expressed GABAARS of defined subunit composition, developing a detailed mechanism of anesthetic actions at the macrocurrent level and determining how structural changes in the putative drug pockets affect anesthetic interactions and channel gating.
Specific Aim 1 is to investigate anesthetic-photolabeled amino acids in aip2/3y2L (synaptic) GABAARS and their roles in channel gating and the affinity/efficacy of etomidate, propofol, and pentobarbital. Mutagenesis, including cysteine mutagenesis and state-dependent modification, together with electrophysiological analysis based on an equilibrium co? agonist gating model will be used.
Specific Aim 2 is to build an allosteric-kinetic model for etomidate modulation of aiP2/3'y2L GABAARS and to develop allosteric co-agonist models for propofol and pentobarbital. Macrocurrent kinetic electrophysiology will be performed using an """"""""artificial synapse"""""""" for rapid application of GABA and anesthetics, and kinetic models will be assessed for their ability to quantitatively account for the data.
Specific Aim 3 is to extend the above approaches to a4p35 (extrasynaptic) GABAARS.
Specific Aim 4 is to collaborate with other PPG projects by assessing new photolabel anesthetics and guiding time-resolved photolabeling experiments.

Public Health Relevance

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM058448-14
Application #
8381233
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
$222,559
Indirect Cost
$70,408
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

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