Some 40 million patients are given general anesthesia each year in the USA using agents that haven't changed for decades. General anesthetics are used as hypnotics during procedures, sedatives in the ICU, and as anticonvulsants. These drugs have low safety margins and are particularly harmful to newborns, the severely ill, and the elderly. The molecular mechanisms of general anesthesia have proved difficult to reveal, hampering the design of improved agents. This interdisciplinary PPG brings together a group of experienced scientists to focus on mechanisms underlying the ability of general anesthetics to enhance the activity of the inhibitory GABAA receptors (GABAAR) and glycine receptors (GlyR), major targets of anesthetics. The overall hypothesis is that the various actions of general anesthetics are mediated by a number of distinct binding sites on these receptors, that their location and affinity varies with the receptor's subunit composition and conformational state. The overall aims of the PPG are to: (i) locate the anesthetic binding sites on heteromeric GABAARs and GlyRs using anesthetic photolabels, (ii) define anesthetic structure-activity relationships at distinct sites, and (iii) define how their occupancy allosterically modulates receptor function using electrophysiology and targeted mutations. Project 1 (PI: Cohen) will locate the unknown binding sites for steroids and propofol anesthetics and a convulsant barbiturate on synaptic (?1?3?2) and extrasynaptic (?4?3?) GABAARs and on ?1? GlyRs. Project 2 (PI: Miller) focuses on exploring the molecular pharmacology of the different anesthetic binding sites that the PPG has discovered on ?1?3?2 and ?4?3? GABAARs, with the objective of understanding how to improve the specificity of anesthetic action on synaptic and extrasynaptic receptors. In addition, new binding sites that have high affinity for the activated states will be sought. Project 3 (PI: Forman) will use mutations and electrophysiology to define the functional roles, including drug specificity, of distinct general anesthetic binding sites, particularly the inter-subunit transmembrane sites on ?1?3?2 and ?4?3? GABAARs and ?1? GlyRs. Photolabeling results and homology models will guide mutagenesis, while allosteric co-agonist modeling will be used as a quantitative analytical framework for electrophysiological data. The Projects are supported by four Core facilities. An Administrative Core (Core A;PI: Miller) coordinates the activities of the components of the PPG, also arranging external oversight through a distinguished Advisory Board. A Synthetic Chemistry Core (Core B;PI: Bruzik) develops and supplies novel general anesthetics for photolabeling and structure activity relationships. A Protein Chemistry Core (Core C;PI: Cohen) locates anesthetic sites of photoincorporation and develops homology models of receptors to guide research. A Protein Production Core (Core D;co-PIs: Miller &Forman) supplies nanomoles of heterologously expressed, purified and functionally reconstituted human neuronal receptors for photolabeling and other studies.

Public Health Relevance

Overall Narrative General anesthetics are essential to modern medicine, yet they have low safety margins especially in sick or elderly patients, requiring delivery by highly trained clinicians. General anesthetics interact with many receptors in the brain to cause both anesthesia and side effects. This Program Project aims to provide new knowledge so that new anesthetics can target just the anesthetic receptors and not those responsible for side effects.

National Institute of Health (NIH)
Research Program Projects (P01)
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Special Emphasis Panel (ZGM1)
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Massachusetts General Hospital
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Forman, Stuart A; Chiara, David C; Miller, Keith W (2015) Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors. Neuropharmacology 96:169-77
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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

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