This application is a competing renewal of R01GM066358 for Years 9-13. The project has focused on computational investigations of the anesthetic action on ion channels with the long-term goal of determining the molecular mechanism of general anesthesia. One of the important discoveries from our computational studies of the 1422 and 17 subtypes of nicotinic acetylcholine receptor (nAChRs) is that anesthetic binding to these two nAChR subtypes are similar regardless of their different functional sensitivity to volatile anesthetics. Given that many anesthetic-sensitive and insensitive receptors within the same superfamily share high structure similarity, it is unlikely that anesthetic binding alone can differentiate functional sensitivities to anesthetics. With the recent success in producing ful-length 17 nAChR and its bacterial pentameric channel analogue GLIC, the availability of photoreactive anesthetics and the significant advancement in supercomputing power, this continuation application proposes to test the central hypothesis that only those anesthetic binding sites capable of shifting the equilibriums among the preexisting global conformations of the receptor will have functional impact. The residues forming such binding sites often involve long-range electrostatic interaction. The three new aims are: (1) to produce high quantity and quality full-length 17, 1722, and 1422 nAChRs, as well as GLIC and its anion-conducting mutants, for functional tests of the theoretical predictions. (2) To determine the anesthetic binding sites in the receptors and mutants produced in Aim 1 using photoreactive anesthetic labeling and fluorescence quenching. (3) To reveal underlying mechanisms of anesthetic action on these ion channels through advanced computations, including long-timescale (up to 5s timescale) molecular dynamics simulations to discover where and why only a sub-class of specific anesthetic binding can have functional impact, manifested as either inhibition or potentiation. The innovation of the proposed research is reflected in the novel hypotheses, integrated aproaches, and multidisciplinary collaborations. The significance of the proposed studies is the paradigm-shifting conceptual framework to quantify the action of low-affinity drugs on proteins, providing a new platform for future rational design of novel anesthetics with reduced side effects.

Public Health Relevance

General anesthesia is a critical component of modern medicine, yet its mechanisms remain undetermined. The computational studies in this project have shown that anesthetics interact directly with the anesthetic- insensitive 17 and anesthetic-supersensitive 1422 nicotinic acetylcholine receptors, suggesting that specific anesthetic binding alone is not sufficient to produce functional impact on proteins. The continuation of the project will combine experimental and computational approaches to dissect types of anesthetic binding that can effectively shift protein conformational equilibriums, leading to changes in channel function. This new approach will lead to a paradigm shift in the rational design of novel anesthetics with reduced side effects.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066358-11
Application #
8466321
Study Section
Special Emphasis Panel (ZRG1-SBIB-E (02))
Program Officer
Cole, Alison E
Project Start
2002-07-01
Project End
2015-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
11
Fiscal Year
2013
Total Cost
$304,062
Indirect Cost
$89,552
Name
University of Pittsburgh
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Tillman, Tommy S; Seyoum, Edom; Mowrey, David D et al. (2014) ELIC-?7 Nicotinic acetylcholine receptor (?7nAChR) chimeras reveal a prominent role of the extracellular-transmembrane domain interface in allosteric modulation. J Biol Chem 289:13851-7
Bondarenko, Vasyl; Mowrey, David D; Tillman, Tommy S et al. (2014) NMR structures of the human ?7 nAChR transmembrane domain and associated anesthetic binding sites. Biochim Biophys Acta 1838:1389-95
Chiara, David C; Gill, Jonathan F; Chen, Qiang et al. (2014) Photoaffinity labeling the propofol binding site in GLIC. Biochemistry 53:135-42
Mowrey, David; Chen, Qiang; Liang, Yuhe et al. (2013) Signal transduction pathways in the pentameric ligand-gated ion channels. PLoS One 8:e64326
Bondarenko, Vasyl; Mowrey, David; Liu, Lu Tian et al. (2013) NMR resolved multiple anesthetic binding sites in the TM domains of the ?4?2 nAChR. Biochim Biophys Acta 1828:398-404
Tillman, Tommy; Cheng, Mary H; Chen, Qiang et al. (2013) Reversal of ion-charge selectivity renders the pentameric ligand-gated ion channel GLIC insensitive to anaesthetics. Biochem J 449:61-8
Mowrey, David; Cheng, Mary Hongying; Liu, Lu Tian et al. (2013) Asymmetric ligand binding facilitates conformational transitions in pentameric ligand-gated ion channels. J Am Chem Soc 135:2172-80
Pan, Jianjun; Chen, Qiang; Willenbring, Dan et al. (2012) Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine. Nat Commun 3:714
Bondarenko, Vasyl; Mowrey, David; Tillman, Tommy et al. (2012) NMR structures of the transmembrane domains of the ýý4ýý2 nAChR. Biochim Biophys Acta 1818:1261-8

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