This is a competing renewal of R01GM49202-09, which has for the past nine years supported the in-depth investigations of the molecular nature of general anesthetic interaction with the neuronal membrane constituents. Recent research efforts have combined the use of modern molecular biology techniques with various biophysical approaches, notably the state-of-the-art, high-resolution, solution- and solid-state NMR, to elucidate the effects of general anesthetics on the structures and dynamics of the transmembrane (TM) segments of the human glyeine receptors (GIyR). Unlike many conventional functional analysis that establishes the protein sequence-function relationship based on inferences from anesthetic sensitivity, this project aims at identifying the structure-function and dynamics-function relationships with direct binding and dynamics analysis at the sub-molecular and atomic levels. The support of this R01 grant has helped (1) to develop the NMR methods for non-destructive quantification of low-affinity anesthetic binding to receptor proteins in the membrane-mimetic environments;(2) to demonstrate that volatile general anesthetics as a molecule group distinguish themselves from the structurally similar nonimmobilizers (nonanesthetics) by their ability to preferentially target the amphipathic sites at the membrane interfacial regions;(3) to illustrate with many lines of experimental evidence that specific binding of volatile anesthetics to TM channels can lead to functional changes without necessarily causing secondary structural changes;(4) to conceptualize a testable hypothesis that the global changes in membrane protein dynamics might underlie a common mechanism for general anesthetic action on a diverse range of anesthetic-sensitive proteins;and (5) to reason with experimental support that the role of interfacial water should not be ignored in analyzing the action of general anesthetics on TM channels. These new insights, which were not fully comprehended at the time of the last competitive renewal 3.5 years ago, will advance the research to a new level. Substantive new structural and dynamical data on the c_l subunits of the human GIyR have been obtained by the investigators to warrant the continuation of the project with the following four new specific aims: 1. To differentiate the effects of anesthetics and nonanesthetics on the water accessibility to the backbone arnide protons of the interfacial and structural residues; 2. To quantify the anesthetic and nonanesthetic effects on the tertiary structure of the TM2+TM3 domains of the human GlyR _tl subunit, along with the extracellular linker, in DPC micelles and in oriented lipid bilayers; 3. To determine the backbone dynamics of the WT TM2+TM3 and two mutants, TM2(S267Y)+TM3 and TM2+TM3(A288W), in DPC micelles in the presence and absence of volatile anesthetics;and 4. To characterize the anesthetic effects on the quaternary structure of the homopentameric channel composed of TMI+TM2+TM3 domains of human GlyR ctl subunit. These new studies will eventually relate the structurally specific molecular events to the functional consequences of the action ofseneral anesthetics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM049202-16
Application #
7843704
Study Section
Special Emphasis Panel (NSS)
Program Officer
Cole, Alison E
Project Start
1995-01-01
Project End
2014-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
16
Fiscal Year
2010
Total Cost
$627,194
Indirect Cost
Name
University of Pittsburgh
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Jia, Li-Jie; Tang, Pei; Brandon, Nicole R et al. (2016) Effects of Propofol General Anesthesia on Olfactory Relearning. Sci Rep 6:33538
Kinde, Monica N; Bondarenko, Vasyl; Granata, Daniele et al. (2016) Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac. Proc Natl Acad Sci U S A 113:13762-13767
Zhou, David W; Mowrey, David D; Tang, Pei et al. (2015) Percolation Model of Sensory Transmission and Loss of Consciousness Under General Anesthesia. Phys Rev Lett 115:108103
Mowrey, David D; Kinde, Monica N; Xu, Yan et al. (2015) Atomistic insights into human Cys-loop receptors by solution NMR. Biochim Biophys Acta 1848:307-14
Wells, Marta M; Tillman, Tommy S; Mowrey, David D et al. (2015) Ensemble-based virtual screening for cannabinoid-like potentiators of the human glycine receptor ?1 for the treatment of pain. J Med Chem 58:2958-2966
Kinde, Monica N; Chen, Qiang; Lawless, Matthew J et al. (2015) Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC. Structure 23:995-1004
Chong, Yuan; Kleinhammes, Alfred; Tang, Pei et al. (2015) Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism. J Phys Chem B 119:5367-75
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
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
Samuelsson, Andrew R; Brandon, Nicole R; Tang, Pei et al. (2014) Cellular registration without behavioral recall of olfactory sensory input under general anesthesia. Anesthesiology 120:890-905

Showing the most recent 10 out of 55 publications