The research proposed in Project 4 will strive to elucidate the mechanisms by which general anesthetics modulate ion channels in the central nervous system to induce reversible immobilization and amnesia. Sensitivity to inhibition, potentiation, or activation by anesthetics is observed in several ion channel families; ligand-gated ion channels (LGIC), and voltage-gated cation channels (VGCCs), which may all serve to transduce the clinical effects of general anesthetics. Project 4 will test the hypothesis that modulation by anesthetics reflects a combination pore and allosteric sites, and that it is the balance of affinities that cor trol the ultimate effects of anesthetics on electrophysiology.
Aim 1 will rely on the unique capacity of computer simulations based on fully atomistic models of the relevant membrane proteins in a hydrated membrane environment to probe structural details and measure affinities of anesthetics for isolated binding sites. To this end, we will measure the binding affinities of isoflurane and propofol to both the channel lumen and two previously identified transmembrane sites on the nicotinic acetylcholine receptor (nAChR) and yaminobutyric acid class A receptor (GABAAr) ofthe Cys-loop superfamily using the alchemical free energy perturbation (FEP) methodology. Additional sites identified from photoaifinity labeling to Cys-loop receptors in Project 1 (Eckenhoff) will also be included in this Aim, resulting in a ranking for likely significance of each site.
In Aim 2 we will employ computer simulations to gain insight into determinants of sensitivity in VGCCs. In detail, we will identify binding sites and compute their affinities of isoflurane and propofol for anestheticsensitive K-Shaw2 and the prokaryotic NaChBac. Our computations will also aid in interpretation of results of Project 2 (Covanrubias), by focusing on the role of the possible allosteric site (S4-S5 linker) in these channels.
Specific Aim 3 will build upon a recently derived molecular dynamics (MD) simulation model for the NaChBac to explore the effects of anesthetics on the various functional states of the channel (e.g., open, closed, resting etc). In detail, we will expose membrane-bound models of NaChBac to clinical concentrations of isoflurane, and propofol (and other interesting anesthetics developed in Project 1) in long-time MD simulations to determine plausible binding sites and thereby link to experimental activities in other projects. The project will also develop a model for sevofluorane.

Public Health Relevance

General anesthetics are used on hundreds of millions of patients annually, even though exposure to these toxic chemicals is one of the highest-risk factors involved in modern surgery. Using computer simulation, this project aims to understand the mechanisms through which anesthetics interact with membrane proteins to confer both desirable and undesirable effects, and thereby ultimately impact the rational design of safer anesthetics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM055876-15
Application #
8740498
Study Section
Special Emphasis Panel (ZGM1-PPBC-5)
Project Start
Project End
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
15
Fiscal Year
2014
Total Cost
$309,171
Indirect Cost
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Carswell, Casey L; Hénault, Camille M; Murlidaran, Sruthi et al. (2015) Role of the Fourth Transmembrane α Helix in the Allosteric Modulation of Pentameric Ligand-Gated Ion Channels. Structure 23:1655-64
Woll, Kellie A; Weiser, Brian P; Liang, Qiansheng et al. (2015) Role for the propofol hydroxyl in anesthetic protein target molecular recognition. ACS Chem Neurosci 6:927-35
Cournia, Zoe; Allen, Toby W; Andricioaei, Ioan et al. (2015) Membrane Protein Structure, Function, and Dynamics: a Perspective from Experiments and Theory. J Membr Biol 248:611-40

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