Membrane protein function is regulated by changes in the host membrane's lipid composition by mechanisms that range from specific (chemical) interactions between proteins and individual lipid molecules, to non-specific (physical) interactions between proteins and the bilayer. The proposed studies focus on the physical regulation of membrane protein function, and explores how biologically active molecules (including drugs and drug candidates) alter lipid bilayer properties, as sensed by bilayer-spanning channels. This bilayer regulation of protein function occurs because hydrophobic interactions between membrane proteins and their host bilayer, couple a protein's conformational preference to the bilayer physical properties. This means that membrane protein conformational transitions (between states I and II) that involve the protein/bilayer boundary will have I?II an associated energetic cost, which becomes the bilayer contribution ( ?Gbilayer ) to the free energy difference for the conformational change. The rationale for the proposed studies is that drug-induced changes in lipid bilayer properties alter membrane I?II I?II protein function through the ensuing changes in ?Gbilayer ). Drug-induced changes in ?Gbilayer will be determined for a well-defined conformational transition, the gramicidin (gA) monomer?dimer equilibrium, using both stopped-flow fluorescence quench experiments to screen libraries of compounds and single-channel experiments to dissect how selected compounds alter bilayer properties. These results will be combined with results of cytotoxicity studies on selected cell lines, to understand how the changes in membrane protein function that arise from drug-induced changes in lipid bilayer properties may alter cell function. Our results to I?II date show that more 90% of molecules that alter ?Gbilayer (for the gA monomer?dimer equilibrium) by more than kBT (where kB is Boltzmann's constant and T is temperature in kelvin) are cytotoxic at the tested I?II concentration, and that all molecules that alter ?Gbilayer by more than 2 kBT are cytotoxic. For comparison, less than 20% of compounds with minimal bilayer-modifying effect are cytotoxic. This provides mechanistic insight into drug toxicity (though a drug may be toxic for any number of reasons not related to changes in bilayer properties). The proposed studies will use select groups of drugs to elucidate the molecular basis for the altered bilayer properties using symmetric and asymmetric bilayers. We will extend these studies in larger- scale studies on chemical libraries and explore the relationship between molecular structure, bilayer-modifying potency and cytotoxicity. The biological implications of this mechanism will be explored in studies on ?real? ion channels (Kv and Nav channels in whole-cell electrophysiological studies and KcsA reconstituted in bilayers of defined composition). We also will pursue the larger scale implications for cell and organism function in toxicity studies on cell lines and zebrafish, to get insight into what compounds that are broadly toxic to multiple tissues versus those that affect mainly one tissue, which will provide insights into the generality of this mechanism.

Public Health Relevance

Many molecules that are used to manipulate biological function, including drugs in clinical use, have physico- chemical properties that cause them to adsorb to lipid bilayers and thereby alter lipid bilayer properties, which will lead to non-specific changes in lipid bilayer properties. The proposed studies are aimed at understanding the bilayer-modifying effects of clinically and biologically important molecules, and how these changes in bilayer properties may cause cytotoxicity. The insights that will be gained will be important for public health because the experimental paradigm will allow for the identification of molecules that are likely to have unacceptable off-target effects, which will be important because it will provide a mechanistic basis for the development of novel screens of drug candidates.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021342-43
Application #
9773213
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Preusch, Peter
Project Start
1977-06-01
Project End
2022-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
43
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Physiology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Dockendorff, Chris; Gandhi, Disha M; Kimball, Ian H et al. (2018) Synthetic Analogues of the Snail Toxin 6-Bromo-2-mercaptotryptamine Dimer (BrMT) Reveal That Lipid Bilayer Perturbation Does Not Underlie Its Modulation of Voltage-Gated Potassium Channels. Biochemistry 57:2733-2743
Zhang, Mike; Peyear, Thasin; Patmanidis, Ilias et al. (2018) Fluorinated Alcohols' Effects on Lipid Bilayer Properties. Biophys J 115:679-689
Posson, David J; Rusinova, Radda; Andersen, Olaf S et al. (2018) Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies. Methods Mol Biol 1684:223-235
Beaven, Andrew H; Sodt, Alexander J; Pastor, Richard W et al. (2017) Characterizing Residue-Bilayer Interactions Using Gramicidin A as a Scaffold and Tryptophan Substitutions as Probes. J Chem Theory Comput 13:5054-5064
O'Donnell, John P; Cooley, Richard B; Kelly, Carolyn M et al. (2017) Timing and Reset Mechanism of GTP Hydrolysis-Driven Conformational Changes of Atlastin. Structure 25:997-1010.e4
Herold, Karl F; Sanford, R Lea; Lee, William et al. (2017) Clinical concentrations of chemically diverse general anesthetics minimally affect lipid bilayer properties. Proc Natl Acad Sci U S A 114:3109-3114
Menny, Anaïs; Lefebvre, Solène N; Schmidpeter, Philipp Am et al. (2017) Identification of a pre-active conformation of a pentameric channel receptor. Elife 6:
Sodt, Alexander J; Beaven, Andrew H; Andersen, Olaf S et al. (2017) Gramicidin A Channel Formation Induces Local Lipid Redistribution II: A 3D Continuum Elastic Model. Biophys J 112:1198-1213
Lum, Kevin; Ingólfsson, Helgi I; Koeppe 2nd, Roger E et al. (2017) Exchange of Gramicidin between Lipid Bilayers: Implications for the Mechanism of Channel Formation. Biophys J 113:1757-1767
Herold, Karl F; Andersen, Olaf S; Hemmings Jr, Hugh C (2017) Divergent effects of anesthetics on lipid bilayer properties and sodium channel function. Eur Biophys J 46:617-626

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