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 ( ΔGbilayer ) 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 ΔGbilayer ). Drug-induced changes in ΔGbilayer 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 ΔGbilayer (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 ΔGbilayer 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 ...