Mechanisms Underlying Drug-Mediated Regulation of Membrane Protein Function

Abstract

The hydrophobic coupling between membrane proteins and their host lipid bilayer provides a mechanism by which bilayer-modifying drugs may alter protein function. The regulation of membrane protein function thus may involve mechanisms ranging from drug binding to specific sites, over more non-specific drug-protein interactions, to drug-induced alteration of bilayer properties. To tease apart these mechanisms, we examine how the prototypical bacterial potassium channel, KcsA, is regulated by bilayer-modifying drugs using a fluorescence-based approach to quantify changes in both KcsA function and lipid bilayer properties (using gramicidin channels as probes). Comparing the relationships between changes in bilayer properties and different KcsA gating steps, and the drugs effects on the bilayer environment reveal that drug-induced regulation of membrane protein function indeed involves a combination of non-specific but direct and bilayer-mediated mechanisms. Both mechanisms are likely to be important whenever there is overlap between a drug's bilayer-modifying and clinical concentration ranges. Using Isothermal Titration Calorimetry (ITC) we determine the binding affinity of the drugs for purified, detergent-solubilized KcsA, which we compare to the affinity for the lipid bilayer in order to separate these direct and bilayer-mediated mechanisms for regulating KcsA function.