Regulation of Ion Channel Function by the Host Lipid Bilayer Examined by a Stopped-Flow Spectrofluorimetric Assay

Abstract

To examine the function of ligand-gated ion channels in a defined membrane environment we developed a robust sequential-push fluorescence-based stopped-flow assay. The method is based on earlier studies (Moore and Raftery, PNAS, 1980, 77, p. 4509) (Karpen et al, Anal. Biochem., 1983, 135, p. 83) (Ingólfsson and Andersen, 2010, Assay Drug Dev. Technol., 8, p. 427), in which channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl+) of a water-soluble fluorophore (ANTS) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted. To validate the method, we explored the activation of wild type (WT) as well as a non-inactivating (E71A) mutant KcsA channels, by extravesicular protons (H+). For either channel type, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is less than 10%. WT and E71A KcsA activation curves are indistinguishable, and the activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology (Thompson et al., 2008, PNAS, 105(19):6900). We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. We found that increasing the acyl chain length (from C18:1 to C22:1 in di-acyl-PC), but not the mole fraction of POPG (above 0.25) in the bilayer-forming phospholipid mixture, alters KcsA proton gating. The bilayer thickness-dependent shift in the activation curve indicates an apparent decrease in H+ affinity and cooperativity. The method's reproducibility, control over bilayer environment and time resolution makes it a powerful assay for exploring ligand-activation and inactivation of ion channels, and how these processes vary with changes in the channels' lipid bilayer environment.