Characterization of Novel Poly(lipid) BLMs for Long-Term Ion Channel Scaffolds Towards the Development of High-Throughput Screening Devices

Abstract

Suspended lipid bilayers, or black lipid membranes (BLMs), have been used to study the electrophysiological properties of ion channels (ICs); however, BLMs assembled from natural, non-polymerizable lipids are inherently unstable due to the non-covalent associations on which they are based. Lifetimes of several hours are commonly observed in BLMs until rupture due to mechanical, thermal, or chemical insults. One potential improvement is the use of polymerizable phospholipids (poly(lipids)). BLMs prepared using dienoyl functionalized poly(lipids) and binary mixtures of fluid, non-polymerizable lipids with poly(lipids) were investigated for IC recordings.poly(BLMs) exhibited enhanced lifetimes from several hours to upwards of 4 weeks while maintaining IC functionality for one week. Activity of ICs that require membrane fluidity was retained using binary phospholipid mixtures of fluid and polymeric phospholipids. IC activity was retained by inducing domain formation, wherein ICs incorporated into the fluid domains. The binary membranes exhibited marked enhancement in stability resulting from fractional poly(lipids) polymerization. Additionally, ICs can be reconstituted into the fluid domains following photopolymerization and subsequent domain formation, a key requirement when UV-sensitive ICs are utilized. Here, the electrical properties, stability, and incorporation of pore-forming ICs, including hemolysin, alamethicin, and gramicidin, into poly(lipid) membranes are reported. Potential applications developing ligand-gated IC based sensors for high throughput screening are being investigated.In parallel to the characterization of poly(lipids) for potential long-term IC membranes, a model ligand-gated IC was expressed, characterized, and reconstituted into non-polymerizable lipids. Mutant KATP channels were expressed in mammalian and yeast systems. The orientations of mutant KATP channels were studied using electrophysiological and immunohistochemical techniques. Large quantities were expressed and purified from Pichia pastoris and functionally reconstituted into BLMs. ATP and long-chaing coenzyme A ester sensitivity was maintained in reconstituted in BLMs. KATP channels will serve as a model system for testing the effect of poly(lipid) BLMs on IC function. Future utilization of poly(lipid) BLMs in combination with ligand-gated ICs offer major advancements to potential increased throughput for IC screening.