Highly Stable Poly(Lipid) Bilayers for Long-Term Ion Channel Recordings

Abstract

Long-term ion channel (IC) screening using cell-based assays is currently limited by throughput and cell to cell variability. ICs isolated and reconstituted into suspended lipid membranes offer an isolated view into IC structure and function, but IC recordings are limited by the short lifetime of the bilayer. Polymerizable lipids (poly(lipids)) offers one potential strategy for long-term investigation and screening of ICs and has been investigated with several model ion channels.The electrical properties and stability of lipid bilayers suspended across glass micropipettes compared natural and polymerized lipids. Breakdown voltage, capacitance, and conductance of the several pure and mixed polymerizable/non-polymerizable lipid bilayers were determined using a patch clamp apparatus.Using, polymerizable phospholipids, we have synthesized membranes with markedly enhanced lifetimes from ca. 3 hours to upwards of 3 weeks. These poly(lipid) bilayers have been used to monitor IC activity of alpha-hemolysin for ca. 1week before loss of alpha-hemolysin. However, poly(lipid) bilayers are rigid and do not support the function of ICs that require membrane fluidity, To address this limitation, binary bilayers composed of poly(lipids) and non-polymerizable lipids were investigated. The resulting mixed bilayers demonstrate markedly enhanced long term stability compared to non-polymerized bilayers and facility IC studies. Alamethicin, a model for voltage-gated ion channels, was shown to be non-functional when reconstituted into homogeneous poly(lipid) bilayers, whereas reconstitution in to mixed bilayers revealed alamethicin activity that and enhanced membrane stability.A functional, truncated form of the KATP channel complex, 6xHis-EGFP-Kir6.2d26, was chosen as a model ligand-gated IC, expressed and purified from yeast. Long-term goals are to reconstitute the truncated version of KATP channels into polymerizable and non-polymerizable bilayers using varying strategies into biomimetric sensing platforms screening ligands and drug candidates for activity.