Highly stable polymerized lipid membranes for ion channel recordings

Suspended lipid membranes, also known as black lipid membranes (BLMs), have long been used to study of ion channel proteins (ICs). However these non-covalently linked assemblies are inherently weak and suffer from short lifetimes due to mechanical and chemical disruptionns, hindering potential development of long-lived, IC/BLM-based sensors. Several methods have been developed to lengthen membrane lifetimes, including miniaturized apertures, polymerized lipid or triblock copolymer membranes, droplet interface bilayers, and encapsulated/supported bilayers. Recent work utilizing dienoyl poly(lipids) to create BLMs with enhanced stability that maintain the activity of incorporated ICs will be highlighted in this chapter. The electrical properties of poly(lipid) BLMs, along with incorporation and characterization of model ICs, will be discussed. We have developed several strategies for reconstitution of ICs, with the degree of BLM stabilization dependent on the composition of the membrane. For ICs that can withstand exposure to polymerization conditions, reconstitution can precede photopolymerization. For example, α-hemolysin (α-HL) was reconstituted prior to photopolymerization of pure poly(lipid) BLMs, and its activity was unaffected by exposure to UV light. Lipid polymerization increased BLM lifetimes from several hours up to several weeks. Other ICs require membrane fluidity for maintenance of function. In these cases, polymerizable lipids can be mixed with non-polymerizable lipids to create fluid domains in a poly(lipid) membrane. Peptide-forming channels such as alamethicin and gramicidin that are stable to UV irradiation can be inserted into the BLM before polymerization, whereas ICs that are susceptible to damage from UV irradiation can be inserted after polymerization. In these mixed BLMs, fluidity is maintained while the membrane lifetime is significantly lengthened relative to an unpolymerized BLM lifetime. For example, a BLM composed of a 1:1 mixture of poly(lipid):non-polymerizable lipid has a lifetime of about 4 days versus only 4 hours before polymerization. In addition, these mixed BLMs (and pure poly(lipid) BLMs) exhibit increased stability to transfers across the air-water interface and application of high electric fields. Overall the results of these studies demonstrate the potential for creating rugged biosensing platforms based on long-lived poly(lipid) membranes functionalized with ICs. Future applications of poly(lipid) membrane sensors and sensing arrays include high-throughput screening of pharmaceutical libraries for activity against ICs that are important in regulating cell functions.