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

Abstract

Membrane active peptides represent a class of soluble proteins that interact and disrupt the plasma membrane. Examples of these include antimicrobial peptides, cancer therapeutics, and cell-penetrating peptides. These peptides are amphipathic and, in a concentration dependent manner, can self assemble to destabilize the lipid bilayer. These peptides are rich in positively charged lysine and arginine residues and thus have a strong preference for negatively charged bilayers. In order to study the insertion mechanism and kinetics of these peptides, we have designed a negatively charged, supported bilayer platform on silicon. The negative charge serves to electrostatically drive peptides to bind to the lipid bilayer interface. Furthermore, this platform is electrically addressable through electrochemical impedance spectroscopy, which yields bilayer resistance, thickness, and structural heterogeneity data. This platform consists of an asymmetrical bilayer with 10 mol% negatively charged POPS, cholesterol, and POPC in the upper leaflet and DPhPC lipids in the lower leaflet, all supported by a PEG cushion on a silicon wafer. Resistances up to 2*10 4 Ohm cm 2 and capacitances of 0.8 yF cm À2 have been measured for the platform. The high resistance allows for high accuracy in the detection of the activity of membrane active peptides of interest.

Nanodomain Formation in Planar Supported Lipid Bilayers Composed of Fluid and Polymerized Dienoyl Lipids

“…For example, in 7:3 DPhPC/poly­(bis-SorbPC) PSLBs, only 22% of the PSLB area was occupied by poly­(bis-SorbPC) domains. This apparent discrepancy is due to the different molecular areas of DPhPC and poly­(bis-SorbPC), for which respective values of 125 Å 2 per molecule and 75 Å 2 per molecule were obtained from Langmuir isotherms . Using these values, the calculated area percentages listed in Table were determined and these are in good agreement with the experimentally determined percentages.…”

“… a Obtained from analysis of three individually prepared samples for each type of PSLB, and three 1 μm × 1 μm spots on each sample. b Calculated from molecular areas of poly­(bis-SorbPC) and DPhPC that were obtained from Langmuir isotherms c Height difference between poly­(bis-SorbPC) and DPhPC domains (Δ h ) calculated as h DPhPC – h poly(bis‑SorbPC) . …”

“… b Calculated from molecular areas of poly­(bis-SorbPC) and DPhPC that were obtained from Langmuir isotherms …”

“…It is well established that for many membrane-associated proteins and peptides, a highly fluid bilayer is required for their bioactivity, − and in these cases, the decreased fluidity accompanying dienoyl lipid polymerization may adversely affect bioactivity. This was observed when gramicidin and alamethicin, peptides that laterally diffuse and associate to form transmembrane ion channels (ICs), were inserted into bis-DenPC BLMs. , UV-initiated lipid polymerization nearly eliminated the IC activity of these peptides. In contrast, in BLMs composed of a mixture of poly­(bis-DenPC) and diphytanoylphosphatidylcholine (DPhPC), a nonpolymerizable fluid lipid commonly used to form BLMs, the IC activity of gramicidin and alamethicin was retained after polymerization. , Consistent with their composition, the stability of these mixed membranes was intermediate between that of poly­(bis-DenPC) BLMs and unpolymerized BLMs.…”

“…This was observed when gramicidin and alamethicin, peptides that laterally diffuse and associate to form transmembrane ion channels (ICs), were inserted into bis-DenPC BLMs. , UV-initiated lipid polymerization nearly eliminated the IC activity of these peptides. In contrast, in BLMs composed of a mixture of poly­(bis-DenPC) and diphytanoylphosphatidylcholine (DPhPC), a nonpolymerizable fluid lipid commonly used to form BLMs, the IC activity of gramicidin and alamethicin was retained after polymerization. , Consistent with their composition, the stability of these mixed membranes was intermediate between that of poly­(bis-DenPC) BLMs and unpolymerized BLMs. It was hypothesized that the polymerization produces a phase-segregated membrane composed of nonpolymerizable fluid lipid and poly­(lipid) domains.…”