1 palmitoyl 2 oleoyl phosphatidylethanolamine

Single-channel recording was performed via a Nanion Orbit-mini planar bilayer system, using 70% 1-palmitoyl-2-oleoyl-phosphatidylethanolamine / 30% 1-palmitoyl-2-oleoyl-phosphatidylglycerol (Avanti Polar Lipids), 5 mg/mL in n-nonane to form bilayers. Single channels were inserted by addition of Ec2-reconstituted liposomes to the 'cis' side of the bilayer, and current was recorded at +200 mV holding potential (cis side defined as zero voltage) in symmetrical solutions containing 300 mM NaF, 10 mM NaCl, 15 mM MOPS-NaOH pH 7.0, with 75–150 nM of monobody S9 added to the cis chamber unless otherwise noted. Recordings were low-pass filtered at 160 Hz, digitized at 1.25 kHz, and analyzed in Clampfit 10 after further digital filtering at 100 Hz. Channel conductance, averaged from 10–14 separate single-channel records under each condition, was measured from the difference between open vs blocked current in each recording. All recordings shown in figures are representative of behavior seen on many channels in multiple reconstitutions.

Porcine brain sphingomyelin (SM); 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE); 1-palmitoyl-2-oleoyl-L-serine (POPS); cholesterol (CHOL) and 1-palmitoyl-2-6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl- phosphatidylcholine (C₆-NBD-PC) were purchased from Avanti Polar Lipids (Alabaster, AL). [³H]-cholesterol was purchased from American Radiolabeled Chemicals, Inc (St. Louis, MO). Lipids were dissolved in chloroform and stored at −20°C. Concentrations of lipids were measured by dry weight. (2-hydroxypropyl)-α-cyclodextrin (HPαCD) average molecular weight 1180, 1 M 2,4,6-trinitrobenzenesulfonic acid in water (TNBS) and sodium hydrosulfite (sodium dithionite) were purchased from Sigma-Aldrich (St. Louis, MO). LW peptide (acetyl-K₂W₂L₈AL₈W₂K₂-amide) and pL4A18 peptide (acetyl-K₂LA₉LWLA₉LK₂-amide) were purchased from Anaspec (San Jose, CA) and used without further purification. High-performance thin-layer chromatography (HP-TLC) plates (Silica Gel 60) were purchased from VWR International (Batavia, IL).