1 palmitoyl 2 oleoyl phosphatidylglycerol

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.

Lipids were purchased from Avanti Polar Lipids: 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol (POPI, PI), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′-phosphate) [PI(4)P], 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′,4′-bisphosphate) [PI(3,4)P₂], 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate) [PI(4,5)P₂], 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′,4′,5′-trisphosphate) [PI(3,4,5)P₃]. All other reagents and chemicals, such as for recording solutions and cell culture media, were purchased from FUJIFILM Wako Pure Chemical or Nacalai Tesque.