Phosphatidylserine

KirBac3.1 proteoliposomes were incorporated into planar lipid bilayers separating two aqueous chambers (cis and trans). Bilayers were produced using the Mueller method 44 . Bilayers were formed across an aperture in a delrin cup with diameter 150-250 µm using a lipid mixture of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine (7:2:1 wt/wt, Avanti Polar Lipids) in n-decane (50 mg ml -1 , ICN Biomedicals). The cis chamber contained 250 mM KCl and 1.0 mM CaCl2 and the trans chamber contained 50 mM KCl and 0.1 mM CaCl2. Proteoliposomal fusion with bilayers was initiated by adding 5 µl aliquots of the proteoliposome preparation (described above) to the cis bath whilst stirring. During experiments, the composition of the cis solution was altered by a perfusion system 45 that allowed exposure of single channels to additions of spermine or KCl/NaCl substitutions within ~ 1 s. All solutions were pH buffered using 10 mM N-tris[hydroxymethyl] methyl-2aminoethanesulfonic acid (TES; ICN Biomedicals) and titrated to pH 7.4 using KOH (ICN Biomedicals). KCl was obtained from Aldrich and CaCl2 from BDH Chemicals. Experiments were carried out at room temperature (23 ± 2 o C).

Lipid analysis of dry spirulina was performed by Avanti Polar Lipids (Alabaster, AL, USA). Modified Bligh and Dyer extractions (Bligh and Dyer 1959) were performed with EquiSPLASH™ Lipidomix™, and Lipidyzer™ standards (Avanti Polar Lipids), and different chain lengths of phosphatidylglycerol, phosphatidylserine, and phosphatidylinositol (Avanti Polar Lipids) were added as internal standards (IS). Total lipids were dried then resolvated in 1 mL of 1:1 methylene chloride:methanol. Samples were injected in triplicate without dilution for LC-MS analysis. The methodology used was hydrophophilic interaction liquid chromatography (HILIC), which separates lipids into classes and subclasses that span a narrow retention time window (Hines et al. 2017) . A standard of 18:1 cholesterol ester was used as an IS for stigmasterols and brassicasterols. Data are presented as ng/mg of sample. Values were calculated using a point calibration determined by multiplying area ratio averages for the analyte to IS and multiplying by the concentration of IS.