Teflon foil

Planar lipid bilayer experiments were done with a vertical bilayer setup (IonoVation) as described previously (Braun et al., 2014 (link)). A 1% hexadecane solution (Merck KGaA) in n-hexane (Carl Roth) was used for pretreating the Teflon foil (Goodfellow GmbH). The hexadecane solution (ca. 0.5 µl) was added to the rim of the hole (100 µm in diameter) in the Teflon foil with a bent Hamilton syringe. The experimental solution contained 100 mM KCl and was buffered to pH 7.0 with 10 mM HEPES/KOH. For bilayer formation, we used the following lipids from Avanti Polar Lipids (Fig. S1): 1,2-diphythanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-diphythanoyl-sn-glycerol-3 phosphatidyl-serine (DPhPS), or n1,2-diphythanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPhPG). Cholesterol (Avanti Polar Lipids) was added to some DPhPC bilayers at a concentration of 0.15–25 mg/ml in n-pentane (Merck KGaA). Ion channel fluctuations were recorded with an EPC7 amplifier (List Medical) and digitized at 5 kHz after 1-kHz filtering with the LIH 1600 from HEKA Electronic. Data were analyzed with KielPatch (version 3.20 ZBM/2011) and Patchmaster (HEKA Electronic).

These experiments were done either on a vertical bilayer set up (IonoVation, Osnabrück, Germany) as described previously [6 (link),22 (link)] or in horizontal bilayers using an eNPR amplifier, equipped with a BLM_chip flowcell (Elements srl, Cesena, Italy). A 1% hexadecane solution (MERCK KGaA, Darmstadt, Germany) in n-hexane (Carl ROTH, Karlsruhe, Germany) was used for pretreating the Teflon foil (Goodfellow GmbH, Hamburg, Germany). The hexadecane solution (ca. 0.5 μL) was added to the rim of the hole (100 μm in diameter) in the Teflon foil with a bent Hamilton syringe (Hamilton Company, Reno, NV, USA). The experimental solution contained 100 mM KCl and was buffered to pH 7.0 with 10 mM HEPES/KOH. As a lipid we used 1,2-diphythanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-diphythanoyl-sn-glycerol-3 phosphatidyl-serine DPhPS, or n1,2-diphytanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) DPhPG.