Asolectin

Purified VDAC1 was reconstituted into a Planar Lipid Bilayer (PLB) apparatus (Warner Instruments, Hamden, CT, USA), previously described46 (link). Bilayers were prepared using asolectin (Sigma) dissolved in decane (Sigma) containing 1% chloroform (Sigma) across a 200 μm hole in a derlin cuvette (Warner Instruments). Experiments were performed in 1 M KCl, 10 mM Hepes, pH 7.040 (link)46 (link). Control experiments using empty membrane and/or detergents were performed to avoid activity in any of the above solutions. Data were acquired using a Bilayer Clamp amplifier (Warner Instruments) at 100 μs/point, filtered at 200 Hz and analyzed offline using Clampfit 10.4 program set (Axon Instruments, Union City, CA, USA). Single channel analysis of VDAC1 was performed in presence or not of 0.2 μM of SOD1 WT or G93A, 15 μM NHK1 or ScNHK1 peptide.

All of the PLB assays were performed using an Orbit mini device (Nanion Technologies GmbH., Munich, Germany) and a MECA 4 Recoding Chip, 100 µm (Ionera, Freiburg, Germany). A potassium-based buffer (150 mM KCl, and 20 mM Tris-HCl (pH7.5)) was injected into the chamber of the MECA 4 chip, and the air was removed from each microcavity by gently pipetting. A planar lipid bilayer was created on each microelectrode cavity by coating them with 1,2-Diphytanoyl-sn-Glycero-3-Phosphatidylcholine (DPhPC, Avanti Pollar Lipids Inc., Alabaster, AL, USA) or asolectin (Sigma-Aldrich, St. Louis, MO, USA) solubilized in decan at a 10 µg/mL concentration. During this step, the condition of the membrane was monitored using Orbit mini with elements data reader software. Finally, 5µ L of purified proteoliposome was injected into the chamber while recording the signal variation of the voltage for ten minutes to one hour.
All of the PLB data were analyzed using Clampfit 10.4 software (Molecular Devices, Palo Alto, CA, USA). First, the obtained signals were compensated for by making the baseline 0 pA and 0 mV, then all points of the signals were analyzed by plotting histograms. For the I-V plot, the average amplitude was calculated by fitting the Gaussian function against all points of the histograms.

Multi-layered vesicles (MLVs) were prepared from films of asolectin (Sigma-Aldrich Co., USA) deposited on the bottom of round flasks after solvent (chloroform) evaporation under N₂ flow, followed by vacuum drying for at least 3 h. Films were hydrated with buffer (citrate/phosphate pH 5.5 containing 150 mM NaF), at 40^°C, to reach a final lipid concentration of 10 mg/mL and vortex mixed. MLVs were submitted to an extrusion process through polycarbonate membranes (Whatman® Nuclepore Track-etch Membrane, Sigma-Aldrich Co., USA) in two steps: first, through 400 nm membranes (6 times), then through two stacked 100 nm membranes, using an Avanti mini-extruder (Avanti Polar Lipids Inc., USA), at 40^°C, rendering LUVs. For ATR-FTIR experiments vesicles were prepared in deuterated buffer and left incubating for 2h prior to spectra acquisition.
Peptide samples were prepared by diluting the stock solution (in D₂O for IR experiments) with buffer/deuterated buffer, or with buffer containing either 40% 2,2,2-trifluoroethanol (or 2,2,2-trifluoroethanol-d3, TFE), or 8 mM sodium dodecyl sulfate (SDS) or asolectin (AZO) LUVs, at 100 and 250 μg/mL.

For ATR-FTIR measurements, detergent-solubilized M₂R was reconstituted into asolectin (Sigma) liposomes with a 20-fold molar excess. The detergent molecule was removed by incubation with Bio-beads SM-2 (Bio-Rad, CA, USA). After removal of Biobeads, the lipid-reconstituted M₂R was collected by ultracentrifugation for 30 min at 222,000×g at 4 °C. After several cycles of wash/spin, lipid-reconstituted M₂R was suspended in a buffer composed of 5 mM phosphate (pH 7.5) and 10 mM KCl.

Asolectin from soybean (Sigma-Aldrich product nr: 11145, lot nr: BCB66221V) was used as received. It contains approximately lecithin (25–33%), cephalin and phosphatidylinositol, saturated fatty acids (24%), mono-unsaturated (14%) and poly-unsaturated fatty acids (62%). Methyl-4-(1-methylethenyl)-cyclohexene (limonene), isooctane, curcumin and vanillin were obtained from Sigma-Aldrich and used as received without further purification.

The lipid bilayer membranes containing KcsA channels were obtained as previously reported^{22 (link),36 (link)}. Briefly, we poured 1.2 ml of an aqueous recording solution (200 mM KNO₃ or KCl, 10 mM MES (pH 4.0)), into a 1.5-ml microtube; subsequently, 150 µl of a lipid solution consisting either of 18 mg/ml 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE, Avanti, Polar Lipids, USA) and 6 mg/ml 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG, Avanti, Polar Lipids, USA) in n-decane or n-hexadecane, or 24 mg/ml asolectin extracted from soybeans (Sigma-Aldrich, USA) in n-decane were layered over the recording solution. The gold probe modified with the PEG layer on which KcsA channels had been attached was made to move into the recording solution through the lipid solution using a manipulator (uM-3A-S1, SENSAPEX, Finland) or an electric actuator (RCP6-SA4R-WA-35P-2, IAI, Japan). As a result, the lipid bilayer membrane was formed at the interface between the two solutions. At this stage, KcsA channels were spontaneously incorporated into the membrane. The membrane capacitance measured using the commercially available gold needle (curvature radius: 450 µm) was approximately 1 nF, which indicated that the bilayer membrane area was estimated to be approximately 0.25 mm², assuming that the electrical capacitance of the bilayer was 0.4 µF/cm²¹³ .