Derlin cuvette

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.

Electrophysiological properties of purified refolded full-length VDAC1 and of the
C-terminally truncated proteins VDAC1(Δβ15-19) and
VDAC1(Δβ16-19) reconstituted into a PLB and subsequently subjected to
single and multiple channel current recordings and data analysis were carried out as
described previously [58 (link), 60 (link)], using a Warner Instruments (Hamden, CT)
planar bilayer apparatus. Bilayers of approximately 150–200 pF capacity were
prepared across a 200 μM hole in a derlin cuvette (Warner Instruments) from a
1% (w/v) solution of DiPhyPC (1,2-diphytanoyl-sn-glycero-3-phosphocoline)
(Avanti Polar-Lipids, Alabaster, AL) in n-decane (Sigma). The volumes of the
cis and trans compartments were 3 ml. Both sides
were connected to the electrodes via salt bridges (1 M KCl) in series with Ag/AgCl
electrodes. All measurements were made in 1 M KCl, 10 mM Hepes, pH 7.0, at room
temperature. Full-length and truncated VDAC were added to the cisside of the chamber from a protein stock solution of 1 mg/ml. Control experiments
with a PLB in the absence of VDAC1 or in the presence of the detergent used for VDAC1
purification showed no currents. Data were acquired using a Bilayer Clamp amplifier
(Warner Instruments) at the 100 μs/point, filtered at 200 Hz and analyzed
offline using pCLAMP Clampfit 10.7 software (Axon Instruments, Union City, CA).

Planar lipid bilayer experiments were performed as described previously [62 (link),63 (link)]. Artificial membranes made of 1% (w/v) diphytanoyl phosphatidylcholine (DiphPC) (Avanti Polar Lipids, Alabaster, AL, USA) in n-decane were painted on a 200 µm hole in a Derlin cuvette (Warner Instruments, Hamden, CT, USA). All the experiments were carried out at RT. Membrane capacitances of 100–150 pF were established for appropriate lipid bilayers. Mutant or native mVDAC3 were added from the protein stock solution of 1 mg/mL to the cis side of the cuvette filled with symmetrical 1 M KCl/10 mM HEPES pH 7.0. The single channel conductance of the pores was measured upon application of a fixed membrane potential (+10 mV) [58 (link)]. The voltage dependence was calculated by applying a triangular voltage ramp from 0 to ±50 mV of 100 ms duration, with a frequency of 10 mHz. At least three independent experiments were performed for each protein. A Bilayer Clamp amplifier (Warner Instruments) at 100 ms/point and filtered at 300 Hz was used for data acquisition. Analyses were performed with the pClamp software (Ver-10; Molecular Devices, San Jose, CA, USA).