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).
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).
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