Lpf 8

Currents across lipid bilayers were recorded with a Planar Lipid Bilayer Workstation (Warner Instruments, Hamden, CT). The cis compartment was connected to the head stage input and the trans compartment was held at virtual ground via a pair of matched Ag/AgCl electrodes. Signals from voltage-clamped BLM were high-pass-filtered at 2.1 kHz using an eight-pole Bessel filter LPF-8 (Warner Instruments), digitized (Data Translation digitizer) and recorded after digitization using homemade analog-to-digital converter acquisition software developed by Elena Pavlova (available upon request). For the statistical analysis data were averaged from at least three independent experiments and analyzed using Origin software. Experiments were performed in three separate trials for each sample. Each recorded trace was analyzed to obtain the mean value of conductance. Results are expressed as mean ± SEM.

Bilayer experiments were performed using very similar instrumentation and methods as described by Zakharian and Reusch (92 (link)). Synthetic diphytanoyl phosphatidylcholine (DphPC; Avanti Polar Lipids, Birmingham, AL) was used to form planar lipid bilayers. Lipids were solubilized in n-Decane at 20 mg/mL, and a glass capillary tube was used to paint a bilayer in an aperture of 200 μM diameter in a Delrin cup (Warner Instruments, Hamden, CT). The bilayer was painted between an aqueous solution of 1 M KCl, 10 mM HEPES, pH 7.1, and capacitance was registered in the range of 66 to 100 pF. Approximately ~40 ng of purified OPOE refolded protein in 1 to 2 μL volume was added to the cis compartment and channel-forming activity was recorded at 30-mV applied potential. The current trace was recorded with a patch-clamp amplifier (BC-535 Bilayer Clamp, Warner Instruments). The trans and cis solutions were connected to the head stage point with Ag-AgCl electrodes. Currents were low-pass filtered at 10 kHz and then digitized through an analog-to-digital converter (Digidata 1550B; Molecular Devices, San Jose, CA). Data filtering was done at 100 Hz through an 8-pole Bessel Filter (Lpf-8; Warner Instruments) and digitized at 1 kHz using pClamp11 software (Molecular Devices). Single-channel conductance events were identified automatically using Clampfit11 from 5 independent membrane recordings.

The lipid bilayers were prepared in a 250 μm aperture in a Derlin cuvette using POPC, POPG, and cholesterol dissolved in n-decane at a 6:3:1 molar ratio. The buffer used was 1 M KCl + 10 mM HEPES, pH 7.4. Bilayer formation was followed by capacitance measurements using a 90-pF minimum threshold. The voltage clamp measurements were conducted using a high-gain electrophysiology amplifier with a resistive feedback headstage (Warner Instruments, BC-535), a digitizer (Digidata 1440 A), and a workstation computer. Data were recorded with a sampling frequency of 10 kHz. An integrated 8-pole low pass Bessel filter was used to filter the data at 1.0 kHz bandwidth. For further filtering and noise reduction, the data were also collected in parallel with another Bessel filter (Warner Instruments, LPF-8) at a cutoff frequency of 60 Hz. Data analysis was carried out with Clampfit (v10.6, Axon Instruments, San Jose, CA).
Peptide samples were prepared by dissolving in 1% NH₄OH and diluting into a working buffer (1 M KCl +10 mM HEPES, pH 7.4). The peptide was added to one of the wells of the cuvette (trans side) at a final concentration of 100 nM, incubated for 5 min, followed by current recordings at various hold voltages, i.e., −100 mV, −50 mV, 50 mV, 100 mV (the sign corresponds to the trans side). Blockade of the channels by Zn²⁺ ions was tested by adding ZnCl₂ to the trans side.