13. Balance the weight of the ultracentrifuge tubes using Buffer 2 to within 0.1 g of each other. Handle the tubes very care-fully so as not to disturb the sucrose cushion.
14. Centrifuge at 30,000×g for 30 min at 4°C.
15. Remove 5–10 ml of the supernatant at the top of the tube using a serological pipette.
16. Collect the cloudy suspension resting on the top of the sucrose cushion using a 10 ml plastic syringe.
17. Transfer the collected interface to a fresh ultracentrifuge tube. Dilute the collected interface with Buffer 4 to fill the ultracentrifuge tube (see Note 3).
18. Balance the weight of the tubes to within 0.1 g of each other and centrifuge at 100,000 g for 45 min at 4°C.
19. Discard the supernatant and resuspend the glassy-appearing pellet in 1.5 ml of Buffer 5 containing the protease inhibitors using a 22-G syringe needle and then a 25-G needle on a 5-ml syringe until the suspension is lump-free.
20. Store in 50–100 µl aliquots at −80°C.
1. Form U-shaped bridges from the capillary glass by melting gently over a Bunsen burner flame.
2. Prepare 4% agar in 2 M KCl by adding 4 g of agar to 100 ml 2 M KCl while heating and stirring.
14.3.2. Membrane Preparation
14.3.3. Agar Bridge Fabrication
3. Fill bridges with molten agar/KCl, ensuring that no bubbles appear.
4. Store bridges in 2 M KCl solution at 4°C.
5. Excess agar/KCl can be stored at 4°C.
Figure 14.2 shows a schematic of the bilayer cups.
1. Reconstitute POPS and POPE in chloroform at 50 mg/ml.
These solutions should be stored in the dark at −20°C.
2. Add 7.5 µl of POPS and 7.5 µl of POPE to a sealable 2 ml glass volumetric flask.
3. Evaporate the chloroform under a stream of nitrogen gas.
Seal the volumetric flask with the stopper to minimise con-tact with air.
4. Resuspend the dried lipid with 25 µl of n-decane.
5. The lipid is ready to use. This preparation is useable for 3–4 h, but avoid contact with air.
6. Place 1 µl of the reconstituted lipid carefully spread around the aperture of the recording cup and over the aperture to form a bilayer (see Note 4).
7. Insert the cup into the chamber holder and add 1 ml of the recording buffer to the cup and 1 ml of the appropriate recording buffer to the chamber holder.
8. Connect the cis and trans side of the bath to the amplifier head stage via the agar bridges as indicates in Fig. 14.2. The cis side should normally be at ground potential.
9. Assess bilayer formation by applying 1–10 mV square or tri-angular voltage pulses (see Note 5).
10. The bilayer can have a voltage clamped between −150 and 150 mV and should have a leak conductance of less than 10 pS and a capacitance of greater than 150 pF (see Note 5).
Once a stable bilayer has formed BKCa channels can be incorpo-rated.
1. Add 2 µl of the membrane preparation to the cis side of the chamber. It is important to stir the bilayer occasionally as membranes can sink to the bottom of the recording cham-ber.
2. Hold the bilayer potential at ± 50 mV and wait for chan-nel insertion by observing single chanchan-nel activity on the oscilloscope.
3. If no channels have inserted after 20 min then a further 2 µl samples of the membrane preparation should be added.
4. Once channels have been inserted it is important to estimate how many channels are present. This can be done by changing 14.3.4. Bilayer
Construction
14.3.5. Channel Incorporation and Modulation by Steroids
the membrane potential to raise the open probability and by observing the number of multiple openings (see Fig. 14.3;
see also Note 6).
5. Once a stable recording of channel activity is observed phar-macological agents such as steroids can be added to either side of the bilayer.
1. Data analysis can be done off-line. It is advisable to get a back-up data onto a portable hard drive.
2. WinEDR can be used to analyse single channel data. Ampli-tude histograms and dwell time histograms, including burst analysis, can be constructed. In addition, data can be exported into other software packages, such as QuB, or a programme to present the data in a statistical form, if required.
1. Channels with a small conductance will not be well suited for this work, as contaminating channels that are endogenous to HEK cells are sometimes observed during recordings.
2. Any other pressure based bursting apparatus may be used, e.g. a French press. Cell lysis by sonication tends to result in higher levels of protein degradation.
3. This is important, as a half-filled tube will collapse.
4. Artificial (or “black”) lipid bilayer formation often needs to be “encouraged”. Painting the lipid onto a cup may fail to 14.3.6. Data Analysis
14.4. Notes
Fig. 14.3.A typical single channel recording. This bilayer has at least three individual BKCa channels inserted, as evi-denced by the multiple downward current levels from the zero current level (dashed line).
form a bilayer. Either the lipid is absent from the aperture and the amplifier can not pass enough current to clamp the voltage, or it is blocked by multiple layers of lipid and the membrane capacitance is too low. There are a number of ways to encourage bilayer formation:
(a) “Lifting a bilayer”: Gently suck up the recording buffer in the cis chamber using a 1-ml pipette and then gently return to the recording chamber. Dragging the surface of the recording solution across the aperture encour-ages a redistribution of lipid and bilayer formation.
(b) “Painting a bilayer”: This is done using a ball of glass formed on the end of capillary tubing by heating the glass with a Bunsen burner. This clean glass probe can then be dipped in the stock lipid and applied directly to the hole and this is often enough to encourage bilayer formation.
5. The passive bilayer properties are easy to evaluate under voltage clamp conditions. The conductance is equal to cur-rent deflection divided by the voltage step (e.g. conductance
= 1 pA/100 mV = 1 × 10−12 A/0.1 V = 1 × 10−11 S or 10 pS).
The capacitance of a membrane is described by the equa-tion C = e A / d, where C is the capacitance in Farads, ε is the dielectric constant of the lipid, A is the area of the bilayer, and d is the thickness. Current flow onto a capacitor (e.g. a lipid bilayer membrane) is given by the equation i = C(dV / dt). We can combine both equations to give i = (e A / d)(dV / dt). A triangular wave form has a con-stant rate of change and if we change the voltage across our bilayer in a triangular manner then dV/dt will be constant.
More importantly the dielectric constant of the lipid is fixed and the thickness of the bilayer is determined by the lipid tail length. Therefore, the equation can be simplified to i = kA, where k = (e / d)(dV / dt). Using a triangular pulse, the current output of the amplifier is therefore directly pro-portional to the area of the bilayer. The current output can be calibrated to capacitance by placing a 100-pF capacitor between the input and ground of the patch clamp amplifier head stage. Take care when handling patch clamp amplifier head-stages as they are extremely sensitive to static electricity and can be easily damaged. Capacitance can also be moni-tored and measured from the capacity transients evoked by square-wave voltage pulses.
6. Changing membrane potential to fully activate channels will only work with those channels that are gated by voltage.
Other stimuli may need to be applied to activate other types of channel, e.g. Ca2+.
References
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2. Orio, P., Roja, P., Rerreira, G., and Latorre, R.
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MaxiK channel beta-subunits. News Physiol.
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