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Reconstitution of Endoplasmic Reticulum InsP3 Receptors into Black Lipid Membranes Ilya Bezprozvanny Cold Spring Harb Protoc; doi: 10.1101/pdb.prot073106 Email Alerting Service Subject Categories
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Protocol
Reconstitution of Endoplasmic Reticulum InsP3 Receptors into Black Lipid Membranes Ilya Bezprozvanny1,2,3 1
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390; 2Laboratory of Molecular Neurodegeneration, St. Petersburg State Polytechnical University, St. Petersburg 195251, Russia
Intracellular ion channels, including endoplasmic reticulum (ER) calcium (Ca2+) channels, are most often studied through their reconstitution into planar lipid bilayers (also called black lipid membranes, or BLMs). General methods for making bilayers and for ion channel reconstitution into BLMs have been extensively detailed elsewhere; thus, here the focus is on specific details relevant for inositol (1,4,5)-trisphosphate receptor (InsP3R) recordings. These procedures describe how to perform single-channel recordings of native or recombinant InsP3Rs in BLMs. Similar procedures are used to study native or recombinant ryanodine receptors (RyanRs) in BLMs.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
ATP (100 mM) Argon gas, purified CaCl2 (100 mM) Ca-EGTA solution Chloroform Cis recording solution n-Decane Heparin solution (1 mM) InsP3 (1 mM) KCl (3M) Methanol Microsomes (see Preparation of Microsomes to Study Ca 2+ Channels [Bezprozvanny 2013]) Petroleum jelly Ruthenium red (1 mM) 3
Correspondence: [email protected]
© 2013 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
1041
I. Bezprozvanny
Synthetic lipids: • Phosphatidylcholine (PC; 850358; Avanti Polar Lipids; PC: 16:1 (Δ9-Cis) PC 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine)
•
Phosphatidylethanolamine (PE; 850725; Avanti Polar Lipids; PE: 18:1 (Δ9-Cis) PE (DOPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)
•
Phosphatidylserine (PS; 840035; Avanti Polar Lipids; PS: 18:1 PS (DOPS) 1,2-dioleoyl-snglycero-3-phospho-L-serine (sodium salt)) Order lipids as 5 mg/mL fractions in chloroform with 2 mL/vial shipped on dry ice. The quality of lipids is extremely important. Store lipids at −80˚C and order a new batch every 3–4 mo.
Trans recording solution Equipment
BLM chambers, flat, custom-made (Lucite; Fig. 1) Commercial cylindrical BLM chambers are also available, such as the Warner Instruments BCH-13A chamber. In these chambers the bilayer is formed on a cylindrical cup made of polystyrene, Delrin (acetyl resin), or polysulfone. The volume of solution in the chamber is 1.2 mL and the standard available aperture diameters (machined by Warner) are 150, 200, or 250 µm. Custom aperture sizes from 50 µm to 1 mm can be custom ordered from Warner.
BLM recording setup as described in Miller (1986) and Smith et al. (1988) Copper wire, thick, with a sharp end Glass micropipettes High-frequency generator tester for leak detection (model BD-10A, Electro-Technic Products, Inc.) Ice bucket Dissecting microscope Lipid storage bottles (dark glass) Parafilm Teflon film, 0.005 inch thickness (Small Parts, Inc.) Vortex METHOD
1. Make a small hole of 100–150 µm in diameter in Teflon film to support BLM formation. Tape the Teflon film to the custom-made frame. Position the tip of the output electrode 1 inch away from the Teflon film (Fig. 2). Position a thick copper wire with a sharp end 0.5 inches away from the other side of the Teflon film (not shown). Connect the copper wire to the ground conductor of an electrical power socket.
FIGURE 1. BLM reconstitution chamber. (A,B) Assembled chamber (top and side views). (C ) Disassembled chamber (top view). Reservoirs for electrodes are attached to the side of the chambers by hot glue. Each chamber has an indentation on the bottom for a magnetic stir bar. Holes drilled in the side of the chamber enable agar bridge connections between the reservoirs for electrodes and solutions in the chamber. The chamber closest to the experimenter is called cis and the opposite chamber is called trans.
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Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
Reconstitution of ER InsP 3 R into BLM
FIGURE 2. Making a hole in Teflon film using BD-10A generator. (A) Side view. (B) Top view.
The BD-10A generator produces 10–50 kV discharge at a frequency of 0.5 MHz at the tip of output electrode.
2. Turn on the BD-10A and slowly move it toward the Teflon film. At a distance of 0.5 inches, electrical discharge will occur between the tip of the electrode and the tip of the copper wire. This electrical discharge will burn a hole in the Teflon film. The initial discharge will make a hole in the Teflon in a random location, depending on the location of local defects in the structure of the film. After the initial hole is made, the tip of the generator and the tip of the copper wire must be repositioned to ensure the hole is located on the straight line connecting the tips. Continuous discharges will increase the diameter of the hole until desired size is achieved.
3. Visually examine the shape and size of each hole under the microscope. Select films with a single round hole of 100–150 µm in diameter. Cut selected film to fit in BLM chamber. Most films will be discarded because holes are not round, too big, too small, or multiple holes are made next to each other. Quality of hole is very important. Save good films!
4. Clamp the selected film (0.005 inches thick) between chambers (Fig. 1C). Achieve a tight fit using the screws that hold the chambers together (Fig. 1A,B). Apply petroleum jelly between Teflon film and the wall of the Lucite chamber to prevent leakage. See Troubleshooting.
5. Prepare working stocks of the PC, PE, and PS lipids. Warm a single vial of each lipid from the −80˚C freezer to room temperature. Transfer the contents of one vial to one dark glass bottle prefilled with argon gas. Tightly close storage bottles and store at −20˚C. Prepare new working stocks each week. To minimize oxidation, make every effort to avoid exposure of lipids to air or moisture.
6. To prepare paint and prepaint solutions: i. Warm working stocks of PC, PE, and PS to room temperature (at least 20 min) ii. From the working stocks, aliquot the indicated lipids with glass micropipettes to glass tubes prefilled with argon. Prepaint solution: 150 µL PC + 50 µL PS Paint solution: 150 µL PE + 50 µL PS iii. Vortex 1 min. Evaporate chloroform under slow argon stream until dry film is formed (15 min) iv. Add 50 μL of n-decane, close with parafilm. Vortex extensively (3 min or more). v. Top with argon (10 sec), close with parafilm. 7. Prepare glass rods for painting bilayers by flaming 100 µL glass pipettes. The tip of the rod should be smooth to avoid making holes in the Teflon film.
8. With the glass rod, prepaint the hole in the Teflon on both sides with prepaint solution (large drops on both sides). Wait until decane completely evaporates (at least 30 min) to allow formation of dry lipid film covering the hole in the Teflon. 9. Fill the cis and trans BLM chambers with 3 mL of cis and trans solutions, respectively. Add stir bars and stir for 30 sec. Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
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I. Bezprozvanny
10. Put 3 M KCl in electrode side chambers and insert agar bridges. 11. Insert Ag/Cl electrodes connected to the BLM amplifier to the electrode side. The cis chamber is connected to a ground electrode and the trans chamber is connected to a signal electrode.
12. Rotate a dissecting microscope 90˚, so the path of sight is parallel to the ground. Under this microscope, remove excess prepaint lipids from the hole with a glass rod until the hole opens and the circuit is shorted. 13. Adjust junction potential on the amplifier. 14. Under the dissecting microscope, begin to paint over the hole with paint solution using a glass rod. Monitor formation of the BLM visually and by capacitance measurements on the BLM amplifier. Capacitance of the BLM should be in the range 150–200 pF. The BLM formed with this procedure must be stable for at least 1 h. There should be no current activity and the resistance of the BLM should be at least 10 GΩ. The capacitance of the BLM should stay in the range >100 pF. See Troubleshooting.
15. To begin the InsP3R reconstitution, take an aliquot of ER microsomes from −80˚C storage and put on ice. 16. Add 3 × 200 µL of 3 M KCl stock (600 mM KCl final concentration) to the cis compartment with constant stirring. After each addition, remove 200 µL from the cis compartment, so volume stays fixed at 3 mL. 17. Add 150 μL of 100 mM CaCl2 (5 mM CaCl2 final concentration) to the cis compartment with stirring. Remove 150 µL from the cis compartment, so volume stays fixed at 3 mL. 18. Add 2 µL microsomes in storage buffer directly to the BLM. Do not stir. Wait 1 min to give ER microsomes a chance to prefuse to the BLM. The density of microsomal storage buffer containing 10% sucrose should be exactly the same as the density of the buffer in cis chamber at this point, which can be confirmed by visual observation. “Milky cloud” of ER microsomes should stay close to the BLM. See Troubleshooting.
19. Start stirring for 1 min. 20. Add 200 µL of 3 M KCl (800 mM KCl final concentration) to the cis compartment with stirring. Remove 200 µL from the cis compartment, so volume stays fixed at 3 mL. The increase in osmolarity on the cis side causes “prefused” microsomes to fuse with the BLM caused by water flux. 21. Wait for fusion (2–5 min). Monitor current at 0 mV holding potential. The fusion should register as appearance of large potassium or chloride currents across the BLM. These currents are very large and can be easily detected by looking at the current reading on the BLM amplifier.
22. If no fusion or insufficient fusion of microsomes to BLM occurs, break and reform BLM with a glass rod. Add a new portion of microsomes directly to the newly formed BLM. 23. Repeat Step 22 until chloride current >100 pA at 0 mV holding potential is recorded by the BLM amplifier. Smaller chloride current or potassium current indicates insufficient fusion. From our experience large chloride currents are the best predictor for successful InsP3R recording.
24. Very slowly perfuse the cis chamber with 2 volumes of cis buffer (6 mL) without stirring using a push–pull 2 syringe system (Fig. 3). Lower density cis buffer is layered on the top and higher density cis +0.8 M KCl buffer is removed from the bottom. Slow and careful perfusion is crucial. Perfusion that is too fast causes buffer mixing and insufficient KCl removal. Fluctuations of solution levels in the cis chamber during perfusion causes the BLM to break. This step requires a lot of patience and practice.
1044
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
Reconstitution of ER InsP 3 R into BLM
FIGURE 3. Push–pull 2 syringe solution exchange system. (A) Position of syringes in the beginning of the perfusion cycle. (B) Position of syringes in the end of the perfusion cycle.
25. Slowly perfuse the cis chamber with 5 volumes of cis buffer (15 mL) with stirring using the push– pull 2 syringe system to completely remove remaining KCl. In experiments with Sf9 microsomes, there should be no current across the BLM at this point. In experiments with cerebellar microsomes, RyanR activity may be observed. See Troubleshooting.
26. Add 136 µL of Ca-EGTA solution to the cis compartment with stirring and remove 136 µL volume. This will result in final concentrations of 1 mM EGTA and 0.707 mM CaCl2, yielding a pCa equal to 6.7. If RyanRs were active, they should close at this point.
27. Add 15 µL of 100 mM ATP to cis compartment with stirring, yielding a final concentration of 0.5 mM ATP. RyanR may get activated in BLM again at this point.
28. Block RyanR activity by adding 6 µL of 1 mM ruthenium red stock (final concentration 2 µM) with stirring. This should cause an immediate and complete block of RyanR activity.
29. Add 6 µL of 1 mM InsP3 stock with stirring to cis compartment, resulting in final concentration of 2 µM InsP3. 30. Observe activity of InsP3R in BLM. Record, analyze, and study InsP3R properties. Example of BLM experiment used to study modulation of recombinant InsP3R1 by PKA and PP1α (Tang et al. 2003) is shown in Figure 4. See Troubleshooting.
31. To verify that recorded channels indeed correspond to InsP3R, add 1 µM (final concentration) heparin solution to the cis chamber at the end of the experiment. This should result in immediate and complete inhibition of InsP3R activity in BLM. If RyanR is present in the BLM, the addition of heparin may activate these channels, even in the presence of ruthenium red.
32. After several days of experiments, take apart the BLM chamber. Soak the Teflon film in a mixture of chloroform and methanol (2:1) to remove all lipids and other organic material. After washing the film, dry with a stream of Argon. The film is reusable. Eventually the film will become deformed or develop cracks. At that point it needs to be replaced.
TROUBLESHOOTING Problem (Step 4): There is petroleum jelly in the recording chambers or a short circuit between
the chambers. Solution: Choose the amount of petroleum jelly used to cover the Teflon film carefully. Too much petroleum jelly results in petroleum jelly getting to the recording chambers, which is not acceptable. Too little petroleum jelly results in a short circuit between the chambers. Problem (Step 14): The capacitance of the BLM is 200 pF. Solution: This means the hole is too large and the Teflon film must be replaced. Problem (Step 14): BLM is unstable, breaks quickly, or has low resistance or “channel-like” activity. Solution: Prepare new lipid stocks (Step 5) taking care to avoid exposure to air and moisture. Problem (Step 18): The “milky cloud” of ER microsomes sinks, indicating that the density of storage
buffer is too high, or the “milky cloud” of ER microsomes floats up, indicating that the density of storage buffer is too low. Solution: Check the storage buffer and sucrose concentration in the microsome preparation. See Preparation of Microsomes to Study Ca 2+ Channels (Bezprozvanny 2013). Problem (Step 25): There is a noisy baseline across the BLM. Solution: Too much KCl is left in the cis compartment; more perfusion is needed. Problem (Step 30): No activity is observed in response to InsP3 addition. Solution: Stop the experiment, wash and rinse BLM chambers with water, air dry, and start over.
RELATED INFORMATION
General methods for making bilayers and for ion channel reconstitution into BLM have been extensively described in an excellent manual (Miller 1986). General bilayer techniques are also described in Cohen and Niles (1993) and Smith et al. (1988). 1046
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
Reconstitution of ER InsP 3 R into BLM
RECIPES Ca-EGTA Solution
Reagent
Volume
CaCl2 (20 mM) EGTA (pH 7.35) (100 mM)
10.6 mL 3.0 mL
Mix, aliquot, and store frozen at −20˚C. Cis Recording Solution
1. Take the amount of HEPES used to make 250 mL of trans recording solution and multiply by 8. Weigh out exactly that amount of dry HEPES powder. 2. Weigh out 30 g of highest purity Tris base. 3. Mix the Tris with the HEPES powder. 4. Add water to make a total volume of 2 L, and stir. 5. Measure the pH. Keep adding dry Tris, with stirring, until the pH reaches 7.35. Trans Recording Solution
1. Weigh out 4.19 g of Ba(OH)2 dry powder to produce a final concentration of 53 mM Ba2+. (Ba(OH)2 is very hydroscopic, so protect it from exposure to air and purchase a new lot periodically.) 2. Add 14 g of HEPES dry powder. Add water to yield a final volume of 250 mL. Stir until the powder dissolves. 3. Measure the pH. Keep adding dry HEPES with stirring until the pH reaches 7.35. 4. Record the final amount of HEPES added. (The total amount of HEPES should be in the range of 14–16 g, yielding a final HEPES concentration of 235–260 mM.) 5. Filter the solution to remove precipitate.
REFERENCES Bezprozvanny I. 2013. Preparation of microsomes to study Ca2+ channels. Cold Spring Harb Protoc doi: 10.1101/pdb.prot073098. Cohen FS, Niles WD. 1993. Reconstituting channels into planar membranes: A conceptual framework and methods for fusing vesicles to planar bilayer phospholipid membranes. Methods Enzymol 220: 50–68. Miller C, ed. 1986. Ion channel reconstitution. Plenum, New York.
Smith JS, Coronado R, Meissner G. 1988. Techniques for observing calcium channels from skeletal muscle sarcoplasmic reticulum in planar lipid bilayers. Methods Enzymol 157: 480–489. Tang TS, Tu H, Wang Z, Bezprozvanny I. 2003. Modulation of type 1 inositol (1,4,5)-trisphosphate receptor function by protein kinase A and protein phosphatase 1α. J Neurosci 23: 403–415.
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
1047
Reconstitution of Endoplasmic Reticulum InsP3 Receptors into Black Lipid Membranes Ilya Bezprozvanny Cold Spring Harb Protoc; doi: 10.1101/pdb.prot073106 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Cell Biology, general (1020 articles) Signal Transduction (29 articles)
To subscribe to Cold Spring Harbor Protocols go to:
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© 2013 Cold Spring Harbor Laboratory Press
Protocol
Reconstitution of Endoplasmic Reticulum InsP3 Receptors into Black Lipid Membranes Ilya Bezprozvanny1,2,3 1
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390; 2Laboratory of Molecular Neurodegeneration, St. Petersburg State Polytechnical University, St. Petersburg 195251, Russia
Intracellular ion channels, including endoplasmic reticulum (ER) calcium (Ca2+) channels, are most often studied through their reconstitution into planar lipid bilayers (also called black lipid membranes, or BLMs). General methods for making bilayers and for ion channel reconstitution into BLMs have been extensively detailed elsewhere; thus, here the focus is on specific details relevant for inositol (1,4,5)-trisphosphate receptor (InsP3R) recordings. These procedures describe how to perform single-channel recordings of native or recombinant InsP3Rs in BLMs. Similar procedures are used to study native or recombinant ryanodine receptors (RyanRs) in BLMs.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
ATP (100 mM) Argon gas, purified CaCl2 (100 mM) Ca-EGTA solution Chloroform Cis recording solution n-Decane Heparin solution (1 mM) InsP3 (1 mM) KCl (3M) Methanol Microsomes (see Preparation of Microsomes to Study Ca 2+ Channels [Bezprozvanny 2013]) Petroleum jelly Ruthenium red (1 mM) 3
Correspondence: [email protected]
© 2013 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
1041
I. Bezprozvanny
Synthetic lipids: • Phosphatidylcholine (PC; 850358; Avanti Polar Lipids; PC: 16:1 (Δ9-Cis) PC 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine)
•
Phosphatidylethanolamine (PE; 850725; Avanti Polar Lipids; PE: 18:1 (Δ9-Cis) PE (DOPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)
•
Phosphatidylserine (PS; 840035; Avanti Polar Lipids; PS: 18:1 PS (DOPS) 1,2-dioleoyl-snglycero-3-phospho-L-serine (sodium salt)) Order lipids as 5 mg/mL fractions in chloroform with 2 mL/vial shipped on dry ice. The quality of lipids is extremely important. Store lipids at −80˚C and order a new batch every 3–4 mo.
Trans recording solution Equipment
BLM chambers, flat, custom-made (Lucite; Fig. 1) Commercial cylindrical BLM chambers are also available, such as the Warner Instruments BCH-13A chamber. In these chambers the bilayer is formed on a cylindrical cup made of polystyrene, Delrin (acetyl resin), or polysulfone. The volume of solution in the chamber is 1.2 mL and the standard available aperture diameters (machined by Warner) are 150, 200, or 250 µm. Custom aperture sizes from 50 µm to 1 mm can be custom ordered from Warner.
BLM recording setup as described in Miller (1986) and Smith et al. (1988) Copper wire, thick, with a sharp end Glass micropipettes High-frequency generator tester for leak detection (model BD-10A, Electro-Technic Products, Inc.) Ice bucket Dissecting microscope Lipid storage bottles (dark glass) Parafilm Teflon film, 0.005 inch thickness (Small Parts, Inc.) Vortex METHOD
1. Make a small hole of 100–150 µm in diameter in Teflon film to support BLM formation. Tape the Teflon film to the custom-made frame. Position the tip of the output electrode 1 inch away from the Teflon film (Fig. 2). Position a thick copper wire with a sharp end 0.5 inches away from the other side of the Teflon film (not shown). Connect the copper wire to the ground conductor of an electrical power socket.
FIGURE 1. BLM reconstitution chamber. (A,B) Assembled chamber (top and side views). (C ) Disassembled chamber (top view). Reservoirs for electrodes are attached to the side of the chambers by hot glue. Each chamber has an indentation on the bottom for a magnetic stir bar. Holes drilled in the side of the chamber enable agar bridge connections between the reservoirs for electrodes and solutions in the chamber. The chamber closest to the experimenter is called cis and the opposite chamber is called trans.
1042
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
Reconstitution of ER InsP 3 R into BLM
FIGURE 2. Making a hole in Teflon film using BD-10A generator. (A) Side view. (B) Top view.
The BD-10A generator produces 10–50 kV discharge at a frequency of 0.5 MHz at the tip of output electrode.
2. Turn on the BD-10A and slowly move it toward the Teflon film. At a distance of 0.5 inches, electrical discharge will occur between the tip of the electrode and the tip of the copper wire. This electrical discharge will burn a hole in the Teflon film. The initial discharge will make a hole in the Teflon in a random location, depending on the location of local defects in the structure of the film. After the initial hole is made, the tip of the generator and the tip of the copper wire must be repositioned to ensure the hole is located on the straight line connecting the tips. Continuous discharges will increase the diameter of the hole until desired size is achieved.
3. Visually examine the shape and size of each hole under the microscope. Select films with a single round hole of 100–150 µm in diameter. Cut selected film to fit in BLM chamber. Most films will be discarded because holes are not round, too big, too small, or multiple holes are made next to each other. Quality of hole is very important. Save good films!
4. Clamp the selected film (0.005 inches thick) between chambers (Fig. 1C). Achieve a tight fit using the screws that hold the chambers together (Fig. 1A,B). Apply petroleum jelly between Teflon film and the wall of the Lucite chamber to prevent leakage. See Troubleshooting.
5. Prepare working stocks of the PC, PE, and PS lipids. Warm a single vial of each lipid from the −80˚C freezer to room temperature. Transfer the contents of one vial to one dark glass bottle prefilled with argon gas. Tightly close storage bottles and store at −20˚C. Prepare new working stocks each week. To minimize oxidation, make every effort to avoid exposure of lipids to air or moisture.
6. To prepare paint and prepaint solutions: i. Warm working stocks of PC, PE, and PS to room temperature (at least 20 min) ii. From the working stocks, aliquot the indicated lipids with glass micropipettes to glass tubes prefilled with argon. Prepaint solution: 150 µL PC + 50 µL PS Paint solution: 150 µL PE + 50 µL PS iii. Vortex 1 min. Evaporate chloroform under slow argon stream until dry film is formed (15 min) iv. Add 50 μL of n-decane, close with parafilm. Vortex extensively (3 min or more). v. Top with argon (10 sec), close with parafilm. 7. Prepare glass rods for painting bilayers by flaming 100 µL glass pipettes. The tip of the rod should be smooth to avoid making holes in the Teflon film.
8. With the glass rod, prepaint the hole in the Teflon on both sides with prepaint solution (large drops on both sides). Wait until decane completely evaporates (at least 30 min) to allow formation of dry lipid film covering the hole in the Teflon. 9. Fill the cis and trans BLM chambers with 3 mL of cis and trans solutions, respectively. Add stir bars and stir for 30 sec. Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
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I. Bezprozvanny
10. Put 3 M KCl in electrode side chambers and insert agar bridges. 11. Insert Ag/Cl electrodes connected to the BLM amplifier to the electrode side. The cis chamber is connected to a ground electrode and the trans chamber is connected to a signal electrode.
12. Rotate a dissecting microscope 90˚, so the path of sight is parallel to the ground. Under this microscope, remove excess prepaint lipids from the hole with a glass rod until the hole opens and the circuit is shorted. 13. Adjust junction potential on the amplifier. 14. Under the dissecting microscope, begin to paint over the hole with paint solution using a glass rod. Monitor formation of the BLM visually and by capacitance measurements on the BLM amplifier. Capacitance of the BLM should be in the range 150–200 pF. The BLM formed with this procedure must be stable for at least 1 h. There should be no current activity and the resistance of the BLM should be at least 10 GΩ. The capacitance of the BLM should stay in the range >100 pF. See Troubleshooting.
15. To begin the InsP3R reconstitution, take an aliquot of ER microsomes from −80˚C storage and put on ice. 16. Add 3 × 200 µL of 3 M KCl stock (600 mM KCl final concentration) to the cis compartment with constant stirring. After each addition, remove 200 µL from the cis compartment, so volume stays fixed at 3 mL. 17. Add 150 μL of 100 mM CaCl2 (5 mM CaCl2 final concentration) to the cis compartment with stirring. Remove 150 µL from the cis compartment, so volume stays fixed at 3 mL. 18. Add 2 µL microsomes in storage buffer directly to the BLM. Do not stir. Wait 1 min to give ER microsomes a chance to prefuse to the BLM. The density of microsomal storage buffer containing 10% sucrose should be exactly the same as the density of the buffer in cis chamber at this point, which can be confirmed by visual observation. “Milky cloud” of ER microsomes should stay close to the BLM. See Troubleshooting.
19. Start stirring for 1 min. 20. Add 200 µL of 3 M KCl (800 mM KCl final concentration) to the cis compartment with stirring. Remove 200 µL from the cis compartment, so volume stays fixed at 3 mL. The increase in osmolarity on the cis side causes “prefused” microsomes to fuse with the BLM caused by water flux. 21. Wait for fusion (2–5 min). Monitor current at 0 mV holding potential. The fusion should register as appearance of large potassium or chloride currents across the BLM. These currents are very large and can be easily detected by looking at the current reading on the BLM amplifier.
22. If no fusion or insufficient fusion of microsomes to BLM occurs, break and reform BLM with a glass rod. Add a new portion of microsomes directly to the newly formed BLM. 23. Repeat Step 22 until chloride current >100 pA at 0 mV holding potential is recorded by the BLM amplifier. Smaller chloride current or potassium current indicates insufficient fusion. From our experience large chloride currents are the best predictor for successful InsP3R recording.
24. Very slowly perfuse the cis chamber with 2 volumes of cis buffer (6 mL) without stirring using a push–pull 2 syringe system (Fig. 3). Lower density cis buffer is layered on the top and higher density cis +0.8 M KCl buffer is removed from the bottom. Slow and careful perfusion is crucial. Perfusion that is too fast causes buffer mixing and insufficient KCl removal. Fluctuations of solution levels in the cis chamber during perfusion causes the BLM to break. This step requires a lot of patience and practice.
1044
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
Reconstitution of ER InsP 3 R into BLM
FIGURE 3. Push–pull 2 syringe solution exchange system. (A) Position of syringes in the beginning of the perfusion cycle. (B) Position of syringes in the end of the perfusion cycle.
25. Slowly perfuse the cis chamber with 5 volumes of cis buffer (15 mL) with stirring using the push– pull 2 syringe system to completely remove remaining KCl. In experiments with Sf9 microsomes, there should be no current across the BLM at this point. In experiments with cerebellar microsomes, RyanR activity may be observed. See Troubleshooting.
26. Add 136 µL of Ca-EGTA solution to the cis compartment with stirring and remove 136 µL volume. This will result in final concentrations of 1 mM EGTA and 0.707 mM CaCl2, yielding a pCa equal to 6.7. If RyanRs were active, they should close at this point.
27. Add 15 µL of 100 mM ATP to cis compartment with stirring, yielding a final concentration of 0.5 mM ATP. RyanR may get activated in BLM again at this point.
28. Block RyanR activity by adding 6 µL of 1 mM ruthenium red stock (final concentration 2 µM) with stirring. This should cause an immediate and complete block of RyanR activity.
29. Add 6 µL of 1 mM InsP3 stock with stirring to cis compartment, resulting in final concentration of 2 µM InsP3. 30. Observe activity of InsP3R in BLM. Record, analyze, and study InsP3R properties. Example of BLM experiment used to study modulation of recombinant InsP3R1 by PKA and PP1α (Tang et al. 2003) is shown in Figure 4. See Troubleshooting.
31. To verify that recorded channels indeed correspond to InsP3R, add 1 µM (final concentration) heparin solution to the cis chamber at the end of the experiment. This should result in immediate and complete inhibition of InsP3R activity in BLM. If RyanR is present in the BLM, the addition of heparin may activate these channels, even in the presence of ruthenium red.
32. After several days of experiments, take apart the BLM chamber. Soak the Teflon film in a mixture of chloroform and methanol (2:1) to remove all lipids and other organic material. After washing the film, dry with a stream of Argon. The film is reusable. Eventually the film will become deformed or develop cracks. At that point it needs to be replaced.
TROUBLESHOOTING Problem (Step 4): There is petroleum jelly in the recording chambers or a short circuit between
the chambers. Solution: Choose the amount of petroleum jelly used to cover the Teflon film carefully. Too much petroleum jelly results in petroleum jelly getting to the recording chambers, which is not acceptable. Too little petroleum jelly results in a short circuit between the chambers. Problem (Step 14): The capacitance of the BLM is 200 pF. Solution: This means the hole is too large and the Teflon film must be replaced. Problem (Step 14): BLM is unstable, breaks quickly, or has low resistance or “channel-like” activity. Solution: Prepare new lipid stocks (Step 5) taking care to avoid exposure to air and moisture. Problem (Step 18): The “milky cloud” of ER microsomes sinks, indicating that the density of storage
buffer is too high, or the “milky cloud” of ER microsomes floats up, indicating that the density of storage buffer is too low. Solution: Check the storage buffer and sucrose concentration in the microsome preparation. See Preparation of Microsomes to Study Ca 2+ Channels (Bezprozvanny 2013). Problem (Step 25): There is a noisy baseline across the BLM. Solution: Too much KCl is left in the cis compartment; more perfusion is needed. Problem (Step 30): No activity is observed in response to InsP3 addition. Solution: Stop the experiment, wash and rinse BLM chambers with water, air dry, and start over.
RELATED INFORMATION
General methods for making bilayers and for ion channel reconstitution into BLM have been extensively described in an excellent manual (Miller 1986). General bilayer techniques are also described in Cohen and Niles (1993) and Smith et al. (1988). 1046
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
Reconstitution of ER InsP 3 R into BLM
RECIPES Ca-EGTA Solution
Reagent
Volume
CaCl2 (20 mM) EGTA (pH 7.35) (100 mM)
10.6 mL 3.0 mL
Mix, aliquot, and store frozen at −20˚C. Cis Recording Solution
1. Take the amount of HEPES used to make 250 mL of trans recording solution and multiply by 8. Weigh out exactly that amount of dry HEPES powder. 2. Weigh out 30 g of highest purity Tris base. 3. Mix the Tris with the HEPES powder. 4. Add water to make a total volume of 2 L, and stir. 5. Measure the pH. Keep adding dry Tris, with stirring, until the pH reaches 7.35. Trans Recording Solution
1. Weigh out 4.19 g of Ba(OH)2 dry powder to produce a final concentration of 53 mM Ba2+. (Ba(OH)2 is very hydroscopic, so protect it from exposure to air and purchase a new lot periodically.) 2. Add 14 g of HEPES dry powder. Add water to yield a final volume of 250 mL. Stir until the powder dissolves. 3. Measure the pH. Keep adding dry HEPES with stirring until the pH reaches 7.35. 4. Record the final amount of HEPES added. (The total amount of HEPES should be in the range of 14–16 g, yielding a final HEPES concentration of 235–260 mM.) 5. Filter the solution to remove precipitate.
REFERENCES Bezprozvanny I. 2013. Preparation of microsomes to study Ca2+ channels. Cold Spring Harb Protoc doi: 10.1101/pdb.prot073098. Cohen FS, Niles WD. 1993. Reconstituting channels into planar membranes: A conceptual framework and methods for fusing vesicles to planar bilayer phospholipid membranes. Methods Enzymol 220: 50–68. Miller C, ed. 1986. Ion channel reconstitution. Plenum, New York.
Smith JS, Coronado R, Meissner G. 1988. Techniques for observing calcium channels from skeletal muscle sarcoplasmic reticulum in planar lipid bilayers. Methods Enzymol 157: 480–489. Tang TS, Tu H, Wang Z, Bezprozvanny I. 2003. Modulation of type 1 inositol (1,4,5)-trisphosphate receptor function by protein kinase A and protein phosphatase 1α. J Neurosci 23: 403–415.
Cite this protocol as Cold Spring Harb Protoc; 2013; doi:10.1101/pdb.prot073106
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