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Reconstitution of Purified VDAC1 into a Lipid Bilayer and Recording of Channel Conductance Danya Ben-Hail and Varda Shoshan-Barmatz Cold Spring Harb Protoc; doi: 10.1101/pdb.prot073148 Email Alerting Service Subject Categories
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Protocol
Reconstitution of Purified VDAC1 into a Lipid Bilayer and Recording of Channel Conductance Danya Ben-Hail and Varda Shoshan-Barmatz1 Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
The functional properties of purified voltage-dependent anion-selective channel protein 1 (VDAC1) have been examined in reconstituted systems based on artificially prepared phospholipid bilayers. The most widespread method for the characterization of the pore-forming activity of the mitochondrial VDAC1 protein requires reconstitution of the channel activity into a planar lipid bilayer (PLB) that separates two aqueous compartments. This system is able to produce a refined and large set of information on channel activity. The activity of the channel is reflected in the flow of ions (i.e., current) through a membrane that otherwise represents a barrier to ion flow. The setup thus requires the use of purified protein and a source of continuous current, as well as a sophisticated detector system able to amplify and record low, picoamper-level currents. This system is so efficient that the activity of even a single channel can be detected, allowing for study of VDAC1 at the molecular level.
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
Agarose solution for building agar salt bridges Buffer for assaying Ca2+ transport by VDAC1 reconstituted into PLBs Chloroform n-Decane KCl (1 M) Nitrogen Soybean asolectin (Sigma-Aldrich 11145) VDAC1 (see Purification of VDAC1 from Rat Liver Mitochondria [Ben-Hail and Shoshan-Barmatz 2014]) Equipment
Ag/AgCl electrodes Agar bridge wells 1
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073148
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VDAC1 Reconstitution and Channel Recording
Amplifier headstage (Warner Instrument Corporation) Bilayer clamp amplifier (Warner Instrument Corporation) Bunsen burner Chamber holder (Warner Instrument Corporation) Digidata 1200/1440 interface board (Warner Instrument Corporation) Faraday cage Glass capillary Glass Pasteur pipettes Glass vial Magnetic stirrer (2 × 5 mm) Perfusion system Pipette (with a gel-loading tip) or syringe (with a needle) (see Step 1) Polystyrene chamber (with 250 µm aperture) Software suitable for data capture and analysis (e.g., pCLAMP 10.2 from Axon Instruments) Vibration isolation table
METHOD PLB membranes are formed across an aperture in a barrier that separates two bath solutions. In this protocol, the design used is that of a polystyrene chamber and a chamber holder (Fig. 1). The solution in the chamber represents one bath, whereas the solution in the holder is considered to be the other bath. After reconstituting VDAC1 into the bilayer membrane, formed in the aperture located in the chamber, currents passing through the channel can be recorded. Electrical connections are made by agar salt bridges placed in each bath. The channel recordings must be made in a Faraday cage to shield them from vibrations and electrical interference.
Preparation for Recording
1. Prepare two agar salt bridges that will serve to connect the electrodes to the chambers. Bend the glass capillary by heating over a Bunsen burner. Fill the capillaries with the agarose (heated to 90oC) using a pipette with a gel-loading tip or a syringe with a needle. The agar must be boiled just before filling the capillaries. The bridge should be as short as possible.
2. Prepare the lipid solution in a glass vial by dissolving soybean asolectin in chloroform (3 mg/mL). Dry the lipids under a nitrogen stream. After they are completely dry, dissolve the lipids in n-decane (30 mg/mL). The lipid solution should always be clear. Lipid solutions contaminated with water will turn milky and cannot be used.
3. Precoat the aperture in the chamber with lipids. Use a glass Pasteur pipette containing a small solid ball at its tip, formed by heating with a Bunsen burner, to coat the outside rim of the hole with the lipid solution. Let the lipid solution dry on the chamber before adding the appropriate solutions to the cup and chamber holder. Assembly of the Bilayer System
4. Place the chamber holder in the Faraday cage. Place the chamber and two agar bridge wells at the appropriate places in the chamber holder. Situate each of the agar salt bridges in one of the wells and in either the cup or the interior of the holder, as appropriate. 5. Connect the Ag/AgCl electrodes through which the transmembrane potential will be applied. Connect the trans side to the ground in the amplifier head-stage by means of an electrode placed in the agar bridge well connected to this chamber. Connect the cis side, where the protein will be added, to the input in the amplifier head-stage using the second electrode. To prevent contamination of silver, the electrodes should not be connected directly to the bath solutions. The agar bridges will close the circuit. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073148
101
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D. Ben-Hail and V. Shoshan-Barmatz
+
Cis
–
Trans
Current
Voltage
Time
FIGURE 1. Schematic representation of planar lipid bilayer (PLB) reconstitution (left) and the channel conductance assay system (right) for assessing the single and multichannel activity of VDAC1. VDAC1 channel activity is measured following its reconstitution into a PLB and measuring the current passing through the channel (green oval) when a salt concentration gradient or voltage is applied across the bilayer. The membrane serves as a capacitor, whereas the ions carry the current. The cis side is defined as the compartment to which VDAC1 was added. Currents can be recorded under voltage-clamp by using a bilayer clamp amplifier and are measured with respect to the cis side of the membrane (ground). The currents can be low-pass filtered at 1 kHz and digitized online using an interface board and pCLAMP software.
6. Attach the perfusion system of choice, according to the manufacturer’s instructions. 7. Carefully add a magnetic stirring bar to the cis chamber. Formation of the Planar Bilayer Membrane
8. Add the experimental buffer to the cis and trans sides. In the Werner system, 1 mL is added to each chamber, although the volumes should be scaled to the chambers used.
9. Into each agar bridge well, add 1 M KCl to cover the tip of the electrodes. 10. Take a clean and dry glass Pasteur pipette, like the one used to precoat the chamber, dip it into the lipid solution, and then lightly touch the aperture. 11. Monitor the bilayer formed using a bilayer clamp amplifier. A straight line at 0 pA should be observed. Check the resistance of the PLB, and if it is between 100 and 120 GΩ, check the membrane for leaks by applying different voltages (±60 mV). If the current remains at 0 pA, the bilayer is stable and intact. See Troubleshooting.
Reconstitution of VDAC1 Into a Planar Lipid Bilayer
12. Add purified VDAC1 (1–2 ng) to one of the compartments (this is then termed the cis side; see Fig. 1) containing the buffer for assaying Ca2+ transport (with the selected CaCl2 concentration), and then turn on the magnetic stirrer. 13. Check that VDAC1 insertion into the membrane is realized within 5–20 min, as reflected by an increase in current. Check for stability of the protein in the PLB by applying +60 mV and verify that you get a typical readout for VDAC1-mediated current at this voltage. The current should first increase due to a greater driving force, but within < 1 sec, the channel should close to a stable low-conducting state (as in Fig. 2A).
102
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VDAC1 Reconstitution and Channel Recording
14. After one or more channels have been inserted into the PLB, remove excess protein by perfusion of the cis chamber with 20 volumes of a solution of the same composition as that used before perfusion, so as to prevent further incorporation. Recording Currents Through VDAC1
15. Record the currents under voltage-clamp using a bilayer clamp amplifier. Measure the currents with respect to the cis side of the membrane (ground). • To measure the currents of identical CaCl2 concentrations (in the range of 100–300 mM) in both the cis and trans compartments or of a CaCl2 concentration gradient (300:150 mM, cis: trans) (see Fig. 2), use voltage steps or voltage ramps between −60 and +60 mV.
•
To assess the Ca2+ permeability of VDAC1 (relative to chloride), determine the reversal potential at different CaCl2 concentration gradients as described previously (Gincel et al. 2001). Obtain an estimate of the Ca2+/Cl− permeability ratio (r) using the reversal potential (Vrev), the CaCl2 activity, and the following equation:
r=
PCa2+ (1 + B) B[Cl− ]0 − [Cl− ]x , = 4 PCl− [Ca2+ ]0 − [Ca2+ ]x B2
A
B
–10 mV
I, pA 90 60 20 pA 1 sec
–40 mV
30 –60
–40
20
–20 –30
40
60
Voltage, mV
–60 –90
Conductance Maximal conductance of control
C 1 0.8 0.6 0.4 0.2 0
–60 –40 –20
0
20 40 60
Voltage, mV
FIGURE 2. VDAC1 channel properties, conductance measurements, and analysis with CaCl2 as the sources of ions carrying the current. (A) Single-channel current in response to voltage steps (0/−10) or (0/–40). The cis and trans chambers contain identical concentrations of 150 mM CaCl2. (B) Single channel currents through VDAC1 in response to voltage ramps (−60 to +60 mV at 60 mV sec−1) in the presence of 150 mM CaCl2 in both chambers (represented in black), or in the presence of a CaCl2 concentration gradient (300/150 mM, cis/trans) (shown in gray). The reverse potential (Vrev) derived from this plot can be used to calculate the relative conductance of Cl−/Ca2+. In this experiment, the calculated ratio is 2:1 Cl−/Ca2+. (C) Channel conductance of PLB-reconstituted VDAC1, as recorded in response to a voltage step from 0 mV to voltages between −60 and +60 mV. The average steady-state conductance at a given voltage was normalized to the conductance at 10 mV (namely, conductance at a given voltage/maximal conductance at 10 mV). Closed circles represent channel conductance measured at identical concentrations of CaCl2 in cis and trans. Open circles represent channel conductance measured with a CaCl2 concentration gradient (300/150 mM, cis/trans).
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D. Ben-Hail and V. Shoshan-Barmatz
where B = eRT Vrev , F is the Faraday constant (9.648 × 104 C mol−1); R is the universal gas constant (8.314 J K−1 mol−1); and T is absolute temperature. F
The currents should be low-pass filtered at 1 kHz (–3 dB). All experiments should be performed at 21˚C–25˚C. A full description of how to use software suitable for data capture and analysis (e.g., pCLAMP from Axon Instruments) is beyond the scope of this article, and can be obtained in the user guide of the software.
TROUBLESHOOTING Problem (Step 11): The membrane is too thick. Solution: Switch the holding voltage from +60 to −60 mV. Repeat this step a few times. If this does not
help to thin out the membrane, break it and make a new membrane. DISCUSSION
The permeability to Ca2+ of VDAC1 can be followed on its reconstitution into a PLB, as a single channel or as multichannels, under voltage-clamp conditions. The PLB is a powerful tool in the study of channels such as VDAC1 and is useful for, among others, simultaneous measurement of ionic charge movement (conductance), channel gating, and studying the effects of inhibitors. When studying the conductance of ions through a channel, the lipid composition of the formed bilayer has to be taken into account, and the lipids associated with the purified protein, such as cholesterol in the case of VDAC1 (Mlayeh et al. 2010; De Pinto et al. 1989), have been shown to modify its channel properties (Rostovtseva et al. 2006; Mlayeh et al. 2010). Moreover, VDAC1 purified in the presence of LDAO has a higher pore-forming activity in lipid bilayer membranes than does VDAC1 isolated in the presence of Triton X-100 (De Pinto et al. 1989). It is well established that VDAC1 is permeable to Ca2+ (Gincel et al. 2001; Bathori et al. 2006; Deniaud et al. 2007; Tan and Colombini 2007). In the presence of CaCl2, the well-defined voltagedependent characteristics of VDAC1 channel activity and selectivity to Cl− over Ca2+ are observed. However, investigators should bear in mind that some differences have been reported with respect to Ca2+ permeability relative to Cl− and voltage gating in the positive and negative potentials (Tan and Colombini 2007). Such differences might result from the nature of the method used for VDAC1 purification, the method used to build the PLB and the concentrations of CaCl2 used in the experiment. RECIPES Agarose Solution for Building Agar Salt Bridges
Reagent
Final concentration
Agarose KCl
2% 1M
Store at 4˚C for up to 2–3 mo. Buffer for Assaying Ca2+ Transport by VDAC1 Reconstituted into PLBs
Reagent CaCl2 Tris–HCl (pH 7.4)
Final concentration 100–300 mM 10 mM
Store at 4˚C. 104
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VDAC1 Reconstitution and Channel Recording
ACKNOWLEDGMENTS
Support from Phil and Sima Needleman is highly acknowledged. This research was partially supported by a grant from the Israel Science Foundation. REFERENCES Bathori G, Csordas G, Garcia-Perez C, Davies E, Hajnoczky G. 2006. Ca2+dependent control of the permeability properties of the mitochondrial outer membrane and voltage-dependent anion-selective channel (VDAC). J Biol Chem 281: 17347–17358. Ben-Hail D, Shoshan-Barmatz V. 2014. Purification of VDAC1 from rat liver mitochondria. Cold Spring Harbor Protoc doi: 10.1101/pdb. prot073130. De Pinto V, Benz R, Palmieri F. 1989. Interaction of non-classical detergents with the mitochondrial porin. A new purification procedure and characterization of the pore-forming unit. Eur J Biochem 183: 179–187. Deniaud A, Rossi C, Berquand A, Homand J, Campagna S, Knoll W, Brenner C, Chopineau J. 2007. Voltage-dependent anion channel transports
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073148
calcium ions through biomimetic membranes. Langmuir 23: 3898– 3905. Gincel D, Zaid H, Shoshan-Barmatz V. 2001. Calcium binding and translocation by the voltage-dependent anion channel: A possible regulatory mechanism in mitochondrial function. Biochem J 358: 147–155. Mlayeh L, Chatkaew S, Leonetti M, Homble F. 2010. Modulation of plant mitochondrial VDAC by phytosterols. Biophys J 99: 2097–2106. Rostovtseva TK, Kazemi N, Weinrich M, Bezrukov SM. 2006. Voltage gating of VDAC is regulated by nonlamellar lipids of mitochondrial membranes. J Biol Chem 281: 37496–37506. Tan W, Colombini M. 2007. VDAC closure increases calcium ion flux. Biochim Biophys Acta 1768: 2510–2515.
105
Reconstitution of Purified VDAC1 into a Lipid Bilayer and Recording of Channel Conductance Danya Ben-Hail and Varda Shoshan-Barmatz Cold Spring Harb Protoc; doi: 10.1101/pdb.prot073148 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. Calcium Imaging (86 articles) Characterization of Proteins (172 articles) Proteins and Proteomics, general (492 articles)
To subscribe to Cold Spring Harbor Protocols go to:
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© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at HSC LIBRARY/SERIALS on November 5, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
Reconstitution of Purified VDAC1 into a Lipid Bilayer and Recording of Channel Conductance Danya Ben-Hail and Varda Shoshan-Barmatz1 Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
The functional properties of purified voltage-dependent anion-selective channel protein 1 (VDAC1) have been examined in reconstituted systems based on artificially prepared phospholipid bilayers. The most widespread method for the characterization of the pore-forming activity of the mitochondrial VDAC1 protein requires reconstitution of the channel activity into a planar lipid bilayer (PLB) that separates two aqueous compartments. This system is able to produce a refined and large set of information on channel activity. The activity of the channel is reflected in the flow of ions (i.e., current) through a membrane that otherwise represents a barrier to ion flow. The setup thus requires the use of purified protein and a source of continuous current, as well as a sophisticated detector system able to amplify and record low, picoamper-level currents. This system is so efficient that the activity of even a single channel can be detected, allowing for study of VDAC1 at the molecular level.
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
Agarose solution for building agar salt bridges Buffer for assaying Ca2+ transport by VDAC1 reconstituted into PLBs Chloroform n-Decane KCl (1 M) Nitrogen Soybean asolectin (Sigma-Aldrich 11145) VDAC1 (see Purification of VDAC1 from Rat Liver Mitochondria [Ben-Hail and Shoshan-Barmatz 2014]) Equipment
Ag/AgCl electrodes Agar bridge wells 1
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073148
100
Downloaded from http://cshprotocols.cshlp.org/ at HSC LIBRARY/SERIALS on November 5, 2014 - Published by Cold Spring Harbor Laboratory Press
VDAC1 Reconstitution and Channel Recording
Amplifier headstage (Warner Instrument Corporation) Bilayer clamp amplifier (Warner Instrument Corporation) Bunsen burner Chamber holder (Warner Instrument Corporation) Digidata 1200/1440 interface board (Warner Instrument Corporation) Faraday cage Glass capillary Glass Pasteur pipettes Glass vial Magnetic stirrer (2 × 5 mm) Perfusion system Pipette (with a gel-loading tip) or syringe (with a needle) (see Step 1) Polystyrene chamber (with 250 µm aperture) Software suitable for data capture and analysis (e.g., pCLAMP 10.2 from Axon Instruments) Vibration isolation table
METHOD PLB membranes are formed across an aperture in a barrier that separates two bath solutions. In this protocol, the design used is that of a polystyrene chamber and a chamber holder (Fig. 1). The solution in the chamber represents one bath, whereas the solution in the holder is considered to be the other bath. After reconstituting VDAC1 into the bilayer membrane, formed in the aperture located in the chamber, currents passing through the channel can be recorded. Electrical connections are made by agar salt bridges placed in each bath. The channel recordings must be made in a Faraday cage to shield them from vibrations and electrical interference.
Preparation for Recording
1. Prepare two agar salt bridges that will serve to connect the electrodes to the chambers. Bend the glass capillary by heating over a Bunsen burner. Fill the capillaries with the agarose (heated to 90oC) using a pipette with a gel-loading tip or a syringe with a needle. The agar must be boiled just before filling the capillaries. The bridge should be as short as possible.
2. Prepare the lipid solution in a glass vial by dissolving soybean asolectin in chloroform (3 mg/mL). Dry the lipids under a nitrogen stream. After they are completely dry, dissolve the lipids in n-decane (30 mg/mL). The lipid solution should always be clear. Lipid solutions contaminated with water will turn milky and cannot be used.
3. Precoat the aperture in the chamber with lipids. Use a glass Pasteur pipette containing a small solid ball at its tip, formed by heating with a Bunsen burner, to coat the outside rim of the hole with the lipid solution. Let the lipid solution dry on the chamber before adding the appropriate solutions to the cup and chamber holder. Assembly of the Bilayer System
4. Place the chamber holder in the Faraday cage. Place the chamber and two agar bridge wells at the appropriate places in the chamber holder. Situate each of the agar salt bridges in one of the wells and in either the cup or the interior of the holder, as appropriate. 5. Connect the Ag/AgCl electrodes through which the transmembrane potential will be applied. Connect the trans side to the ground in the amplifier head-stage by means of an electrode placed in the agar bridge well connected to this chamber. Connect the cis side, where the protein will be added, to the input in the amplifier head-stage using the second electrode. To prevent contamination of silver, the electrodes should not be connected directly to the bath solutions. The agar bridges will close the circuit. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073148
101
Downloaded from http://cshprotocols.cshlp.org/ at HSC LIBRARY/SERIALS on November 5, 2014 - Published by Cold Spring Harbor Laboratory Press
D. Ben-Hail and V. Shoshan-Barmatz
+
Cis
–
Trans
Current
Voltage
Time
FIGURE 1. Schematic representation of planar lipid bilayer (PLB) reconstitution (left) and the channel conductance assay system (right) for assessing the single and multichannel activity of VDAC1. VDAC1 channel activity is measured following its reconstitution into a PLB and measuring the current passing through the channel (green oval) when a salt concentration gradient or voltage is applied across the bilayer. The membrane serves as a capacitor, whereas the ions carry the current. The cis side is defined as the compartment to which VDAC1 was added. Currents can be recorded under voltage-clamp by using a bilayer clamp amplifier and are measured with respect to the cis side of the membrane (ground). The currents can be low-pass filtered at 1 kHz and digitized online using an interface board and pCLAMP software.
6. Attach the perfusion system of choice, according to the manufacturer’s instructions. 7. Carefully add a magnetic stirring bar to the cis chamber. Formation of the Planar Bilayer Membrane
8. Add the experimental buffer to the cis and trans sides. In the Werner system, 1 mL is added to each chamber, although the volumes should be scaled to the chambers used.
9. Into each agar bridge well, add 1 M KCl to cover the tip of the electrodes. 10. Take a clean and dry glass Pasteur pipette, like the one used to precoat the chamber, dip it into the lipid solution, and then lightly touch the aperture. 11. Monitor the bilayer formed using a bilayer clamp amplifier. A straight line at 0 pA should be observed. Check the resistance of the PLB, and if it is between 100 and 120 GΩ, check the membrane for leaks by applying different voltages (±60 mV). If the current remains at 0 pA, the bilayer is stable and intact. See Troubleshooting.
Reconstitution of VDAC1 Into a Planar Lipid Bilayer
12. Add purified VDAC1 (1–2 ng) to one of the compartments (this is then termed the cis side; see Fig. 1) containing the buffer for assaying Ca2+ transport (with the selected CaCl2 concentration), and then turn on the magnetic stirrer. 13. Check that VDAC1 insertion into the membrane is realized within 5–20 min, as reflected by an increase in current. Check for stability of the protein in the PLB by applying +60 mV and verify that you get a typical readout for VDAC1-mediated current at this voltage. The current should first increase due to a greater driving force, but within < 1 sec, the channel should close to a stable low-conducting state (as in Fig. 2A).
102
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VDAC1 Reconstitution and Channel Recording
14. After one or more channels have been inserted into the PLB, remove excess protein by perfusion of the cis chamber with 20 volumes of a solution of the same composition as that used before perfusion, so as to prevent further incorporation. Recording Currents Through VDAC1
15. Record the currents under voltage-clamp using a bilayer clamp amplifier. Measure the currents with respect to the cis side of the membrane (ground). • To measure the currents of identical CaCl2 concentrations (in the range of 100–300 mM) in both the cis and trans compartments or of a CaCl2 concentration gradient (300:150 mM, cis: trans) (see Fig. 2), use voltage steps or voltage ramps between −60 and +60 mV.
•
To assess the Ca2+ permeability of VDAC1 (relative to chloride), determine the reversal potential at different CaCl2 concentration gradients as described previously (Gincel et al. 2001). Obtain an estimate of the Ca2+/Cl− permeability ratio (r) using the reversal potential (Vrev), the CaCl2 activity, and the following equation:
r=
PCa2+ (1 + B) B[Cl− ]0 − [Cl− ]x , = 4 PCl− [Ca2+ ]0 − [Ca2+ ]x B2
A
B
–10 mV
I, pA 90 60 20 pA 1 sec
–40 mV
30 –60
–40
20
–20 –30
40
60
Voltage, mV
–60 –90
Conductance Maximal conductance of control
C 1 0.8 0.6 0.4 0.2 0
–60 –40 –20
0
20 40 60
Voltage, mV
FIGURE 2. VDAC1 channel properties, conductance measurements, and analysis with CaCl2 as the sources of ions carrying the current. (A) Single-channel current in response to voltage steps (0/−10) or (0/–40). The cis and trans chambers contain identical concentrations of 150 mM CaCl2. (B) Single channel currents through VDAC1 in response to voltage ramps (−60 to +60 mV at 60 mV sec−1) in the presence of 150 mM CaCl2 in both chambers (represented in black), or in the presence of a CaCl2 concentration gradient (300/150 mM, cis/trans) (shown in gray). The reverse potential (Vrev) derived from this plot can be used to calculate the relative conductance of Cl−/Ca2+. In this experiment, the calculated ratio is 2:1 Cl−/Ca2+. (C) Channel conductance of PLB-reconstituted VDAC1, as recorded in response to a voltage step from 0 mV to voltages between −60 and +60 mV. The average steady-state conductance at a given voltage was normalized to the conductance at 10 mV (namely, conductance at a given voltage/maximal conductance at 10 mV). Closed circles represent channel conductance measured at identical concentrations of CaCl2 in cis and trans. Open circles represent channel conductance measured with a CaCl2 concentration gradient (300/150 mM, cis/trans).
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D. Ben-Hail and V. Shoshan-Barmatz
where B = eRT Vrev , F is the Faraday constant (9.648 × 104 C mol−1); R is the universal gas constant (8.314 J K−1 mol−1); and T is absolute temperature. F
The currents should be low-pass filtered at 1 kHz (–3 dB). All experiments should be performed at 21˚C–25˚C. A full description of how to use software suitable for data capture and analysis (e.g., pCLAMP from Axon Instruments) is beyond the scope of this article, and can be obtained in the user guide of the software.
TROUBLESHOOTING Problem (Step 11): The membrane is too thick. Solution: Switch the holding voltage from +60 to −60 mV. Repeat this step a few times. If this does not
help to thin out the membrane, break it and make a new membrane. DISCUSSION
The permeability to Ca2+ of VDAC1 can be followed on its reconstitution into a PLB, as a single channel or as multichannels, under voltage-clamp conditions. The PLB is a powerful tool in the study of channels such as VDAC1 and is useful for, among others, simultaneous measurement of ionic charge movement (conductance), channel gating, and studying the effects of inhibitors. When studying the conductance of ions through a channel, the lipid composition of the formed bilayer has to be taken into account, and the lipids associated with the purified protein, such as cholesterol in the case of VDAC1 (Mlayeh et al. 2010; De Pinto et al. 1989), have been shown to modify its channel properties (Rostovtseva et al. 2006; Mlayeh et al. 2010). Moreover, VDAC1 purified in the presence of LDAO has a higher pore-forming activity in lipid bilayer membranes than does VDAC1 isolated in the presence of Triton X-100 (De Pinto et al. 1989). It is well established that VDAC1 is permeable to Ca2+ (Gincel et al. 2001; Bathori et al. 2006; Deniaud et al. 2007; Tan and Colombini 2007). In the presence of CaCl2, the well-defined voltagedependent characteristics of VDAC1 channel activity and selectivity to Cl− over Ca2+ are observed. However, investigators should bear in mind that some differences have been reported with respect to Ca2+ permeability relative to Cl− and voltage gating in the positive and negative potentials (Tan and Colombini 2007). Such differences might result from the nature of the method used for VDAC1 purification, the method used to build the PLB and the concentrations of CaCl2 used in the experiment. RECIPES Agarose Solution for Building Agar Salt Bridges
Reagent
Final concentration
Agarose KCl
2% 1M
Store at 4˚C for up to 2–3 mo. Buffer for Assaying Ca2+ Transport by VDAC1 Reconstituted into PLBs
Reagent CaCl2 Tris–HCl (pH 7.4)
Final concentration 100–300 mM 10 mM
Store at 4˚C. 104
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VDAC1 Reconstitution and Channel Recording
ACKNOWLEDGMENTS
Support from Phil and Sima Needleman is highly acknowledged. This research was partially supported by a grant from the Israel Science Foundation. REFERENCES Bathori G, Csordas G, Garcia-Perez C, Davies E, Hajnoczky G. 2006. Ca2+dependent control of the permeability properties of the mitochondrial outer membrane and voltage-dependent anion-selective channel (VDAC). J Biol Chem 281: 17347–17358. Ben-Hail D, Shoshan-Barmatz V. 2014. Purification of VDAC1 from rat liver mitochondria. Cold Spring Harbor Protoc doi: 10.1101/pdb. prot073130. De Pinto V, Benz R, Palmieri F. 1989. Interaction of non-classical detergents with the mitochondrial porin. A new purification procedure and characterization of the pore-forming unit. Eur J Biochem 183: 179–187. Deniaud A, Rossi C, Berquand A, Homand J, Campagna S, Knoll W, Brenner C, Chopineau J. 2007. Voltage-dependent anion channel transports
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot073148
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