[30]
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447
[30] I n s e r t i o n o f I o n C h a n n e l s i n t o P l a n a r L i p i d B i l a y e r s by Vesicle Fusion
By P E D R O
LABARCA and RAM6N
LATORRE
Introduction Model or "artificial" lipid bilayers have become common tools in the study of the molecular mechanisms that undedy the ion-translocating properties of cell membranes. The assembly in model lipid bilayers of the molecular entities engaged in ion translocation permits their study in convenient isolation from the rest of the cell machinery. In the extreme case, a transport protein is isolated to purity from its native environment and functionally reconstituted in a model bilayer allowing for the establishment of detailed structure-function correlates. In fact, the ultimate criterion to qualify a given molecular entity as responsible for an iontranslocating function still is its functional reconstitution in a model lipid bilayer. The study of membrane transport in model lipid bilayers has been rightly defined as an extreme reductionist approach, a quality to which it owes much success. Among the variety of transport mechanisms exhibited by cell membranes, ion channels are the ones that have profited the most from model bilayers. The simplicity and current resolution capabilities of current-measuring devices available and the fantastic rates at which ion channels catalyze ion fluxes through lipid bilayers make it possible to monitor the currents flowing through a single channel molecule inserted or reconstituted in a special type of model lipid membrane, the planar lipid bilayer. In this chapter we summarize the experimental procedures used to insert ion channels derived from cell membranes into planar lipid bilayers using fusion approaches, l-3 An attempt has also been made to introduce the most general concepts that usually guide preliminary studies of ion channels in planar lipid bilayers. Some of the limitations of the technique as well as some less explored avenues of research are introduced.
C. Miller and E. Racker, J. Membr. Biol. 30, 283 (1976). 2 F. S. Cohen, M. H. Akabas, J. Zimmemberg, and A. Finkelstein, Science 217, 458 (1982). 3 W. Hanke, H. Eibl, and G. Boheim, Biophys. Struct. Mech. 7, 131 (1981).
METHODS IN ENZYMOLOGY, VOL. 207
Copyright© 1992by AcademicPttl, Inc. All rightsof relmxiuctionin any form reserved.
448
RECONSTITUTION OF ION CHANNELS
[30]
Insertion of Ion Channels into Planar Lipid Bilayers Using Fusion Technique Planar Lipid Bilayers
Planar lipid bilayers amenable to ion channel insertion can be produced either by the "painting" technique4 or from two lipid monolayers spread at the air-water interface. 5 In the "painting" variant a small aliquot of a solution of lipid in decane (10-50 mg/ml) is applied in the aperture (0.1-0.5 mm diameter) of a plastic septum separating two aqueous chambers. A short time after deposition of a drop of lipid in the aperture a lipid bilayer develops in the center of the aperture. The lipid bilayer so formed hangs from a solvent torus which is adsorbed to the edge of the aperture. The resulting bilayers have capacitances of the order of 0.30.6/xF/cm 2 and conductances of about 10 pS, with peak-to-peak electrical noise of 0.5-2 pA at 1-kHz low-pass filtering. Bilayer thinning can be monitored optically through a microscope and/or by capacitance measurements. A thin lipid bilayer looks black to the eye since it reflects little light. For this reason the planar bilayer is often referred to as a "black membrane." After thinning, the bilayer should occupy 75-80% of the aperture area. It is worth noting that "painted" bilayers, in addition to lipid, contain solvent. The presence of solvent means that painted bilayers possess electrical capacitances that are notoriously smaller than those of cell membranes. The technique to assemble lipid bilayers from monolayers spread at the air-water interface was developed by Montal and Mueller, 5 following the original suggestion of Langmuifs and the experiences of Takagi et al. 7 In this method two lipid monolayers are spread in each compartment of a chamber separated by a septum in which a small hole 50-300 g m in diameter has been punctured. Previous to bilayer assembly the aperture is doped with petroleum jelly or squalene, providing the system with a torus to support the bilayer. A few minutes after spreading the monolayers to allow for solvent (usually pentane) evaporation, the buffer in each chamber is sequentially raised above the aperture resulting in the formation of a flat lipid film exhibiting capacitances of the order of 0.6-0.8/zF/cm and high electrical resistances. Bilayers assembled by this procedure are virtually 4 p. Mueller, D. Rudin, H. T. Tien, and W. C. Wescott, Circulation 26, 1167 (1962). 5 M. Montal and P. MueIler, Proc. Natl. Acad. Sci. U.S.A. 69, 3561 (1972). I. Langmuir, J. Chem. Phys. 1, 756 (1933). M. Takagi, K. Azuma, and U. Kishimoto, Annu. Rep. Biol. Works Fac. Sci. Osaka Univ. 13, 107 (1965).
[30]
INSERTION OF ION CHANNELS BY VESICLE FUSION
449
"solvent free, ''s providing a lipid matrix devoid o f solvent in which the insertion o f ion channel molecules can be attempted. Because bilayers made from monolayers do not thin, bilayer formation in this case is followed only through capacitance measurements. Lipid Requirements
The experimenter engaged in preliminary attempts to fuse vesicular fragments derived from cell membranes or lipid vesicles into which purified channel proteins have been reconstituted should benefit from the proper choice o f lipids used in bilayer formation. Experience indicates the convenience of including an acidic lipid in the lipid mixture used to form bilayers since they increase the chances o f fusion. The most widely used acidic lipid is phosphatidylserine (PS), which is mixed with a neutral lipid such as phosphatidylethanolamine (PE) at various ratios, although other acidic lipids like phosphatidic acid and cardiolipin have also been reported to yield satisfactory results. (The reader should be aware that the surface charge contributed by acidic lipid can influence ion channel properties?) Table I shows the lipid composition o f planar bilayers in where ion channels were inserted using the fusion m e t h o d ? °-24 Vesicle fusion to planar lipid bilayers can also be obtained in planar bilayers made o f neutral lipids. A variety o f natural and synthetic lipids are commercially available from Avanti Polar Lipids (Birmingham, AL), or, alternatively, lipids of 8 S. H. White, In "Ion Channel Reconstitution" (C. Miller, ed.), p. 3. Plenum, New York and London, 1986. 9 R. Latorre, P. Labarca, and D. Naranjo, this volume [32]. ,o j. C. Tanaka, R. E. Furman, and R. L. Barchi, In "Ion Channel Reconstitution" (C. Miller, ed.), p. 277. Plenum, New York and London, 1986. ,l R. P. Hatshorne, B. U. Keller, J. A. Talvenheimo, W. A. Caterall, and M. Montal, Proc. Natl. Acad. Sci. U.S.A. 82, 240 0985). ,2 I. K. Krueger, F. W. Jennings, and R. J. French, Nature (London) 303, 172 (1983). 13E. Moczydlowski,S. S. Garber, and C. Miller, J. Gen. Physiol. 84, 665 (1984). L4j. S. Smith, T. Imagawa, M. Jianjie, M. Fill, K. P. Campbell, and R. Coronado, J. Gen. Physiol. 92, 1 (1986). ,5 B. A. Suarez-Isla, V. Iribarra, A. Oberhanser, L. Larralde, C. Hidalgo, and E. Jaimovich, Biophys. J. 54, 737 (1988). t6 H. Affolterand R. Coronado, Biophys. J. 48, 341 (1985). 17A. Lievano, E. C. Vega-Saenzde Micra, and A. Darszon, J. Gen. Physiol. 95, 273 (1990). 18M. T. Nelson, R. J. French, and K. K_rueger,Nature (London) 308, 77 (1984). t9 E. Moczydlowski,O. Alvarez, C. Vergara, and R. Latorre, J. Membr. Biol. 82, 273 (1985). 2oj. Bell and C. Miller, Biophys. J. 45, 279 (1984). 2~p. Labarca, Ph.D. Thesis, Brandeis University, Waltham, Massachusetts 0980). 22p. Labarca, R. Coronado, and C. Miller, J. Gen. Physiol. 76, 396 (1980). 23j. A. Hill, R. Coronado, and H. C. Strauss, Circ. Res. 62, 411 0988). 24M. M. White and C. Miller, J. Biol. Chem. 254, 10161 (1979).
450
RECONSTITUTION OF ION CHANNELS
[30]
TABLE I LIPIDS MOST COMMONLYUSED TO FORM BILAYERSIN ION CHANNELRECONSTITUTION STUDIES Channel (source) Purified Na+ channel (skeletal muscle) Purified Na+ channel (brain) Na+ channel (rat brain homogenates) Na+ channel (rat skeletal muscle plasma membrane) Ca~+ channel (purified from skeletal muscle sarcoplasmic reticulum) Ca2+ channel (skeletal sarcoplasmic reticulum vesicular fragments) Ca2+ channel (T-tubule membranes) Cae+ channel (sperm plasma membranes) Ca2+ channel (brain homogenates) Ca2+-activated K + channel (T-tubule membranes) K + channels (skeletal muscle sarcoplasmic reticulum membranes)
K + channel (cardiac muscle sarcoplasmic reticulum membranes) CI- channel (fish electric organ) Cation-selective pore (peroxisomal membranes)
Type of lipid
Ref.
Phosphatidylethanolamine, phosphatidylcholine Phosphatidylethanolamine, phosphafidylcholine Phosphatidylsedne, phosphatidylethanolamine Phosphafidylethanolamine, phosphatidylcholine
10
Phosphatidylethanolamine, phosphatidylserine
14
Phosphatidylethanolamine phosphatidylcholine Phosphatidylethanolamine phosphatidylserine Phosphatidylethanolamine phosphatidylserine Phosphatidylethanolamine pbosphatidylserine Phosphatidylethanolamine phosphatidylserine Phosphatidylethanolamine phosphatidylsedne, phosphatidylcholine Phosphatidylserine, phosphafidylethanolamine Phosphatidylethanolamine, phosphatidylcholine Asolectin, cardiolipin Asolectin, phosphatidic acid Phosphatidylserine, phosphatidylethanolamine
15
Phosphatidylethanolanine, phosphatidylglycerol Phosphatidylethanolamine, phosphatidyleholine
11 12 13
16 17 18 19 20
21 22 23 24
[30]
INSERTION OF ION CHANNELS BY VESICLE FUSION
451
bilayer quality can be purified in the laboratory. Stock lipid solutions are kept at - 2 0 ° to - 8 0 ° under a nitrogen atmosphere, and lipid used in bilayer work must be prepared fresh from stocks each day. Bilayer chambers and glass material used in bilayer work should be immaculate.
Fusion of Vesicular Membrane Fractions to Planar Lipid Bilayers The main criterion that makes a given membrane preparation suitable for fusion into planar lipid bilayers is its purity, apart from the obvious interest of a researcher in studying the conductance properties of a given cell membrane at the single-channel level. Use of a highly purified membrane fraction provides the potential reproducibility that is so important in this kind of experimental work. However, crude membrane extracts have also been used in some cases. 2~ For reasons that have not been completely understood vesicle fusion in painted bilayers is more easily achieved than fusion in bilayers made from monolayers. However, the experimental conditions needed to fuse lipid vesicles are identical for both types of model planar bilayers.2~,zz,26 For operational purposes the two aqueous compartments separated by the lipid film are defined as cis and trans compartments. The cis compartment is the one to which a voltage generator is connected to the bilayer through an Ag/AgCI electrode. The trans compartment is connected to the input of the current=measuring amplifier through a second Ag/AgCI electrode. It is recommended that both electrodes be connected to the chambers through 3 M KCI agar bridges that, ideally, should be made each working day to avoid bacterial and fungal contamination. Both chambers must be provided with stirring facilities. It is also necessary to provide the setup with a peffusion system. After a bilayer is formed under the buffer solution of choice, the Ca 2+ concentration in the cis compartment must be raised by adding an aliquot from a concentrated stock solution. From now on all operations are made under stirring in the cis compartment. (The precise divalent ion concentration in the cis side must be empirically determined; fusion requires the presence of Ca 2+ in the I - I0 m M range.) An aliquot of vesicles is added to the cis chamber. The membrane vesicles should be loaded with a buffer (usually a sucrose buffer) at 0.3-0.5 M solute concentration prior to their use in fusion experiments. Because sucrose is an impermeable solute, the vesicles swell when added into the bilayer chamber, which helps in pro25I. B. Le~tan, in"Ion Channel Reconsfitufion"(C. Miller, ed.),p. 523. Plenum, New York and London, 1986. 26 F. S. Cohen, i n " I o n Channel Reconstitution"(C. MiUer, ed.),p. 131. Plenum New York and London, 1986.
452
RECONSTITUTION OF ION CHANNELS
[30]
moting fusion. The amount of vesicles to be added depends on the fusogenic properties of each particular preparation, but it ranges from 0.2 to 50 #g protein/ml (final concentration). Using bilayer chambers that hold small volumes (0.3-0.5 ml) is important when the purified membranes are scarce. Under the experimental conditions that we presented (vesicles and Ca 2+ in the cis side, and the same ionic buffer in both compartments), vesicle fusion to planar lipid bilayers is infrequent. However, fusion can now be induced in a controlled fashion by establishing an osmotic gradient between the cis and the trans bilayer sides. For this, an aliquot of a concentrated solute (e.g., KC1 or LiC1) is added to the cis chamber. The magnitude of the osmotic gradient necessary to trigger fusion is variable, but a 3 : 1 gradient should be used to start. Larger osmotic gradients result in more fusion. The researcher should spend some time becoming familiar with the experimental conditions needed to achieve fusion of a particular membrane preparation in order to monitor single-channel currents. Experimental conditions must be found that are reproducible and efficient in obtaining the insertion of single ion channel molecules into the planar bilayer. Conditions leading to massive fusion of vesicles to planar bilayers should be avoided. Bilayer conductance is followed throughout these procedures by applying a potential difference and by measuring current. At the beginning of the experiment the bare bilayer conductance should be low (
INSERTION OF ION CHANNELS BY VESICLE FUSION
447
[30] I n s e r t i o n o f I o n C h a n n e l s i n t o P l a n a r L i p i d B i l a y e r s by Vesicle Fusion
By P E D R O
LABARCA and RAM6N
LATORRE
Introduction Model or "artificial" lipid bilayers have become common tools in the study of the molecular mechanisms that undedy the ion-translocating properties of cell membranes. The assembly in model lipid bilayers of the molecular entities engaged in ion translocation permits their study in convenient isolation from the rest of the cell machinery. In the extreme case, a transport protein is isolated to purity from its native environment and functionally reconstituted in a model bilayer allowing for the establishment of detailed structure-function correlates. In fact, the ultimate criterion to qualify a given molecular entity as responsible for an iontranslocating function still is its functional reconstitution in a model lipid bilayer. The study of membrane transport in model lipid bilayers has been rightly defined as an extreme reductionist approach, a quality to which it owes much success. Among the variety of transport mechanisms exhibited by cell membranes, ion channels are the ones that have profited the most from model bilayers. The simplicity and current resolution capabilities of current-measuring devices available and the fantastic rates at which ion channels catalyze ion fluxes through lipid bilayers make it possible to monitor the currents flowing through a single channel molecule inserted or reconstituted in a special type of model lipid membrane, the planar lipid bilayer. In this chapter we summarize the experimental procedures used to insert ion channels derived from cell membranes into planar lipid bilayers using fusion approaches, l-3 An attempt has also been made to introduce the most general concepts that usually guide preliminary studies of ion channels in planar lipid bilayers. Some of the limitations of the technique as well as some less explored avenues of research are introduced.
C. Miller and E. Racker, J. Membr. Biol. 30, 283 (1976). 2 F. S. Cohen, M. H. Akabas, J. Zimmemberg, and A. Finkelstein, Science 217, 458 (1982). 3 W. Hanke, H. Eibl, and G. Boheim, Biophys. Struct. Mech. 7, 131 (1981).
METHODS IN ENZYMOLOGY, VOL. 207
Copyright© 1992by AcademicPttl, Inc. All rightsof relmxiuctionin any form reserved.
448
RECONSTITUTION OF ION CHANNELS
[30]
Insertion of Ion Channels into Planar Lipid Bilayers Using Fusion Technique Planar Lipid Bilayers
Planar lipid bilayers amenable to ion channel insertion can be produced either by the "painting" technique4 or from two lipid monolayers spread at the air-water interface. 5 In the "painting" variant a small aliquot of a solution of lipid in decane (10-50 mg/ml) is applied in the aperture (0.1-0.5 mm diameter) of a plastic septum separating two aqueous chambers. A short time after deposition of a drop of lipid in the aperture a lipid bilayer develops in the center of the aperture. The lipid bilayer so formed hangs from a solvent torus which is adsorbed to the edge of the aperture. The resulting bilayers have capacitances of the order of 0.30.6/xF/cm 2 and conductances of about 10 pS, with peak-to-peak electrical noise of 0.5-2 pA at 1-kHz low-pass filtering. Bilayer thinning can be monitored optically through a microscope and/or by capacitance measurements. A thin lipid bilayer looks black to the eye since it reflects little light. For this reason the planar bilayer is often referred to as a "black membrane." After thinning, the bilayer should occupy 75-80% of the aperture area. It is worth noting that "painted" bilayers, in addition to lipid, contain solvent. The presence of solvent means that painted bilayers possess electrical capacitances that are notoriously smaller than those of cell membranes. The technique to assemble lipid bilayers from monolayers spread at the air-water interface was developed by Montal and Mueller, 5 following the original suggestion of Langmuifs and the experiences of Takagi et al. 7 In this method two lipid monolayers are spread in each compartment of a chamber separated by a septum in which a small hole 50-300 g m in diameter has been punctured. Previous to bilayer assembly the aperture is doped with petroleum jelly or squalene, providing the system with a torus to support the bilayer. A few minutes after spreading the monolayers to allow for solvent (usually pentane) evaporation, the buffer in each chamber is sequentially raised above the aperture resulting in the formation of a flat lipid film exhibiting capacitances of the order of 0.6-0.8/zF/cm and high electrical resistances. Bilayers assembled by this procedure are virtually 4 p. Mueller, D. Rudin, H. T. Tien, and W. C. Wescott, Circulation 26, 1167 (1962). 5 M. Montal and P. MueIler, Proc. Natl. Acad. Sci. U.S.A. 69, 3561 (1972). I. Langmuir, J. Chem. Phys. 1, 756 (1933). M. Takagi, K. Azuma, and U. Kishimoto, Annu. Rep. Biol. Works Fac. Sci. Osaka Univ. 13, 107 (1965).
[30]
INSERTION OF ION CHANNELS BY VESICLE FUSION
449
"solvent free, ''s providing a lipid matrix devoid o f solvent in which the insertion o f ion channel molecules can be attempted. Because bilayers made from monolayers do not thin, bilayer formation in this case is followed only through capacitance measurements. Lipid Requirements
The experimenter engaged in preliminary attempts to fuse vesicular fragments derived from cell membranes or lipid vesicles into which purified channel proteins have been reconstituted should benefit from the proper choice o f lipids used in bilayer formation. Experience indicates the convenience of including an acidic lipid in the lipid mixture used to form bilayers since they increase the chances o f fusion. The most widely used acidic lipid is phosphatidylserine (PS), which is mixed with a neutral lipid such as phosphatidylethanolamine (PE) at various ratios, although other acidic lipids like phosphatidic acid and cardiolipin have also been reported to yield satisfactory results. (The reader should be aware that the surface charge contributed by acidic lipid can influence ion channel properties?) Table I shows the lipid composition o f planar bilayers in where ion channels were inserted using the fusion m e t h o d ? °-24 Vesicle fusion to planar lipid bilayers can also be obtained in planar bilayers made o f neutral lipids. A variety o f natural and synthetic lipids are commercially available from Avanti Polar Lipids (Birmingham, AL), or, alternatively, lipids of 8 S. H. White, In "Ion Channel Reconstitution" (C. Miller, ed.), p. 3. Plenum, New York and London, 1986. 9 R. Latorre, P. Labarca, and D. Naranjo, this volume [32]. ,o j. C. Tanaka, R. E. Furman, and R. L. Barchi, In "Ion Channel Reconstitution" (C. Miller, ed.), p. 277. Plenum, New York and London, 1986. ,l R. P. Hatshorne, B. U. Keller, J. A. Talvenheimo, W. A. Caterall, and M. Montal, Proc. Natl. Acad. Sci. U.S.A. 82, 240 0985). ,2 I. K. Krueger, F. W. Jennings, and R. J. French, Nature (London) 303, 172 (1983). 13E. Moczydlowski,S. S. Garber, and C. Miller, J. Gen. Physiol. 84, 665 (1984). L4j. S. Smith, T. Imagawa, M. Jianjie, M. Fill, K. P. Campbell, and R. Coronado, J. Gen. Physiol. 92, 1 (1986). ,5 B. A. Suarez-Isla, V. Iribarra, A. Oberhanser, L. Larralde, C. Hidalgo, and E. Jaimovich, Biophys. J. 54, 737 (1988). t6 H. Affolterand R. Coronado, Biophys. J. 48, 341 (1985). 17A. Lievano, E. C. Vega-Saenzde Micra, and A. Darszon, J. Gen. Physiol. 95, 273 (1990). 18M. T. Nelson, R. J. French, and K. K_rueger,Nature (London) 308, 77 (1984). t9 E. Moczydlowski,O. Alvarez, C. Vergara, and R. Latorre, J. Membr. Biol. 82, 273 (1985). 2oj. Bell and C. Miller, Biophys. J. 45, 279 (1984). 2~p. Labarca, Ph.D. Thesis, Brandeis University, Waltham, Massachusetts 0980). 22p. Labarca, R. Coronado, and C. Miller, J. Gen. Physiol. 76, 396 (1980). 23j. A. Hill, R. Coronado, and H. C. Strauss, Circ. Res. 62, 411 0988). 24M. M. White and C. Miller, J. Biol. Chem. 254, 10161 (1979).
450
RECONSTITUTION OF ION CHANNELS
[30]
TABLE I LIPIDS MOST COMMONLYUSED TO FORM BILAYERSIN ION CHANNELRECONSTITUTION STUDIES Channel (source) Purified Na+ channel (skeletal muscle) Purified Na+ channel (brain) Na+ channel (rat brain homogenates) Na+ channel (rat skeletal muscle plasma membrane) Ca~+ channel (purified from skeletal muscle sarcoplasmic reticulum) Ca2+ channel (skeletal sarcoplasmic reticulum vesicular fragments) Ca2+ channel (T-tubule membranes) Cae+ channel (sperm plasma membranes) Ca2+ channel (brain homogenates) Ca2+-activated K + channel (T-tubule membranes) K + channels (skeletal muscle sarcoplasmic reticulum membranes)
K + channel (cardiac muscle sarcoplasmic reticulum membranes) CI- channel (fish electric organ) Cation-selective pore (peroxisomal membranes)
Type of lipid
Ref.
Phosphatidylethanolamine, phosphatidylcholine Phosphatidylethanolamine, phosphafidylcholine Phosphatidylsedne, phosphatidylethanolamine Phosphafidylethanolamine, phosphatidylcholine
10
Phosphatidylethanolamine, phosphatidylserine
14
Phosphatidylethanolamine phosphatidylcholine Phosphatidylethanolamine phosphatidylserine Phosphatidylethanolamine phosphatidylserine Phosphatidylethanolamine pbosphatidylserine Phosphatidylethanolamine phosphatidylserine Phosphatidylethanolamine phosphatidylsedne, phosphatidylcholine Phosphatidylserine, phosphafidylethanolamine Phosphatidylethanolamine, phosphatidylcholine Asolectin, cardiolipin Asolectin, phosphatidic acid Phosphatidylserine, phosphatidylethanolamine
15
Phosphatidylethanolanine, phosphatidylglycerol Phosphatidylethanolamine, phosphatidyleholine
11 12 13
16 17 18 19 20
21 22 23 24
[30]
INSERTION OF ION CHANNELS BY VESICLE FUSION
451
bilayer quality can be purified in the laboratory. Stock lipid solutions are kept at - 2 0 ° to - 8 0 ° under a nitrogen atmosphere, and lipid used in bilayer work must be prepared fresh from stocks each day. Bilayer chambers and glass material used in bilayer work should be immaculate.
Fusion of Vesicular Membrane Fractions to Planar Lipid Bilayers The main criterion that makes a given membrane preparation suitable for fusion into planar lipid bilayers is its purity, apart from the obvious interest of a researcher in studying the conductance properties of a given cell membrane at the single-channel level. Use of a highly purified membrane fraction provides the potential reproducibility that is so important in this kind of experimental work. However, crude membrane extracts have also been used in some cases. 2~ For reasons that have not been completely understood vesicle fusion in painted bilayers is more easily achieved than fusion in bilayers made from monolayers. However, the experimental conditions needed to fuse lipid vesicles are identical for both types of model planar bilayers.2~,zz,26 For operational purposes the two aqueous compartments separated by the lipid film are defined as cis and trans compartments. The cis compartment is the one to which a voltage generator is connected to the bilayer through an Ag/AgCI electrode. The trans compartment is connected to the input of the current=measuring amplifier through a second Ag/AgCI electrode. It is recommended that both electrodes be connected to the chambers through 3 M KCI agar bridges that, ideally, should be made each working day to avoid bacterial and fungal contamination. Both chambers must be provided with stirring facilities. It is also necessary to provide the setup with a peffusion system. After a bilayer is formed under the buffer solution of choice, the Ca 2+ concentration in the cis compartment must be raised by adding an aliquot from a concentrated stock solution. From now on all operations are made under stirring in the cis compartment. (The precise divalent ion concentration in the cis side must be empirically determined; fusion requires the presence of Ca 2+ in the I - I0 m M range.) An aliquot of vesicles is added to the cis chamber. The membrane vesicles should be loaded with a buffer (usually a sucrose buffer) at 0.3-0.5 M solute concentration prior to their use in fusion experiments. Because sucrose is an impermeable solute, the vesicles swell when added into the bilayer chamber, which helps in pro25I. B. Le~tan, in"Ion Channel Reconsfitufion"(C. Miller, ed.),p. 523. Plenum, New York and London, 1986. 26 F. S. Cohen, i n " I o n Channel Reconstitution"(C. MiUer, ed.),p. 131. Plenum New York and London, 1986.
452
RECONSTITUTION OF ION CHANNELS
[30]
moting fusion. The amount of vesicles to be added depends on the fusogenic properties of each particular preparation, but it ranges from 0.2 to 50 #g protein/ml (final concentration). Using bilayer chambers that hold small volumes (0.3-0.5 ml) is important when the purified membranes are scarce. Under the experimental conditions that we presented (vesicles and Ca 2+ in the cis side, and the same ionic buffer in both compartments), vesicle fusion to planar lipid bilayers is infrequent. However, fusion can now be induced in a controlled fashion by establishing an osmotic gradient between the cis and the trans bilayer sides. For this, an aliquot of a concentrated solute (e.g., KC1 or LiC1) is added to the cis chamber. The magnitude of the osmotic gradient necessary to trigger fusion is variable, but a 3 : 1 gradient should be used to start. Larger osmotic gradients result in more fusion. The researcher should spend some time becoming familiar with the experimental conditions needed to achieve fusion of a particular membrane preparation in order to monitor single-channel currents. Experimental conditions must be found that are reproducible and efficient in obtaining the insertion of single ion channel molecules into the planar bilayer. Conditions leading to massive fusion of vesicles to planar bilayers should be avoided. Bilayer conductance is followed throughout these procedures by applying a potential difference and by measuring current. At the beginning of the experiment the bare bilayer conductance should be low (
