[3 1 ]
PLANAR LIPID BILAYERS ON PATCH PIPETTES
463
culties should lead the experimenter to avoid the use o f such lipids and to improve techniques in handling planar bilayers. O t h e r M e t h o d s to I n s e r t I o n C h a n n e l s into P l a n a r L i p i d B i l a y e r s Fusion o f lipid vesicles to "solvent-free" planar bilayers made of 1stearoyl-3-myristoyl/glycero-2-phosphocholine, a mixed-chain lecithin, at temperatures below the lipid phase transition has been reported by Boheim and collaborators (see e.g., Ref. 3). This m e t h o d is not widely used since it requires the use of a particular lipid at restricted temperatures. The m e t h o d of Schindler 42 to transfer proteins from lipid vesicles to a planar bilayer has been successfully used to insert channels into planar bilayers 27,2a,43 and to form bilayers from monolayers in the tip o f a patch pipette. 44-*sAcknowledgments This work was supported by grants from the Fondo Nacional de lnvestigacion (11671988, 451-1988), the National Institutes of Health (GM-35981),and the Tinker Foundation. R.L. is a recipient of a John S. GuggenheimFellowship. He also wishes to thank the Dreyfus Bank (Switzerland)for generous support from a private foundation that they made available. 42H. Schindler, FEBSLett. 122, 77 (1980). 43H. Schindler and U. Quast, Proc. Natl. Acad. Sci. U.S.A. 77, 3052 (1980). 44W. Hanke, C. Methfessel, H. U. Wilsem, and G. Boheim, Biochem. Bioeng. J. 12, 329 (1984). 45R. Coronado and R. Latorre, Biophys. J. 43, 231 (1983). 46B. Suarez-Isla,K. Wan, J. Lindstrom, and M. Montal, Biochemistry 22, 2319 (1983).
[31] Planar Lipid Bilayers on Patch Pipettes: Bilayer Formation and Ion Channel Incorporation B y B A R B A R A E. E H R L I C H
Introduction This chapter describes how to make bilayers on the tip o f patch-style pipettes and then incorporate channels into these bilayers. As there have been several reports describing the specific techniques needed for the formation o f this type of bilayer, 1-4 and even more reports describing how t R. Coronado and R. Latorre, Biophys J. 43, 231 (1983). 2 T. Schuerholzand H. Schindler,FEBS Lett. 152, 187 (1983). 3 B. A. Suarez-Isla, K. Wan, J. Lindstrom, and M. Montal, Biochemistry 22, 2319 (1983). 4 W. Hanke, C. Methfessel, H. V. Wilmsen, and G. Boheim, Biochem. Bioeng. J. 12, 329 (1984).
METHODS IN ENZYMOLOGY, VOL 207
Copyright© 1992by AcademicPress,Inc. All rightsof reproductionin any form reserved.
464
RECONSTITUTIONOF ION CHANNELS
[31 ]
to insert channels into membranes, 5 the emphasis of this chapter will be to transcribe some of the "oral lore and traditions" that accompany the formation of bilayers on the tip of patch-style pipettes and the study of channels in these bilayers. After a short overview, I describe the method for making these bilayers and outline the assorted techniques that have been developed to incorporate channels into the membrane. As appropriate, I shall include some of the pitfalls that need to be avoided. Overview Tip-dip membranes, double dip membranes, and patch membranes are bilayers whose name refers to the method of membrane formation. In other words, bilayers are made at the tip of patch-style pipettes by passing the tip of the pipette through a monolayer of lipid two times. Tip-dip bilayers represent the combination of two other techniques, patch damping and planar lipid bilayers. Using these techniques an artificial membrane is formed that allows for alterations in the lipid and salt composition, the ionic strength, and the presence of cofactors, agonists, and the like. In addition, the small size of the membrane means low current noise, which allows rapid changes in the voltage and good resolution of small currents, especially those from rapidly gating channels. Although tip-dip bilayers provided the best way to make small, high resolution bilayers for many years, more recently a technique that makes small, traditional, decane-containing bilayers has been described that allows high frequency resolution. 6 An early example of making small bilayers on the tip of glass pipettes used a glass pipette "patched" onto a standard black lipid bilayer. 7 The large membrane is constructed from lipid dissolved in n-decane. Then a fire-polished, silanized glass pipette is brought up to the membrane as if it were a cell, and, after waiting 0.5 to 5 min, a small membrane is formed on the tip and the large membrane seals around the pipette. With this configuration it is possible to measure both macroscopic and single-channel currents. Disadvantages of this method are that two bilayers must remain intact and that lipid is dissolved in n-decane. Using decane probably is not a serious disadvantage, but there are some reports of differences in channel kinetics when compared in painted bilayers and in solvent-free bilayers.8 Subsequently, methods to make solvent-free bilayers on the tip of glass 5C. Miller,ed., "Ion ChannelReconstitution."Plenum,New York, 1986. 6W. Wonderlin,A. Finkel,and R. French,Biophys. J. 58, 289 (1990). 7Andersonand Muller,J. Gen. Physiol. 80, 403 (1982). s W. Hanke, in "Ion Channel Reconstitution"(C. Miller, ed.), p. 141. Plenum,New York, 1986.
[31 ]
PLANAR LIPID BILAYERS ON PATCH PIPETTES
465
pipettes were developed. ~-4 It is this type of tip-dip bilayer that will be described in this chapter. Even though the basic concept for making bilayers at the tip of patch pipettes is simple, construction of these membranes has some requirements that differ from those used in standard bilayer formation. Fabrication of Tip-Dip Bilayers
Starting Materials The basic equipment needed for any bilayer setup includes an oscilloscope, a Faraday cage, a vibration-isolation table, a stimulator for pulsing the bilayer and for setting the holding potential, a signal generator for capacitance measurements if the stimulator cannot make a triangle wave, a stirrer, and chart and tape recorders.9 To make tip-dip bilayers a patch clamp amplifier, a pipette puller, and a micromanipulator are needed in addition to the basic electronic components used to make painted or solvent-free membranes. Optional equipment includes a computer with software and an analog-digital interface for taking, storing, and analyzing the data. High-quality lipids should be obtained from a supplier such as Avanti Polar Lipids (Birmingham, AL). I have had good success in storing the lipids in chloroform at - 70 °. When ready to use, the lipid is brought to room temperature, the desired volume is put in a clean glass tube, the chloroform is removed by a stream of nitrogen, and the lipid is resuspended in the desired solvent. In all cases where I suggest hexane as the solvent, pentane can be substituted. The final component needed is membrane vesicles or a protein to study. Numerous proteins can or have been studied in bilayers.
Fabrication of Pipettes The membrane is literally made at the end of a glass micropipette. The pipettes such as Kimax-51 capillary tubes (0.8- 1.1 × 100 ram) and microhematocrit capillary tubes (without heparin), are constructed as if they were to be used for patch clamping a cell. Standard methods for pulling the pipettes are employed, l° Briefly, the pipettes are pulled in two stages: the first stage thins the glass over about 10 m m and the second stage pulls the 90. Alvarez, in "Ion Channel Reconstitution" (C. Miller, ed.), p. 115. Plenum, New York, 1986. ~00. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, PfluegersArch. 391, 85 (1981).
466
RECONSTITUTIONOF ION CHANNELS
[31]
recentered capillary into two pipettes. It is not necessary to coat the shank of the pipette with Sylgard, nor to heat polish the tip of the pipette. It is useful, however, to determine the size of the pipette tip by attaching the pipette shank to a 10-cma syring with a Touhy-Borst adaptor with the syringe plunger set at 10 cm. a The tip of the pipette is then dipped into a scintillation vial filled with methanol. The syringe plunger is depressed until the first bubbles are formed, and the location of the plunger is noted. Pipettes that form bubbles between approximately 1.5 and 2 atm pressure (between 3.5 and 5 ml on the syringe) usually give good results. Many pipettes (5 to 20 acceptable ones) should be made and kept in a clean, dry location before the experiment (a small, square desiccator is a good holder). New pipettes should be made each day. Solution should be added to the pipette just before use, as is the custom in patch clamp experiments. The easiest way to make a device to fill the pipette is to pull a 1-cm3 syringe to a fine tube by gently heating while turning over a small flame. This device has several advantages: it fits into the shank of the pipette, there are no metal parts to leach divalent cations into the pipette solution (which will alter the pH), and it is easy to clean or replace.
Preparation of Bilayer Chamber A 96-well microtiter plate, preferably with flat bottoms, is used. The flat bottoms make it possible to stir the solution in each well with a 3 × 5 m m magnetic flea without disturbing the surface. To utilize the microtiter plate to the fullest extent, cover it with a piece of Parafilm to keep the unused wells clean. To begin an experiment four (or more) wells are uncovered; one well is for the ground wire, and the other wells are for experimental solutions. The wells are connected by agar bridges. Because it is important that the bridges do not move and do not interfere with the path of the patch pipette during the experiment, make the bridges from short lengths of glass tubing that have been bent in as LI shape to span the wells without projecting more than a few millimeters above the level of the microtiter plate. The tubes are filled with 2% agar dissolved in 0.5 M CsC1. With this type of setup each well can have a different solution. To change solutions during the experiment the pipette is moved to the appropriate well, rather than perfusing a new solution into the well.
Making Bilayers A buffered salt solution is added to each well (usually 0.3 ml) and a solution of lipid in hexane is layered on top of the solution. One to 2/tl of a lipid (10 mg/ml) is used even though this is much more than is needed to form a monolayer. After the hexane is evaporated, the pipette is passed
[31 ]
PLANAR LIPID BILAYERS ON PATCH PIPETTES
467
through the monolayer several times, starting with the pipette in the air, then into the solution, into the air, and back into the solution. It is best to have the tip just below the surface to minimize the capacitance due to the submerged glass. Alternatively, the glass could be coated with Sylgard, but it seemed easier to just keep the tip close to the surface. During the dipping procedure it is useful to pulse the membrane with a small voltage ( - 10 mV) to monitor the formation of the membrane. There are several common outcomes of the dipping procedure. (1) Formation of a membrane is not possible, that is, the tip resistance remains low. The only way to deal with this outcome is to discard the pipette and take another pipette. Often the tip has broken and is too large to hold a membrane. (2) The membrane resistance is extremely high (> 10 u f~), suggesting that there is a glob of lipid dogging the tip of the pipette. Again, discard the clogged pipette and take another pipette. (3) The membrane is formed, but the resistance is too low to be useful. Usually the membrane resistance can be increased by applying suction via a tube attached to the pipette holder as if one were patching onto a cell. Satisfactory results are usually obtained with membrane resistances of 2 - 10 × 10 ~° f~. Because there is no cytoskeleton, too much pressure will rupture the membrane and the procedure must be started again. It is possible to form a membrane on a pipette more than once if too much suction is applied. Once the bilayer is formed, the membrane is ready for the experiment (Fig. 1). To change solutions lift the pipette out of the solution and move the pipette to another well with a different solution composition. Sufficient solution adheres to the pipette to maintain the bilayer, but not so much solution that there is concern of contaminating the new solution. The use of a micromanipulator makes this procedure more convenient.
Advantages and Disadvantages The main advantage of tip-dip membranes is that they are very small (1- 5/zm) so the drawbacks associated with larger bilayers are avoided. Fast, small events can be detected with tip-dip membranes. The main reasons many investigators have avoided tip-dip membranes are that access to the solution inside the pipette is difficult and some channel types just will not insert into this type of membrane. It may be possible to perfuse the inside of the pipette as has been demonstrated in patchclamped cells, 1~(see also articles 10 and 48 this volume by Tang et al.), but we have not tried this. There are no obvious solutions to the lack of channel insertion. Another problem is that there can be cation-selective channellike events in the absence of added protein, possibly owing to an 1~ M. Soejima and A. Noma,
Pfluegers Arch. 400, 424
(1984).
468
RECONSTITUTIONOF ION CHANNELS
[31 ]
J
FIG. 1. Schematic representation of tip-dip bilayer formation. See text for details.
interaction between the lipid and the glass. Using high divalent cation concentrations (> 25 mM), especially in the pipette solution, and synthetic lipids (rather than extracted lipids) and taking extra care in cleaning the solutions and all associated paraphernalia often reduce the appearance of these events. As in making all types of bilayers, it is useful to be obssessive about cleanliness.
[31 ]
PLANAR LIPID BILAYERS ON PATCH PIPETTES
469
Incorporation of Channels Although the method to construct tip-dip bilayers is different from more traditional methods used for making lipid bilayers, channel incorporation occurs by the same techniques developed previously. Of the two main techniques, the first uses proteoliposomes as the source of lipid for the solvent-free or the tip-dip membranes. The protein of interest is reconstituted into vesicles (or native membrane vesicles are used), and these vesicles are layered on top of the saline solution. Vesicles at the air-water interface will lyse and form a monolayer with protein embedded at the density found in the vesicles. When the membrane is formed the protein is already in the monolayer and will, therefore, be in the bilayer. The membrane protein at the air- water interface is subjected to the same forces that induce the lysing of the vesicles and therefore, probably denature the protein. Nonetheless, some membrane proteins have been incorporated into bilayers with this method. It is conceivable that the proteins at the interface are denatured and that the proteins incorporated into the bilayer actually get there by fusion of the vesicles below and not at the interface. This is the first technique. In the majority of work that studies channels after they have been incorporated into bilayers, the vesicles have been fused to the bilayer. It is generally assumed that fusion occurs by a mechanism that imitates cellular exocytosis. Either native membrane vesicles or purified protein reconstituted into lipid vesicles can be used. Fusion requires addition of calcium to the vesicle-containing bath and an osmotic gradient across the bilayer, where the hyperosmotic solution is the vesicle-containing bath. Actually, if both the phospholipid vesicles and the bilayer contain no negatively charged lipids, calcium is not required. ~2 In practice, however, calcium is necessary because many applications use native membrane vesicles which contain negatively charged lipids, or negatively charged lipids are added to the phospholipid vesicles to allow the control of fusion by calcium. The calcium is needed to allow the vesicles to "adhere" to the bilayer. ~3 The osmotic gradient is needed to promote vesicle swelling and eventual lysis by stimulating diffusion of water across the membrane? 4 If the vesicle is very close to the bilayer ("adhered"), then swelling increases the area of contact between the vesicle and the bilayer. It is theorized that vesicles swollen to the maximum volume will interact with the bilayer, and this interaction initiates fusion between the two bilayers. Some degree of control can be obtained by varying the magnitude of ~2F. S. Cohen, M. H. Akabas, J. Zimmerberg, and A. Finkelstein, J. Cell Biol. 98, 1054 (1984). 13 M. H. Akabas, F. S. Cohen, and A. Finkelstein, J. Cell Biol. 98, 1063 (1984). ,4 F. S. Cohen, M. H. Akabas, and A. Finkelstein, Science 217, 458 (1982).
470
RECONSTITUTIONOF ION CHANNELS
[31 ]
the osmotic gradient and the number of vesicles. For example, to terminate fusion, the osmotic gradient can be neutralized and the vesicles removed from the bath by perfusion. Nonetheless, additional vesicles often will fuse in the absence of an osmotic gradient. This phenomenon is probably due to the observation that vesicles completely coat the bilayer and pcrfusion will not remove all the "adhered" vesicles. The fact that the vesicles coat the bilayer also explains why adding more vesicles often will not increase the number of fusion events or decrease the lag between vesicle addition and the first fusion event. Conclusion It is important to remember that although the methods outlined here and in other reviews appear to be straightforward, there are numerous steps that cannot be explained theoretically at the present time. A better understanding of the physical processes that govern protein-lipid and lipidlipid interactions will assist in future efforts in bilayer formation and channel reconstitution. A final word of caution was given by Finkelstein 15 in a chapter on planar lipid bilayer formation in an earlier volume of this series. This piece of advice can be applied to tip-dip bilayers. He noted, " . . . that some manipulation of variables is required before everything is working properly. Then, one can make stable membranes quickly and reliably week after week until, as happens to everyone I know who works with this system, one day a stable membrane cannot be formed. After a few agonizing days of changing and permuting the lipid, the septa, the brush, the distilled water source, and your socks, everything works properly again. Most likely, the conditions are the same as before." I have been asked many times if it is truly necessary to change your socks. The answer is, yes.
Acknowledgments I thank Dr. llyaBczprozvanny for comments on the manuscript. This work was supported by National Institutesof Health Grants HL-33026 and GM-39029. B.E.E. isa Pew Scholar in the Biomedical Sciences.
15 A. Finkelstein, this series, VoL 32, p. 489.
PLANAR LIPID BILAYERS ON PATCH PIPETTES
463
culties should lead the experimenter to avoid the use o f such lipids and to improve techniques in handling planar bilayers. O t h e r M e t h o d s to I n s e r t I o n C h a n n e l s into P l a n a r L i p i d B i l a y e r s Fusion o f lipid vesicles to "solvent-free" planar bilayers made of 1stearoyl-3-myristoyl/glycero-2-phosphocholine, a mixed-chain lecithin, at temperatures below the lipid phase transition has been reported by Boheim and collaborators (see e.g., Ref. 3). This m e t h o d is not widely used since it requires the use of a particular lipid at restricted temperatures. The m e t h o d of Schindler 42 to transfer proteins from lipid vesicles to a planar bilayer has been successfully used to insert channels into planar bilayers 27,2a,43 and to form bilayers from monolayers in the tip o f a patch pipette. 44-*sAcknowledgments This work was supported by grants from the Fondo Nacional de lnvestigacion (11671988, 451-1988), the National Institutes of Health (GM-35981),and the Tinker Foundation. R.L. is a recipient of a John S. GuggenheimFellowship. He also wishes to thank the Dreyfus Bank (Switzerland)for generous support from a private foundation that they made available. 42H. Schindler, FEBSLett. 122, 77 (1980). 43H. Schindler and U. Quast, Proc. Natl. Acad. Sci. U.S.A. 77, 3052 (1980). 44W. Hanke, C. Methfessel, H. U. Wilsem, and G. Boheim, Biochem. Bioeng. J. 12, 329 (1984). 45R. Coronado and R. Latorre, Biophys. J. 43, 231 (1983). 46B. Suarez-Isla,K. Wan, J. Lindstrom, and M. Montal, Biochemistry 22, 2319 (1983).
[31] Planar Lipid Bilayers on Patch Pipettes: Bilayer Formation and Ion Channel Incorporation B y B A R B A R A E. E H R L I C H
Introduction This chapter describes how to make bilayers on the tip o f patch-style pipettes and then incorporate channels into these bilayers. As there have been several reports describing the specific techniques needed for the formation o f this type of bilayer, 1-4 and even more reports describing how t R. Coronado and R. Latorre, Biophys J. 43, 231 (1983). 2 T. Schuerholzand H. Schindler,FEBS Lett. 152, 187 (1983). 3 B. A. Suarez-Isla, K. Wan, J. Lindstrom, and M. Montal, Biochemistry 22, 2319 (1983). 4 W. Hanke, C. Methfessel, H. V. Wilmsen, and G. Boheim, Biochem. Bioeng. J. 12, 329 (1984).
METHODS IN ENZYMOLOGY, VOL 207
Copyright© 1992by AcademicPress,Inc. All rightsof reproductionin any form reserved.
464
RECONSTITUTIONOF ION CHANNELS
[31 ]
to insert channels into membranes, 5 the emphasis of this chapter will be to transcribe some of the "oral lore and traditions" that accompany the formation of bilayers on the tip of patch-style pipettes and the study of channels in these bilayers. After a short overview, I describe the method for making these bilayers and outline the assorted techniques that have been developed to incorporate channels into the membrane. As appropriate, I shall include some of the pitfalls that need to be avoided. Overview Tip-dip membranes, double dip membranes, and patch membranes are bilayers whose name refers to the method of membrane formation. In other words, bilayers are made at the tip of patch-style pipettes by passing the tip of the pipette through a monolayer of lipid two times. Tip-dip bilayers represent the combination of two other techniques, patch damping and planar lipid bilayers. Using these techniques an artificial membrane is formed that allows for alterations in the lipid and salt composition, the ionic strength, and the presence of cofactors, agonists, and the like. In addition, the small size of the membrane means low current noise, which allows rapid changes in the voltage and good resolution of small currents, especially those from rapidly gating channels. Although tip-dip bilayers provided the best way to make small, high resolution bilayers for many years, more recently a technique that makes small, traditional, decane-containing bilayers has been described that allows high frequency resolution. 6 An early example of making small bilayers on the tip of glass pipettes used a glass pipette "patched" onto a standard black lipid bilayer. 7 The large membrane is constructed from lipid dissolved in n-decane. Then a fire-polished, silanized glass pipette is brought up to the membrane as if it were a cell, and, after waiting 0.5 to 5 min, a small membrane is formed on the tip and the large membrane seals around the pipette. With this configuration it is possible to measure both macroscopic and single-channel currents. Disadvantages of this method are that two bilayers must remain intact and that lipid is dissolved in n-decane. Using decane probably is not a serious disadvantage, but there are some reports of differences in channel kinetics when compared in painted bilayers and in solvent-free bilayers.8 Subsequently, methods to make solvent-free bilayers on the tip of glass 5C. Miller,ed., "Ion ChannelReconstitution."Plenum,New York, 1986. 6W. Wonderlin,A. Finkel,and R. French,Biophys. J. 58, 289 (1990). 7Andersonand Muller,J. Gen. Physiol. 80, 403 (1982). s W. Hanke, in "Ion Channel Reconstitution"(C. Miller, ed.), p. 141. Plenum,New York, 1986.
[31 ]
PLANAR LIPID BILAYERS ON PATCH PIPETTES
465
pipettes were developed. ~-4 It is this type of tip-dip bilayer that will be described in this chapter. Even though the basic concept for making bilayers at the tip of patch pipettes is simple, construction of these membranes has some requirements that differ from those used in standard bilayer formation. Fabrication of Tip-Dip Bilayers
Starting Materials The basic equipment needed for any bilayer setup includes an oscilloscope, a Faraday cage, a vibration-isolation table, a stimulator for pulsing the bilayer and for setting the holding potential, a signal generator for capacitance measurements if the stimulator cannot make a triangle wave, a stirrer, and chart and tape recorders.9 To make tip-dip bilayers a patch clamp amplifier, a pipette puller, and a micromanipulator are needed in addition to the basic electronic components used to make painted or solvent-free membranes. Optional equipment includes a computer with software and an analog-digital interface for taking, storing, and analyzing the data. High-quality lipids should be obtained from a supplier such as Avanti Polar Lipids (Birmingham, AL). I have had good success in storing the lipids in chloroform at - 70 °. When ready to use, the lipid is brought to room temperature, the desired volume is put in a clean glass tube, the chloroform is removed by a stream of nitrogen, and the lipid is resuspended in the desired solvent. In all cases where I suggest hexane as the solvent, pentane can be substituted. The final component needed is membrane vesicles or a protein to study. Numerous proteins can or have been studied in bilayers.
Fabrication of Pipettes The membrane is literally made at the end of a glass micropipette. The pipettes such as Kimax-51 capillary tubes (0.8- 1.1 × 100 ram) and microhematocrit capillary tubes (without heparin), are constructed as if they were to be used for patch clamping a cell. Standard methods for pulling the pipettes are employed, l° Briefly, the pipettes are pulled in two stages: the first stage thins the glass over about 10 m m and the second stage pulls the 90. Alvarez, in "Ion Channel Reconstitution" (C. Miller, ed.), p. 115. Plenum, New York, 1986. ~00. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, PfluegersArch. 391, 85 (1981).
466
RECONSTITUTIONOF ION CHANNELS
[31]
recentered capillary into two pipettes. It is not necessary to coat the shank of the pipette with Sylgard, nor to heat polish the tip of the pipette. It is useful, however, to determine the size of the pipette tip by attaching the pipette shank to a 10-cma syring with a Touhy-Borst adaptor with the syringe plunger set at 10 cm. a The tip of the pipette is then dipped into a scintillation vial filled with methanol. The syringe plunger is depressed until the first bubbles are formed, and the location of the plunger is noted. Pipettes that form bubbles between approximately 1.5 and 2 atm pressure (between 3.5 and 5 ml on the syringe) usually give good results. Many pipettes (5 to 20 acceptable ones) should be made and kept in a clean, dry location before the experiment (a small, square desiccator is a good holder). New pipettes should be made each day. Solution should be added to the pipette just before use, as is the custom in patch clamp experiments. The easiest way to make a device to fill the pipette is to pull a 1-cm3 syringe to a fine tube by gently heating while turning over a small flame. This device has several advantages: it fits into the shank of the pipette, there are no metal parts to leach divalent cations into the pipette solution (which will alter the pH), and it is easy to clean or replace.
Preparation of Bilayer Chamber A 96-well microtiter plate, preferably with flat bottoms, is used. The flat bottoms make it possible to stir the solution in each well with a 3 × 5 m m magnetic flea without disturbing the surface. To utilize the microtiter plate to the fullest extent, cover it with a piece of Parafilm to keep the unused wells clean. To begin an experiment four (or more) wells are uncovered; one well is for the ground wire, and the other wells are for experimental solutions. The wells are connected by agar bridges. Because it is important that the bridges do not move and do not interfere with the path of the patch pipette during the experiment, make the bridges from short lengths of glass tubing that have been bent in as LI shape to span the wells without projecting more than a few millimeters above the level of the microtiter plate. The tubes are filled with 2% agar dissolved in 0.5 M CsC1. With this type of setup each well can have a different solution. To change solutions during the experiment the pipette is moved to the appropriate well, rather than perfusing a new solution into the well.
Making Bilayers A buffered salt solution is added to each well (usually 0.3 ml) and a solution of lipid in hexane is layered on top of the solution. One to 2/tl of a lipid (10 mg/ml) is used even though this is much more than is needed to form a monolayer. After the hexane is evaporated, the pipette is passed
[31 ]
PLANAR LIPID BILAYERS ON PATCH PIPETTES
467
through the monolayer several times, starting with the pipette in the air, then into the solution, into the air, and back into the solution. It is best to have the tip just below the surface to minimize the capacitance due to the submerged glass. Alternatively, the glass could be coated with Sylgard, but it seemed easier to just keep the tip close to the surface. During the dipping procedure it is useful to pulse the membrane with a small voltage ( - 10 mV) to monitor the formation of the membrane. There are several common outcomes of the dipping procedure. (1) Formation of a membrane is not possible, that is, the tip resistance remains low. The only way to deal with this outcome is to discard the pipette and take another pipette. Often the tip has broken and is too large to hold a membrane. (2) The membrane resistance is extremely high (> 10 u f~), suggesting that there is a glob of lipid dogging the tip of the pipette. Again, discard the clogged pipette and take another pipette. (3) The membrane is formed, but the resistance is too low to be useful. Usually the membrane resistance can be increased by applying suction via a tube attached to the pipette holder as if one were patching onto a cell. Satisfactory results are usually obtained with membrane resistances of 2 - 10 × 10 ~° f~. Because there is no cytoskeleton, too much pressure will rupture the membrane and the procedure must be started again. It is possible to form a membrane on a pipette more than once if too much suction is applied. Once the bilayer is formed, the membrane is ready for the experiment (Fig. 1). To change solutions lift the pipette out of the solution and move the pipette to another well with a different solution composition. Sufficient solution adheres to the pipette to maintain the bilayer, but not so much solution that there is concern of contaminating the new solution. The use of a micromanipulator makes this procedure more convenient.
Advantages and Disadvantages The main advantage of tip-dip membranes is that they are very small (1- 5/zm) so the drawbacks associated with larger bilayers are avoided. Fast, small events can be detected with tip-dip membranes. The main reasons many investigators have avoided tip-dip membranes are that access to the solution inside the pipette is difficult and some channel types just will not insert into this type of membrane. It may be possible to perfuse the inside of the pipette as has been demonstrated in patchclamped cells, 1~(see also articles 10 and 48 this volume by Tang et al.), but we have not tried this. There are no obvious solutions to the lack of channel insertion. Another problem is that there can be cation-selective channellike events in the absence of added protein, possibly owing to an 1~ M. Soejima and A. Noma,
Pfluegers Arch. 400, 424
(1984).
468
RECONSTITUTIONOF ION CHANNELS
[31 ]
J
FIG. 1. Schematic representation of tip-dip bilayer formation. See text for details.
interaction between the lipid and the glass. Using high divalent cation concentrations (> 25 mM), especially in the pipette solution, and synthetic lipids (rather than extracted lipids) and taking extra care in cleaning the solutions and all associated paraphernalia often reduce the appearance of these events. As in making all types of bilayers, it is useful to be obssessive about cleanliness.
[31 ]
PLANAR LIPID BILAYERS ON PATCH PIPETTES
469
Incorporation of Channels Although the method to construct tip-dip bilayers is different from more traditional methods used for making lipid bilayers, channel incorporation occurs by the same techniques developed previously. Of the two main techniques, the first uses proteoliposomes as the source of lipid for the solvent-free or the tip-dip membranes. The protein of interest is reconstituted into vesicles (or native membrane vesicles are used), and these vesicles are layered on top of the saline solution. Vesicles at the air-water interface will lyse and form a monolayer with protein embedded at the density found in the vesicles. When the membrane is formed the protein is already in the monolayer and will, therefore, be in the bilayer. The membrane protein at the air- water interface is subjected to the same forces that induce the lysing of the vesicles and therefore, probably denature the protein. Nonetheless, some membrane proteins have been incorporated into bilayers with this method. It is conceivable that the proteins at the interface are denatured and that the proteins incorporated into the bilayer actually get there by fusion of the vesicles below and not at the interface. This is the first technique. In the majority of work that studies channels after they have been incorporated into bilayers, the vesicles have been fused to the bilayer. It is generally assumed that fusion occurs by a mechanism that imitates cellular exocytosis. Either native membrane vesicles or purified protein reconstituted into lipid vesicles can be used. Fusion requires addition of calcium to the vesicle-containing bath and an osmotic gradient across the bilayer, where the hyperosmotic solution is the vesicle-containing bath. Actually, if both the phospholipid vesicles and the bilayer contain no negatively charged lipids, calcium is not required. ~2 In practice, however, calcium is necessary because many applications use native membrane vesicles which contain negatively charged lipids, or negatively charged lipids are added to the phospholipid vesicles to allow the control of fusion by calcium. The calcium is needed to allow the vesicles to "adhere" to the bilayer. ~3 The osmotic gradient is needed to promote vesicle swelling and eventual lysis by stimulating diffusion of water across the membrane? 4 If the vesicle is very close to the bilayer ("adhered"), then swelling increases the area of contact between the vesicle and the bilayer. It is theorized that vesicles swollen to the maximum volume will interact with the bilayer, and this interaction initiates fusion between the two bilayers. Some degree of control can be obtained by varying the magnitude of ~2F. S. Cohen, M. H. Akabas, J. Zimmerberg, and A. Finkelstein, J. Cell Biol. 98, 1054 (1984). 13 M. H. Akabas, F. S. Cohen, and A. Finkelstein, J. Cell Biol. 98, 1063 (1984). ,4 F. S. Cohen, M. H. Akabas, and A. Finkelstein, Science 217, 458 (1982).
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the osmotic gradient and the number of vesicles. For example, to terminate fusion, the osmotic gradient can be neutralized and the vesicles removed from the bath by perfusion. Nonetheless, additional vesicles often will fuse in the absence of an osmotic gradient. This phenomenon is probably due to the observation that vesicles completely coat the bilayer and pcrfusion will not remove all the "adhered" vesicles. The fact that the vesicles coat the bilayer also explains why adding more vesicles often will not increase the number of fusion events or decrease the lag between vesicle addition and the first fusion event. Conclusion It is important to remember that although the methods outlined here and in other reviews appear to be straightforward, there are numerous steps that cannot be explained theoretically at the present time. A better understanding of the physical processes that govern protein-lipid and lipidlipid interactions will assist in future efforts in bilayer formation and channel reconstitution. A final word of caution was given by Finkelstein 15 in a chapter on planar lipid bilayer formation in an earlier volume of this series. This piece of advice can be applied to tip-dip bilayers. He noted, " . . . that some manipulation of variables is required before everything is working properly. Then, one can make stable membranes quickly and reliably week after week until, as happens to everyone I know who works with this system, one day a stable membrane cannot be formed. After a few agonizing days of changing and permuting the lipid, the septa, the brush, the distilled water source, and your socks, everything works properly again. Most likely, the conditions are the same as before." I have been asked many times if it is truly necessary to change your socks. The answer is, yes.
Acknowledgments I thank Dr. llyaBczprozvanny for comments on the manuscript. This work was supported by National Institutesof Health Grants HL-33026 and GM-39029. B.E.E. isa Pew Scholar in the Biomedical Sciences.
15 A. Finkelstein, this series, VoL 32, p. 489.
