[49]
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designed to mimic intracellular conditions and have composition and pH close to lobster musclefl6,37 The relaxing solution is designed to mimic resting intracellular conditions. The loading solution is designed to facilitate accumulation of calcium in the SR. The release solution is designed to induce calcium release from the SR and is made by adding caffeine as the dry powder to a volume of basic solution. The actual salts and their concentrations are shown in Table II. Conclusion The utility of this preparation and these procedures is documented by the results obtained.2°-23,3s The skinned lobster remotor muscle seems as viable as most skinned preparations as judged by usual criteria, namely, the tension generated by caffeine: responses are vigorous, and the SR can be reloaded many times. In addition, single K+ and Ca2+ channels can be studied in their native membrane by the patch clamp technique. The skinned lobster remotor preparation can be studied with an unusually powerful combination of techniques and so perhaps can yield some unusual information. a6 p. B. Dunham and H. Gainer, Biochim. Biophys. Acta 150, 488 (1968). 37 j. D. Robertson, J. Exp. Biol. 38, 707 (1961). as j. M. Tang, Ph.D thesis (1991).
[49] P l a n a r B i l a y e r R e c o r d i n g o f R y a n o d i n e R e c e p t o r s of Sarcoplasmic Reticulum B y ROBERTO CORONADO, SEIKO K A W A N O , CHEOL J. LEE, CARMEN VALDIVIA,
and H E C T O R H . VALDIVIA
Introduction Three situations in ion channel analysis require a ceU-free recording or planar bilayer technique: (1) in the case of channels confined to regions of the cell which are largely inaccessible to patch electrodes such as narrow tubules and intracellular organelles~; (2) to control solutions on both faces of a channel with analytical precision3; and (3) to test the ionophoric 1 H. Valdivia and R. Coronado, J. Gen. Physiol. 95, 1 (1990). 2 C. Valdivia, H. Valdivia, B. V. L. Potter, and R. Coronado, Biophys. J. 57, 1233 (1990). J. S. Smith, T. Imagawa, J. Ma, M. Fill, K. P. Campbell, and R. Coronado, J. Gen. Physiol. 92, 1 (1988). METHODS IN ENZYMOLOGY,VOL. 207
Copyright© 1992by AcademicPress,Inc. All fightsof reproductionin any formreserved.
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[49]
properties of purified proteins. 4 In this chapter we describe methods to incorporate sarcoplasmic reticulum (SR) Ca 2+ channels, also called ryanodine receptors, into planar bilayers. Decane-containing bilayers are described exclusively since, in our experience, they are the most reproducible for this application. The technique is illustrated with recordings of ryanodine receptors from rabbit skeletal muscle and bovine heart. 5 Bilayer Hardware Planar bilayers useful for recording SR channels6 are formed spontaneously after depositing a small volume of phospholipids dissolved in decane in a pinhole or aperture under water. The thermodynamic forces that govern thinning of the originally thick film into a bimoleeular film have been described in detail by White. 7 Lipid films formed in a mechanical support require a transition zone or annulus between the macroscopicsized support and the bilayer film. Critical to the stability of planar bilayers is the thickness and composition of the aperture in contact with the annulus. A lipid-wetting surface such as that of TPFE fluorocarbon (Teflon) is not adequate since this material is too soft for drilling clean holes. A hardened version of PTFE called Delrin AF blend (VT Central Plastics, Inc., Chicago, IL) has excellent machinability and provides an acceptable surface for bilayer assembly. Compared side by side, bilayers formed in Delrin holes last 2 - 5 times longer than those assembled in Teflon holes. A cup serving as inner chamber s is machined from a i inch diameter Delrin rod into a cylinder with approximate dimensions 2.2 cm outer diameter, 1.9 cm inner diameter, and 1.9 cm height. One side of the cup wall is shaved down to about 25 g m in its thinnest section. A 0.0135 inch diameter driU is used to perforate a hole at the center of the thinnest section. The hole should be free from plastic burrs or residue on inspection under low power magnification and should have a diameter no less than 300 a m and no more than 400/zm. For convenience, the hole should lie approximately 0.7 cm from the bottom of the cup. A block machined from PVC or from any other dark PV-type material such as Acetron Acetal (VT Central 4 T. Imagawa, J. S. Smith, R. Coronado, and K. P. Campbell, J. Biol. Chem. 262, 16636 (1987). s M. Fill and R. Coronado, Trends Neurosci. 11, 453 (1988). 6 C. Miller, J. Membr. Biol. 40, 1 (1978). S. H. White, in "Ion Channel Reconstitution" (C. Miller, ed.), p. 3. Plenum, New York, 1986. 8 j. S. Smith, R. Coronado, and G. Meissner, this series, "Col. 157, p. 480.
[49]
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Plastics, Inc.) serves as outer chamber and should provide a snug fit for the Delrin cup. The PVC block fits on a brass block close to the electrode inputs of a head stage amplifier. A metal box with a lid serves as grounding shield for the head stage amplifier, electrodes, and solutions. To avoid condensation of water in the vicinity of the head-stage input that causes drift, this box should be no smaller than 30 cm wide by 30 cm deep by 15 cm high. The box is machined from 1.2 m m thick stainless steel and is bolted to a 2 cm thick lead base that provides mechanical stability. A 12 V dc, 125 rpm motor under the box moves a magnet, in turn used to spin a small magnetic stir bar (3 m m diameter, 12.7 m m length) placed inside the bilayer cup. Magnetic bars can be purchased from Fisher Scientific (Pittsburgh, PA). The dc motor can be purchased from Edmund Scientific (Barrington, NJ). The box rests in an air-lifted table to insulate it from mechanical vibration. Homemade circuits for monitoring planar bilayer currents at high gain, built from standard electronic components, have been described in detail elsewhere,s However, a regular patch clamp amplifier is also adequate. Software packages for single-channel analysis such as PClamp 5.5 distributed by Axon Instruments (Fullerton, CA) is highly desirable since it serves for data acquisition and for delivering voltage pulses. Electrodes and Noise Macro Ag/AgC1 electrodes are encased in agar-fiUed polyethylene tubing to minimize liquid junction potentials and to avoid contamination of solutions with Ag+ ions. Electrodes are made from 0.25 m m diameter silver wire ( - 5 cm) soldered to a miniature gold-plated pin for hookup to amplifier (a diagram appears in Ref. 8). The silver metal is coated with silver chloride in an electrolytic cell filled with 0.1 N HCI. A coated electrode is inserted into 1 m m diameter polyethylene tubing which has been filled with 0.2 M KC1 in 2% agar. Carboxylate cement is used to glue the tubing to the pin connector. This assembly is durable and may last for several weeks if the electrode tip is always kept in a 0.2 M KC1 solution. Peak-to-peak noise of an assembled bilayer should be no more than 10 pA at 2 kHz or 0.2 pA at 400 Hz low-pass filtering for the recommended aperture size. Appropriate grounding of the bilayer box is sufficient to eliminate the 60 cycles noise common to most electrophysiological setups. Other sources typical of planar bilayers are mechanical and microphonic noise. Both are the result of small changes in area (capacitance) produced when the solutions vibrate and the bilayer film wobbles. Mechanical noise is usually of low frequency and many times erratic. The
702
CHANNEL RECORDING IN ORGANELLES AND MICROBES
[49]
bilayer setup should be isolated from other fixtures in the room. Ideally, the setup should rest on an air-lifted table (see list of equipment). Electrodes should be fitted tightly to the head stage. The head stage should be glued or screwed to the bilayer box. Microphonic noise increases as the bilayer capacitance increases. Avoid placing a setup next to an elevator shaft, equipment room, or cold room. Bilayer Stability The quality of the pinhole is the number one source of day-to-day technical difficulty in assembling long-lasting stable bflayers. In an acceptable cup, a thin bilayer should last no less than 30 rain at 0 mV applied potential in the absence of added protein. Cups should be inspected regularly to ensure that the hole is free from dirt and that cracks have not formed along the edges of the hole. Cups are rinsed only with high quality methanol. Avoid the use of detergents or chloroform. Users should have a set of no less than 20 machined cups at their disposal at any given time. Cups showing signs of wear should be discarded. A good commercial source of phospholipids is Avanti Polar Lipids (Birmingham, AL). Lipids should be purchased dissolved in chloroform and shipped in N2-sealed vials in aliquots of I ml of 5 mg/ml phospholipid. Phosphatidylethanolamine (PE) and phosphatidylserine (PS) purified from brain are ideal for bilayer stability and single-channel recording. Lipids are mixed at a 1 : 1 weight ratio, dried under a stream of N2, and resuspended in ultrapure n-decane (Aldrich Chemical Co., Milwaukee, WI) at a concentration of 20 mg/ml phospholipid. A 100 gl lipid solution in decane should be prepared daily. Fresh vials of PE and PS should be opened every 2 weeks and old lipids discarded. Lipid solution is applied to the aperture with a 0.2 cm diameter Delrin stick cut diagonally at the tip. Bilayer thinning, from a thick to a thin film, is monitored by capacitance measurement. Capacitance rises from approximately 100 to 500 pF in the apertures of the size recommended. Ultrapure grade chloride salts of monovalents or divalents are recommended for planar bilayer recording. A good source is Johnson Matthey Chemicals Ltd. (Hereforshire, England) distributed in the United States by Alpha Products (Morthon Thiokol, Inc., Danvers, MA). The recommended concentration of EDTA (ethyleneAiaminetetraacetic acid) for Ca 2+ buffering is 1 mM. pH buffers such as HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), MES (2-N-morpholinoethanesulfonic acid), or Tris [Tris(hydroxymethyl)aminomethane] should not be used in excess of l0 mM. Water should be distilled in the laboratory in an all-glass still.
[49]
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Fractionation of Sarcoplasmic Reticulum Light and heavy SR contains K + and Cl- channels,5 but ryanodinc receptors arc only found in heavy SR. 9 Procedures that use Ca2+-precipitaring agents such as oxalatc or phosphate for loading Ca 2+ into the SR (transport-specific fractionation) should bc avoided since intravcsicular Ca 2+ impairs SR incorporation into bilayers. A procedure to fractionatc light and heavy SR of skclctal or cardiac muscle, which yiclds active ryanodinc receptor channels in planar bilaycrs,is as follows. Tissue taken from the animal is immersed in ice-cooled saline and taken to a cold room where the homogenization step is carried out. White muscle from the back and hind legs of one adult N e w Zealand rabbit or one fresh bovine heart collected no more than 20 rain after sacrificeis minced and ground in a food processor. Each of 4-5 portions of 40 g arc homogenized for 60 scc in 300 ml of 0.3 M sucrose, 0.5 m M EGTA, 20 m M Na4P2OT, 20 r n M NaH2PO4, 1.0 m M MgCI2, p H 7.1, using a Waxing blender at m a x i m u m speed. The following protcasc inhibitorsarc added during homogenization: pcpstatin A (I ~tM), iodoacctamidc (I raM), phenylmethylsulfonyl fluoride (PMSF) (0.I raM), Icupcptin (I/~M), and bcnzamidinc (I raM). Thc homogcnatc is spun for 15 rain at 9000 rpm in a Sorvall G S A rotor. The supernatant is liltcredthrough four layers of gauze and centrifuged at 14,000 rpm for 30 rain in the same rotor. Pelletsarc resuspended to a finalvolume of 60 ml in the same medium used for homogenization and layered on top of a stcp sucrose gradient composed of 7 ml of 27% (w/v) sucrose, 7 ml of 32% sucrose, 14 ml of 38% sucrose, in 20 r n M Na4P2OT, 20 m M NaH2PO4, 1 m/14MgCI2, pH 7.1. Gradients are centrifuged for 16 hr at 20,000 rpm in a Beckman SW 28 rotor at 4*. The heavy microsome fraction is collected from the 32-38% sucrose interface. After gentle dilution with 3 volumes of homogenization medium without sucrose, a pellet is obtained by centrifugation at 30,000 rpm for 40 rain using a Beckman 35 rotor. Microsomes are resuspended in 0.3 M sucrose, 5 m M HEPES-KOH, pH 7.0, aliquoted, frozen in liquid nitrogen, and stored at - 8 0 * for up to 3 months. When prepared from rabbit skeletal muscle,2'1° this preparation has a [3H]PN200-1 l0 binding capacity of 12 pmol/mg protein, a [3H]ryanodine binding capacity of 9 pmol/mg protein, and an ATP-dependent Ca 2+ uptake capacity of 15 nmol/mg protein.
9G. Meissner,J. Biol. Chem. 259, 2365 (1984). ~oH. Valdivia,C. Valdivia,J. Ma, and R. Coronado,Biophys. J. in press(1990).
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Solutions and Incorporation of Channels Recordings of ryanodine receptors are best made using CsCI as current carrier ~-3,~0,~ instead of the Ca-HEPES and Tris-HEPES solutions suggested earlier. 8,n Insights into the mechanism of SR vesicle incorporation into bilayers may be found in Ref. 13. The previous recording technique assumed that only Ca2+ was permeable through ryanodine receptors, and therefore large concentrations of divalents (Ca 2+ or Ba2+) were used to increase current amplitude. This assumption turned out to be incorrect: we now know that the channel is highly nonselective.3 Divalent cations can be thus kept at physiological levels without loss of conductance if a monovalent cation is present in both chambers (Fig. 114). Other complications in the previous technique arose from the use of large cations and anions like Tris+ and HEPES- to block SR K + and CIchannels. Because SR incorporation into bilayers is rare in the absence of CI-, bilayer chambers had to be perfused with C1--free solutions after SR incorporation in CI- media. The perfusion maneuver tended to break bilayers even when a circuit was developed to bypass the electronics,s This made the procedure tedious, and the number of successful recordings was limited. The use of CsCI instead of Ca-HEPES and Tris-HEPES eliminated the need for perfusion and eliminated the need for large and unphysiological gradients of Ca 2÷, which severely inactivate the channel (in high trans Ca 2+, the open probability is around 10-fold lower than when the trans free Ca 2+ is kept at 10/tM). Finally, Cs+ blocks SR K + channels, and because it has a higher conductance than Ca 2+ or Na t through ryanodine receptors (gc,/gc~ = 2), 3 it improved the signal-to-noise ratio. Sarcoplasmic reticulum C1- channels can be separated from ryanodine receptors on the basis of reversal potential. In the solutions recommended, E o is + 59 mV and Ec, is - 5 9 mV. The reversal potential for SR CI- channels is approximately + 3 0 inV. Recordings in the presence of CI- channels can thus be made by holding the membrane potential close to the CI- equilibrium potential (more positive than + 20 mV). Sarcoplasmic reticulum preparations are thawed by hand and kept on ice. Approximately 100 pg of skeletal SR or 500/zg of cardiac SR protein are added to the cup-side solution, designated the cis side. Cis solution is n M. Fill, R. Coronado, J. R. Mickelson, J. Vilven, J. Ma, B. A. Jacobson, and C. F. Louis, Biophys. J. 50, 471 (1987). 12j. S. Smith, R. Coronado, and G. Meissner, Nature (London) 316, 446 (1985). ~3F. S. Cohen, M. H. Akabcs, J. Zimmerberg, and A. Filkenstein, J. Cell Biol. 98, 1054 (1984). ~4S. Kawano and R. Coronado, J. Physiol. (1992) In press.
[49]
A
RYANODINE RECEPTORS OF SARCOPLASMICRETICULUM
705
E3
400
ms
1 O0 m s
25
ms
|
I 40 pA FIG. 1. Planar bilayer recording of cardiac and skeletal ryanodine receptor. (A) Cardiac ryanodine receptor from bovine heart at + 20 mV holding potential in cis 0.5 M CsO, trans 50 mM CsCI. Cis and trans Ca2+ was 3.5/zM. (B) Skeletal ryanodine receptor from rabbit skeletal muscle under same conditions. (From Ref. 14 with permission.)
typically composed of 3 ml of 0.5 M CsC1, 10 to 100#M CaC12, and 10 mM HEPES-Tris, pH 7.5. The bath-side solution, designated trans side, is the same except the CsCl is 50 raM. In the recordings of Fig. l, a List L/M EPC 7 amplifier (List Electronics, Eberstadt, Germany) was connected to the interior of the cup; the bath side was held at ground. Recordings were filtered through a low-pass Bessel filter (Frequency Devices, Haverhill, MA) at a front panel setting of 1 kHz and digitized at 100 to 250/~sec per point. The protocol to incorporate SR channels is according to points (1) through (4) as follows. (1) Bilayers are first formed in symmetrical 50 mM CsCl buffer (the trans solution). Wetting the dry hole with a drop of lipid, before pouring a solution into the cup, considerably helps the formation of the first bilayer. Bilayer capacitance is continuously monitored to ensure an appropriate degree of thinning. The increase in bilayer capacitance or "thinning of the bilayer" is favored by large positive or negative potentials (--4-_100 mV). After thinning is completed, the CsC1 in the cis solution is increased to the final value. (2) Sarcoplasmic reticulum protein is the last component added after adjustment of free Ca 2+ with CaEGTA buffers. The cis chamber is stirred for 30 to 60 see. Membrane potential is kept
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constant at 0 mV for cardiac SR or between 0 mV and + 2 0 mV for skeletal SR. Cardiac SR makes bilayers unstable more often than skeletal SR does. Less applied voltage is recommended when using cardiac SR protein. When a bilayer breaks, or there is a shift in baseline indicative of a leak, or when the capacitance decreases spontaneously, a new membrane should be made in the same solutions. If this maneuver is repeated too many times, however, there will be a substantial mixing of the cis and trans solutions. (3) In an typical case, C1- channels incorporate within the first 10 min and ryanodine receptors incorporate afterward within 30 min. Recordings of C1- channels should be continued until ryanodine receptors appear. The frequency of presentation of ryanodine receptors without C1channels is 1 out of 5 recordings. (4) When channels do not incorporate, the bilayer is broken and a new one is reformed in the same solutions. Breakage and reformation is repeated up to four times. Afterward, the cup is removed and rinsed with methanol before repeating the protocol. The polarity of channels incorporated into the bilayer is constant, in the majority of cases. The myoplasmic end of the receptor faces into the cis solution, and the intravesicular end faces into the trans solution. Polarity can be easily confirmed by the cis-activation of channels by ATP and micromolar Ca 2+, which are myoplasmic activators of ryanodine receptors. 9 Four useful characteristics that serve to identify ryanodine receptors are the following: (1) a linear current-voltage relationship with a slope conductance of 600 pS for cardiac SR and 700 pS for skeletal SR and reversal at potentials more negative than - 5 0 mV in the solutions recommended~4; (2) an activatory effect of adenine nucleotides and Ca 2+ and the inhibition by Mg2+. n,t4 Cis 5 m M ATP should increase the open probability about 2- to 5-fold; cis Ca 2+ should increase open probability from virtually null at pCa 9 to typical values of 0.03 for skeletal and 0.2 for cardiac receptors at pCa 6; cis 1 m M free Mg 2+ should decreases activity around 10-fold; (3) an inhibition by cis I g M ruthenium red which decreases activity 100-foldH,t2; (4) an activation by ryanodine, which at a concentration of 100 n M should produce an irreversible decrease in conductance and an increase in mean open time. 3
List of E q u i p m e n t A minimal list of equipment for planar bilayer recording and suggested sources are given below. Preparative biochemical instruments for microsome purification are not included. Bilayer cups, PVC block, brass block, and steel box with lead base, to be machined at a local shop
[49]
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Vibration-isolation table (Technical Manufacturing Corporation, Peabody, MA, Model Micro-G) Patch clamp amplifier (Axon Instruments, Burlingame, CA, Model Axopatch 1D with head stage for planar bilayers) VCR-based instrumentation recorder (Medical Systems Corporation, Greenvale, NY, Model PCM-2 Recorder adapter, VCR not included) 286-based PC with MS DOS, full memory, coprocessor, 40 Mbyte hard disk drive and EGA graphics (Standard Brand Products, Austin, TX, model Standard 286) Acquisition software (Axon Instruments, Model TL-125 A/D and D/A interface, PClamp software version 5.5) Laser printer (Hewlett Packard, Sunnyvale, CA, Model Laserjet series II)
RYANODINE RECEPTORS OF SARCOPLASMIC RETICULUM
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designed to mimic intracellular conditions and have composition and pH close to lobster musclefl6,37 The relaxing solution is designed to mimic resting intracellular conditions. The loading solution is designed to facilitate accumulation of calcium in the SR. The release solution is designed to induce calcium release from the SR and is made by adding caffeine as the dry powder to a volume of basic solution. The actual salts and their concentrations are shown in Table II. Conclusion The utility of this preparation and these procedures is documented by the results obtained.2°-23,3s The skinned lobster remotor muscle seems as viable as most skinned preparations as judged by usual criteria, namely, the tension generated by caffeine: responses are vigorous, and the SR can be reloaded many times. In addition, single K+ and Ca2+ channels can be studied in their native membrane by the patch clamp technique. The skinned lobster remotor preparation can be studied with an unusually powerful combination of techniques and so perhaps can yield some unusual information. a6 p. B. Dunham and H. Gainer, Biochim. Biophys. Acta 150, 488 (1968). 37 j. D. Robertson, J. Exp. Biol. 38, 707 (1961). as j. M. Tang, Ph.D thesis (1991).
[49] P l a n a r B i l a y e r R e c o r d i n g o f R y a n o d i n e R e c e p t o r s of Sarcoplasmic Reticulum B y ROBERTO CORONADO, SEIKO K A W A N O , CHEOL J. LEE, CARMEN VALDIVIA,
and H E C T O R H . VALDIVIA
Introduction Three situations in ion channel analysis require a ceU-free recording or planar bilayer technique: (1) in the case of channels confined to regions of the cell which are largely inaccessible to patch electrodes such as narrow tubules and intracellular organelles~; (2) to control solutions on both faces of a channel with analytical precision3; and (3) to test the ionophoric 1 H. Valdivia and R. Coronado, J. Gen. Physiol. 95, 1 (1990). 2 C. Valdivia, H. Valdivia, B. V. L. Potter, and R. Coronado, Biophys. J. 57, 1233 (1990). J. S. Smith, T. Imagawa, J. Ma, M. Fill, K. P. Campbell, and R. Coronado, J. Gen. Physiol. 92, 1 (1988). METHODS IN ENZYMOLOGY,VOL. 207
Copyright© 1992by AcademicPress,Inc. All fightsof reproductionin any formreserved.
700
CHANNEL RECORDING IN ORGANELLES AND MICROBES
[49]
properties of purified proteins. 4 In this chapter we describe methods to incorporate sarcoplasmic reticulum (SR) Ca 2+ channels, also called ryanodine receptors, into planar bilayers. Decane-containing bilayers are described exclusively since, in our experience, they are the most reproducible for this application. The technique is illustrated with recordings of ryanodine receptors from rabbit skeletal muscle and bovine heart. 5 Bilayer Hardware Planar bilayers useful for recording SR channels6 are formed spontaneously after depositing a small volume of phospholipids dissolved in decane in a pinhole or aperture under water. The thermodynamic forces that govern thinning of the originally thick film into a bimoleeular film have been described in detail by White. 7 Lipid films formed in a mechanical support require a transition zone or annulus between the macroscopicsized support and the bilayer film. Critical to the stability of planar bilayers is the thickness and composition of the aperture in contact with the annulus. A lipid-wetting surface such as that of TPFE fluorocarbon (Teflon) is not adequate since this material is too soft for drilling clean holes. A hardened version of PTFE called Delrin AF blend (VT Central Plastics, Inc., Chicago, IL) has excellent machinability and provides an acceptable surface for bilayer assembly. Compared side by side, bilayers formed in Delrin holes last 2 - 5 times longer than those assembled in Teflon holes. A cup serving as inner chamber s is machined from a i inch diameter Delrin rod into a cylinder with approximate dimensions 2.2 cm outer diameter, 1.9 cm inner diameter, and 1.9 cm height. One side of the cup wall is shaved down to about 25 g m in its thinnest section. A 0.0135 inch diameter driU is used to perforate a hole at the center of the thinnest section. The hole should be free from plastic burrs or residue on inspection under low power magnification and should have a diameter no less than 300 a m and no more than 400/zm. For convenience, the hole should lie approximately 0.7 cm from the bottom of the cup. A block machined from PVC or from any other dark PV-type material such as Acetron Acetal (VT Central 4 T. Imagawa, J. S. Smith, R. Coronado, and K. P. Campbell, J. Biol. Chem. 262, 16636 (1987). s M. Fill and R. Coronado, Trends Neurosci. 11, 453 (1988). 6 C. Miller, J. Membr. Biol. 40, 1 (1978). S. H. White, in "Ion Channel Reconstitution" (C. Miller, ed.), p. 3. Plenum, New York, 1986. 8 j. S. Smith, R. Coronado, and G. Meissner, this series, "Col. 157, p. 480.
[49]
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Plastics, Inc.) serves as outer chamber and should provide a snug fit for the Delrin cup. The PVC block fits on a brass block close to the electrode inputs of a head stage amplifier. A metal box with a lid serves as grounding shield for the head stage amplifier, electrodes, and solutions. To avoid condensation of water in the vicinity of the head-stage input that causes drift, this box should be no smaller than 30 cm wide by 30 cm deep by 15 cm high. The box is machined from 1.2 m m thick stainless steel and is bolted to a 2 cm thick lead base that provides mechanical stability. A 12 V dc, 125 rpm motor under the box moves a magnet, in turn used to spin a small magnetic stir bar (3 m m diameter, 12.7 m m length) placed inside the bilayer cup. Magnetic bars can be purchased from Fisher Scientific (Pittsburgh, PA). The dc motor can be purchased from Edmund Scientific (Barrington, NJ). The box rests in an air-lifted table to insulate it from mechanical vibration. Homemade circuits for monitoring planar bilayer currents at high gain, built from standard electronic components, have been described in detail elsewhere,s However, a regular patch clamp amplifier is also adequate. Software packages for single-channel analysis such as PClamp 5.5 distributed by Axon Instruments (Fullerton, CA) is highly desirable since it serves for data acquisition and for delivering voltage pulses. Electrodes and Noise Macro Ag/AgC1 electrodes are encased in agar-fiUed polyethylene tubing to minimize liquid junction potentials and to avoid contamination of solutions with Ag+ ions. Electrodes are made from 0.25 m m diameter silver wire ( - 5 cm) soldered to a miniature gold-plated pin for hookup to amplifier (a diagram appears in Ref. 8). The silver metal is coated with silver chloride in an electrolytic cell filled with 0.1 N HCI. A coated electrode is inserted into 1 m m diameter polyethylene tubing which has been filled with 0.2 M KC1 in 2% agar. Carboxylate cement is used to glue the tubing to the pin connector. This assembly is durable and may last for several weeks if the electrode tip is always kept in a 0.2 M KC1 solution. Peak-to-peak noise of an assembled bilayer should be no more than 10 pA at 2 kHz or 0.2 pA at 400 Hz low-pass filtering for the recommended aperture size. Appropriate grounding of the bilayer box is sufficient to eliminate the 60 cycles noise common to most electrophysiological setups. Other sources typical of planar bilayers are mechanical and microphonic noise. Both are the result of small changes in area (capacitance) produced when the solutions vibrate and the bilayer film wobbles. Mechanical noise is usually of low frequency and many times erratic. The
702
CHANNEL RECORDING IN ORGANELLES AND MICROBES
[49]
bilayer setup should be isolated from other fixtures in the room. Ideally, the setup should rest on an air-lifted table (see list of equipment). Electrodes should be fitted tightly to the head stage. The head stage should be glued or screwed to the bilayer box. Microphonic noise increases as the bilayer capacitance increases. Avoid placing a setup next to an elevator shaft, equipment room, or cold room. Bilayer Stability The quality of the pinhole is the number one source of day-to-day technical difficulty in assembling long-lasting stable bflayers. In an acceptable cup, a thin bilayer should last no less than 30 rain at 0 mV applied potential in the absence of added protein. Cups should be inspected regularly to ensure that the hole is free from dirt and that cracks have not formed along the edges of the hole. Cups are rinsed only with high quality methanol. Avoid the use of detergents or chloroform. Users should have a set of no less than 20 machined cups at their disposal at any given time. Cups showing signs of wear should be discarded. A good commercial source of phospholipids is Avanti Polar Lipids (Birmingham, AL). Lipids should be purchased dissolved in chloroform and shipped in N2-sealed vials in aliquots of I ml of 5 mg/ml phospholipid. Phosphatidylethanolamine (PE) and phosphatidylserine (PS) purified from brain are ideal for bilayer stability and single-channel recording. Lipids are mixed at a 1 : 1 weight ratio, dried under a stream of N2, and resuspended in ultrapure n-decane (Aldrich Chemical Co., Milwaukee, WI) at a concentration of 20 mg/ml phospholipid. A 100 gl lipid solution in decane should be prepared daily. Fresh vials of PE and PS should be opened every 2 weeks and old lipids discarded. Lipid solution is applied to the aperture with a 0.2 cm diameter Delrin stick cut diagonally at the tip. Bilayer thinning, from a thick to a thin film, is monitored by capacitance measurement. Capacitance rises from approximately 100 to 500 pF in the apertures of the size recommended. Ultrapure grade chloride salts of monovalents or divalents are recommended for planar bilayer recording. A good source is Johnson Matthey Chemicals Ltd. (Hereforshire, England) distributed in the United States by Alpha Products (Morthon Thiokol, Inc., Danvers, MA). The recommended concentration of EDTA (ethyleneAiaminetetraacetic acid) for Ca 2+ buffering is 1 mM. pH buffers such as HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), MES (2-N-morpholinoethanesulfonic acid), or Tris [Tris(hydroxymethyl)aminomethane] should not be used in excess of l0 mM. Water should be distilled in the laboratory in an all-glass still.
[49]
RYANODINE RECEPTORS OF SARCOPLASMIC RETICULUM
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Fractionation of Sarcoplasmic Reticulum Light and heavy SR contains K + and Cl- channels,5 but ryanodinc receptors arc only found in heavy SR. 9 Procedures that use Ca2+-precipitaring agents such as oxalatc or phosphate for loading Ca 2+ into the SR (transport-specific fractionation) should bc avoided since intravcsicular Ca 2+ impairs SR incorporation into bilayers. A procedure to fractionatc light and heavy SR of skclctal or cardiac muscle, which yiclds active ryanodinc receptor channels in planar bilaycrs,is as follows. Tissue taken from the animal is immersed in ice-cooled saline and taken to a cold room where the homogenization step is carried out. White muscle from the back and hind legs of one adult N e w Zealand rabbit or one fresh bovine heart collected no more than 20 rain after sacrificeis minced and ground in a food processor. Each of 4-5 portions of 40 g arc homogenized for 60 scc in 300 ml of 0.3 M sucrose, 0.5 m M EGTA, 20 m M Na4P2OT, 20 r n M NaH2PO4, 1.0 m M MgCI2, p H 7.1, using a Waxing blender at m a x i m u m speed. The following protcasc inhibitorsarc added during homogenization: pcpstatin A (I ~tM), iodoacctamidc (I raM), phenylmethylsulfonyl fluoride (PMSF) (0.I raM), Icupcptin (I/~M), and bcnzamidinc (I raM). Thc homogcnatc is spun for 15 rain at 9000 rpm in a Sorvall G S A rotor. The supernatant is liltcredthrough four layers of gauze and centrifuged at 14,000 rpm for 30 rain in the same rotor. Pelletsarc resuspended to a finalvolume of 60 ml in the same medium used for homogenization and layered on top of a stcp sucrose gradient composed of 7 ml of 27% (w/v) sucrose, 7 ml of 32% sucrose, 14 ml of 38% sucrose, in 20 r n M Na4P2OT, 20 m M NaH2PO4, 1 m/14MgCI2, pH 7.1. Gradients are centrifuged for 16 hr at 20,000 rpm in a Beckman SW 28 rotor at 4*. The heavy microsome fraction is collected from the 32-38% sucrose interface. After gentle dilution with 3 volumes of homogenization medium without sucrose, a pellet is obtained by centrifugation at 30,000 rpm for 40 rain using a Beckman 35 rotor. Microsomes are resuspended in 0.3 M sucrose, 5 m M HEPES-KOH, pH 7.0, aliquoted, frozen in liquid nitrogen, and stored at - 8 0 * for up to 3 months. When prepared from rabbit skeletal muscle,2'1° this preparation has a [3H]PN200-1 l0 binding capacity of 12 pmol/mg protein, a [3H]ryanodine binding capacity of 9 pmol/mg protein, and an ATP-dependent Ca 2+ uptake capacity of 15 nmol/mg protein.
9G. Meissner,J. Biol. Chem. 259, 2365 (1984). ~oH. Valdivia,C. Valdivia,J. Ma, and R. Coronado,Biophys. J. in press(1990).
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CHANNEL RECORDING IN ORGANELLES AND MICROBES
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Solutions and Incorporation of Channels Recordings of ryanodine receptors are best made using CsCI as current carrier ~-3,~0,~ instead of the Ca-HEPES and Tris-HEPES solutions suggested earlier. 8,n Insights into the mechanism of SR vesicle incorporation into bilayers may be found in Ref. 13. The previous recording technique assumed that only Ca2+ was permeable through ryanodine receptors, and therefore large concentrations of divalents (Ca 2+ or Ba2+) were used to increase current amplitude. This assumption turned out to be incorrect: we now know that the channel is highly nonselective.3 Divalent cations can be thus kept at physiological levels without loss of conductance if a monovalent cation is present in both chambers (Fig. 114). Other complications in the previous technique arose from the use of large cations and anions like Tris+ and HEPES- to block SR K + and CIchannels. Because SR incorporation into bilayers is rare in the absence of CI-, bilayer chambers had to be perfused with C1--free solutions after SR incorporation in CI- media. The perfusion maneuver tended to break bilayers even when a circuit was developed to bypass the electronics,s This made the procedure tedious, and the number of successful recordings was limited. The use of CsCI instead of Ca-HEPES and Tris-HEPES eliminated the need for perfusion and eliminated the need for large and unphysiological gradients of Ca 2÷, which severely inactivate the channel (in high trans Ca 2+, the open probability is around 10-fold lower than when the trans free Ca 2+ is kept at 10/tM). Finally, Cs+ blocks SR K + channels, and because it has a higher conductance than Ca 2+ or Na t through ryanodine receptors (gc,/gc~ = 2), 3 it improved the signal-to-noise ratio. Sarcoplasmic reticulum C1- channels can be separated from ryanodine receptors on the basis of reversal potential. In the solutions recommended, E o is + 59 mV and Ec, is - 5 9 mV. The reversal potential for SR CI- channels is approximately + 3 0 inV. Recordings in the presence of CI- channels can thus be made by holding the membrane potential close to the CI- equilibrium potential (more positive than + 20 mV). Sarcoplasmic reticulum preparations are thawed by hand and kept on ice. Approximately 100 pg of skeletal SR or 500/zg of cardiac SR protein are added to the cup-side solution, designated the cis side. Cis solution is n M. Fill, R. Coronado, J. R. Mickelson, J. Vilven, J. Ma, B. A. Jacobson, and C. F. Louis, Biophys. J. 50, 471 (1987). 12j. S. Smith, R. Coronado, and G. Meissner, Nature (London) 316, 446 (1985). ~3F. S. Cohen, M. H. Akabcs, J. Zimmerberg, and A. Filkenstein, J. Cell Biol. 98, 1054 (1984). ~4S. Kawano and R. Coronado, J. Physiol. (1992) In press.
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A
RYANODINE RECEPTORS OF SARCOPLASMICRETICULUM
705
E3
400
ms
1 O0 m s
25
ms
|
I 40 pA FIG. 1. Planar bilayer recording of cardiac and skeletal ryanodine receptor. (A) Cardiac ryanodine receptor from bovine heart at + 20 mV holding potential in cis 0.5 M CsO, trans 50 mM CsCI. Cis and trans Ca2+ was 3.5/zM. (B) Skeletal ryanodine receptor from rabbit skeletal muscle under same conditions. (From Ref. 14 with permission.)
typically composed of 3 ml of 0.5 M CsC1, 10 to 100#M CaC12, and 10 mM HEPES-Tris, pH 7.5. The bath-side solution, designated trans side, is the same except the CsCl is 50 raM. In the recordings of Fig. l, a List L/M EPC 7 amplifier (List Electronics, Eberstadt, Germany) was connected to the interior of the cup; the bath side was held at ground. Recordings were filtered through a low-pass Bessel filter (Frequency Devices, Haverhill, MA) at a front panel setting of 1 kHz and digitized at 100 to 250/~sec per point. The protocol to incorporate SR channels is according to points (1) through (4) as follows. (1) Bilayers are first formed in symmetrical 50 mM CsCl buffer (the trans solution). Wetting the dry hole with a drop of lipid, before pouring a solution into the cup, considerably helps the formation of the first bilayer. Bilayer capacitance is continuously monitored to ensure an appropriate degree of thinning. The increase in bilayer capacitance or "thinning of the bilayer" is favored by large positive or negative potentials (--4-_100 mV). After thinning is completed, the CsC1 in the cis solution is increased to the final value. (2) Sarcoplasmic reticulum protein is the last component added after adjustment of free Ca 2+ with CaEGTA buffers. The cis chamber is stirred for 30 to 60 see. Membrane potential is kept
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CHANNEL RECORDING IN ORGANELLES AND MICROBES
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constant at 0 mV for cardiac SR or between 0 mV and + 2 0 mV for skeletal SR. Cardiac SR makes bilayers unstable more often than skeletal SR does. Less applied voltage is recommended when using cardiac SR protein. When a bilayer breaks, or there is a shift in baseline indicative of a leak, or when the capacitance decreases spontaneously, a new membrane should be made in the same solutions. If this maneuver is repeated too many times, however, there will be a substantial mixing of the cis and trans solutions. (3) In an typical case, C1- channels incorporate within the first 10 min and ryanodine receptors incorporate afterward within 30 min. Recordings of C1- channels should be continued until ryanodine receptors appear. The frequency of presentation of ryanodine receptors without C1channels is 1 out of 5 recordings. (4) When channels do not incorporate, the bilayer is broken and a new one is reformed in the same solutions. Breakage and reformation is repeated up to four times. Afterward, the cup is removed and rinsed with methanol before repeating the protocol. The polarity of channels incorporated into the bilayer is constant, in the majority of cases. The myoplasmic end of the receptor faces into the cis solution, and the intravesicular end faces into the trans solution. Polarity can be easily confirmed by the cis-activation of channels by ATP and micromolar Ca 2+, which are myoplasmic activators of ryanodine receptors. 9 Four useful characteristics that serve to identify ryanodine receptors are the following: (1) a linear current-voltage relationship with a slope conductance of 600 pS for cardiac SR and 700 pS for skeletal SR and reversal at potentials more negative than - 5 0 mV in the solutions recommended~4; (2) an activatory effect of adenine nucleotides and Ca 2+ and the inhibition by Mg2+. n,t4 Cis 5 m M ATP should increase the open probability about 2- to 5-fold; cis Ca 2+ should increase open probability from virtually null at pCa 9 to typical values of 0.03 for skeletal and 0.2 for cardiac receptors at pCa 6; cis 1 m M free Mg 2+ should decreases activity around 10-fold; (3) an inhibition by cis I g M ruthenium red which decreases activity 100-foldH,t2; (4) an activation by ryanodine, which at a concentration of 100 n M should produce an irreversible decrease in conductance and an increase in mean open time. 3
List of E q u i p m e n t A minimal list of equipment for planar bilayer recording and suggested sources are given below. Preparative biochemical instruments for microsome purification are not included. Bilayer cups, PVC block, brass block, and steel box with lead base, to be machined at a local shop
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RYANODINE RECEPTORS OF SARCOPLASMIC RETICULUM
707
Vibration-isolation table (Technical Manufacturing Corporation, Peabody, MA, Model Micro-G) Patch clamp amplifier (Axon Instruments, Burlingame, CA, Model Axopatch 1D with head stage for planar bilayers) VCR-based instrumentation recorder (Medical Systems Corporation, Greenvale, NY, Model PCM-2 Recorder adapter, VCR not included) 286-based PC with MS DOS, full memory, coprocessor, 40 Mbyte hard disk drive and EGA graphics (Standard Brand Products, Austin, TX, model Standard 286) Acquisition software (Axon Instruments, Model TL-125 A/D and D/A interface, PClamp software version 5.5) Laser printer (Hewlett Packard, Sunnyvale, CA, Model Laserjet series II)
