INTRACELLULAR PERFUSIONOFXenopusOOCYTES
[21 ]
345
be useful for expression of neurotransmitter receptors coupled to phospholipase C, for such receptors are often monitored by activation of the C1current. The injections of RNA are technically more difficult in stage II-III oocytes. This can, however, be overcome with some simple modifications of the injection apparatus, mainly inclusion of a pump or similar device for actual injections.
[21] I n t r a c e l l u l a r P e r f u s i o n o f Xenopus O o c y t e s .By N A T H A N DASCAL, G A V I N CHILCOTT,
and HENRY A. LESTER
Introduction This chapter describes a method for recording macroscopic voltage clamp currents through the membrane of intracellularly perfused Xenopus oocytes. The method is similar to other internal perfusion methods described for ascidian eggs,~-3 with several modifications, notably the addition of a perfusion tube that allows exchange of the intracellular solution within a few seconds. The extracellular solution can also be rapidly exchanged. Thus, this method allows full control of the solutions bathing the membrane. Moreover, sequential addition of transmitters, second messengers, regulatory proteins, and other agents becomes possible. The internal perfusion of large volumes of solution assures homogeneous distribution of the intracellular environment, in contrast to the spatial gradients that occur when the substances are injected (by pressure or iontophoresis) into intact oocytes. The technique has been applied in a limited number of experiments,4 but it will presumably be exploited in the future for detailed studies of (1) ion channel permeation and (2) modulation of ion channels by second messengers and intracellular proteins. The potential importance of the internal perfusion method is enhanced by the widespread use of Xenopus oocytes for the heterologous expression of wild-type and mutated ion channels.5-7 K. Takahashi and M. Yoshii, J. Physiol. (London) 279, 519 (1978). 2 S. Hagiwara and M. Yoshii, J. Physiol. (London) 279, 251 (1979). 3 M. Yoshii and K. Takahashi, in "Intracellular Perfusion of Excitable Cells" (P. G. Kostyuk and O. A. Krishtal, eels.), p. 77. Wiley, New York, 1984. 4 N. Dascal, G. Chilcott, and H. A. Lester, J. Neurosci. Meth. 39, 29 (1991). 5 N. Dascal, Crit. Rev. Biochem. 22, 317 (1987). 6 T. P. Snutch, Trends Neurosci. 11, 250 (1988). 7 H. A. Lester, Science 241, 1057 (1988). METHODS IN ENZYMOLOGY, VOL. 207
Copyright© 1992by AcademicPrtm,Inc. All rightsof reproductionin any formreserved.
346
EXPRESSION OF ION CHANNELS
[21 ]
Intracellular Perfusion of Oocytes
General Description of Method The setup is presented schematically in Fig. I. The oocyte is placed in a fire-polished Pasteur pipette (upper chamber), filled with the extracellular solution. The upper chamber is lowered into the "internal solution" chamber. The recording employs a two-electrode voltage clamp configuration. The ground electrode is in the extracellular solution; current and voltage electrodes are in the intracellular solution. Not shown in Fig. 1 are the oocyte internal perfusion tube and the perfusion apparatus of the upper chamber (see Fig. 3).
Internal Perfusion Setup The internal solution chamber is made of transparent polystyrene (e.g., the lower third of a 25-cm 2 tissue culture flask) and should be large enough to accommodate the upper chamber, voltage and current electrodes, the perfusion tubes used to change the solution in this chamber (Fig. 1), and the internal perfusion tube (Fig. 3). The experiment is performed under visual observation using a standard stereomicroscope. If the microscope is on a vertical mount, a mirror is placed behind the chamber to redirect the line of sight (Fig. 2); if the microscope can be mounted horizontally, the mirror is unnecessary. The upper chamber, current and voltage electrodes, and the perfusion tubes are permanently mounted on steady rods with crocodile holders. The upper chamber holder should allow adjustment along the vertical axis. The upper chamber is made of a Pasteur pipette cut at both ends. The
ground electrode\
voltage
electro
upper chamber .(Pasteurpipette)
current trode P
oocyte Internal solution chamber / Fie. 1. Schematic presentation of the experimental arrangement for internal perfusion of an oocyte.
[21 ]
INTRACELLULAR PERFUSION OF Xenopus OOCYTES
EYEPIECJE~ ~ .
~
347
/SCOPE
LINE OF SIG~ REFLECTEr THROUGH MIR
CHAMBER
( FIG. 2. Side view of the microscope and the lower chamber.
wide part is cut to a length of about 4 cm; the narrow part is cut where the outer diameter tapers to 1.2-1.8 mm. The narrow edge is then fire polished until the desired inner diameter (300-400/~m) is obtained (the oocyte passes through larger openings). The pipette serving as upper chamber can be used for only one oocyte. Both soft and borosilicate glass provides a good seal. The perfusion tubes of the upper chamber and the ground electrode are arranged as shown in Fig. 3. The perfusion tubes must be provided with vertical movement, allowing change of the extracellular solution level. Note that the edge of the infusion tube protrudes 2 - 3 m m below the level of the meniscus, assuring that the perfusate will not drop on the meniscus and agitate the oocyte. The actual position of the ground electrode is of little importance as long as it comes in contact with the solution; therefore, it can be mounted separately from the perfusion tubes. All perfusion tubes, as well as the voltage and current electrodes, are made of 1.2-2 m m (outer diameter) glass tubes. The internal perfusion tube is made as follows (Fig. 3). First, the tip is pulled manually or with a microelectrode puller, then broken to an outer diameter of 50- 100/zm. Then the tube is bent, in a Bunsen burner, to give the shape shown in Fig. 3. The tube is mounted on a micromanipulator, to allow precise position-
348
EXPRESSION OF ION CHANNELS
[21 ]
wire or agar.
Ag
. ~ IILOOCVTE ~ !jl Pn'ETTE
/ ~
OOCYTE
PEP~-USION PIPETTE
INTERNAL.I- I--PERFUSATE /,
FIG. 3. Upper chamber perfusion devices and the internal perfusion pipette.
ing under the oocyte and upward-downward movement afterward. The wide end of the perfusion pipette is connected through polyethylene tubing to a syringe containing the desired internal perfusion solution. The electrodes are filled with 3 M KCI in 1.5% agar. The ground electrode is made in the same way, or a simple Ag/AgCI wire can be used if no changes of C1- concentration in the extracellular solution are planned. The electrical resistance of the KC1/agar electrodes is a few tens of kiloohms (at most). The electrodes are connected to a negative feedback amplifier in the usual two-electrode voltage clamp configuration. Note that most commercially available voltage clamp amplifiers are designed for stability with electrodes of megohm resistances and may not perform optimally with low-resistance electrodes.
Preparation of Oocytes Frogs are maintained and dissected as described) The oocytes are defolliculated by treatment with 1.5-2 mg/ml collagenase in a Ca2+-free s A. L. Goldin, this volume [15].
[21 ]
INTRACELLULAR PERFUSION OF Xenopus OOCYTES 1
2
349
3
_J
350 ~m OOCYTE INTRODUCED INTO PIPETTE
MEMBRANE SEALS AGAINST GLASS
LOWER MEMBRANE IS RUPTURED
FIG. 4. Xenopus oocyte in a pipette.
solution, 9 transferred to a normal physiological solution (e.g., ND 969), and treated as desired (injected with RNA, incubated under different conditions, etc.). Just before the internal perfusion, the vitelline membrane is stripped, essentially as described. L° In brief, the oocyte is placed for 2 0 30 min into a high-osmolarity solution (e.g., ND 96 with the addition of 50 m M NaC1 or 100 m M sucrose). The oocyte shrinks, and the viteUine membrane detaches from the surface at some points and can be mechanically removed with a pair of fine forceps. Do not expose the deviteUinized oocyte to air as it will explode immediately; transfer it inside a horizontally positioned Pasteur pipette (to prevent sliding to the tip and exposure to air).
Succession of Events in Internal Perfusion The succession of events in internal perfusion is shown schematically in Fig. 4. The lower and upper chambers are filled with a normal physiological solution (all solutions used in the study should be filtered through 0.29 N. Dascal, T. P. Snutch, H. Lubbert, N. Davidson, and H. A. Lester, Science 231, 1147 (1986). to C. Methfessel, V. Witzemann, B. Sakmann, T. Takahashi, M. Mishina, and S. Numa, PfluegersArch. 407, 577 (1986).
350
EXPRESSION OF ION CHANNELS
[21 ]
or 0.45-~m filters). Solutions containing C a 2+ appear to promote the formation of a seal between the glass and the membrane and are therefore recommended. The oocyte is dropped into the upper chamber and settles over the bottom opening. From this moment on, the oocyte should not be moved or mechanically disturbed until the seal has formed completely, for movements would permanently destroy the seal. The solution level in the upper chamber is set 4 - 8 m m higher than that in the lower chamber; this positive pressure on the oocyte helps the formation of the seal. At this stage, there is no potential difference between the two chambers. The resistance of the oocyte and the seal (connected in parallel) should be continuously monitored. To do so, the feedback loop is closed, the voltage between the chambers is clamped at 0 mV, and rectangular pulses of 10 to 20-mV amplitude are applied periodically at intervals of several seconds. 4 The development of the seal is indicated by a steady increase of the measured resistance from 50-200 k.Q to a constant level of 2 - 1 0 Mr2 over 15- 30 min (Fig. 5). Resistances significantly lower than 2 M ~ usu"extemar'-> ND 96 L . . . . . . . B..a acetate "internal"-> ND 96 ]High K+, EGTA,I High K +, EGTA, Cs +, ATP Cs +
E2 Ilmembrane broken
o-o
.1.
o.-~
--o-o
~oo____o.~O-----..o..~" 0
0
A
i
20
40
i
i
60 80 time, min
i
1oo
•
120
FIG. 5. Time course of changes in ~ as a function of time, membrane rupture, solution changes, and the addition of 1.5 m M ATP in an oocyte injected with cardiac RNA 3 days before the recording (see Ref. 9 for details). The two rows at the top indicate the solutions used: "external," the solution in the Pasteur pipene; "internal," the solution in the internal solution chamber. The vertical bars show the times of solution changes. The composition of the solutions was as follows: ND 96, 96 m M NaCI, 2 m M KCI, 1 m M MgCI2, 1.8 m M CaC12, 5 m M HEPES (pH 7.5); Ba acetate solution, 40 m M barium acetate, 2 m M potassium acetate, 60 m M N-methyl-D-glucamine (acetate salt), 5 m M HEPES (pH 7.5); high K + solution, 86 m M KC1, 12 m M NaCI, 5 m M HEPES (pH 7.4), 1 m M EGTA, and 5 m M CsC1.
[21 ]
INTRACELLULAR PERFUSIONOF Xenopus OOCYTES
351
ally suggest low seal quality; such cells should be abandoned. The formation of a stable seal can be verified by the fact that changes in the upper chamber solution level do not affect the measured resistance. The actual resistance of the seal cannot be measured directly, but calculations show that it is at least an order of magnitude or more higher than the resistance of half of the oocyte membrane.4,H; the latter is usually between 0.5 and 2 Mfl in normal physiological solution. Following the formation of a seal, the upper chamber solution level is reduced until it exceeds that of the lower chamber by no more than 1-3 m m n (excessive positive pressure may cause collapse of the oocyte after the lower membrane is ruptured). The lower membrane (facing the internal solution chamber) is ruptured by the tip of the internal perfusion pipette, which can be inserted a few hundred microns into the oocyte and moved in and out, to promote efflux of yolk. The yolk, however, still leaks out of the oocyte for tens of minutes even after extensive internal washings. At this time, the internal chamber solution can be changed to the desired intracellular solution. The solutions used are designed in the same way as for experiments done in the whole-cell configuration of the patch clamp, 13,~4for example, containing a high concentration of K ÷, low Na ÷, Cs ÷, and EGTA (Fig. 5). The voltage protocol is changed to one designed to measure the ionic currents under study, and the extracellular solution can be changed to one specially designed to measure currents through a particular class of channel; for example, currents through Ca 2+ channels can be recorded with Ba 2÷ as the charge carrier (see Fig. 5). Constituents of the internal chamber solution diffuse into the oocyte extremely slowly. ~-4 However, the internal perfusion tube allows one to introduce various substances and, apparently, change the actual ionic composition of the intracellular solution in just tens of seconds. The perfusion is accomplished by applying positive pressure from the syringe to the oocyte perfusion tube and blowing the solution into the oocyte. The oocyte expands visibly under the pressure of the inflowing solution and 11 Assuming an average resistance of the seal formed between a normal patch pipette of 1 #m diameter with a cell membrane to be 3 G~, the seal of a 300-/zm pipette with the oocyte membrane will be approximately 10 Mfl, assuming a similar contact between the cell surface and the glass. However, because the surface area of the contact per unit diameter is much larger than in the usual patch (see, e.g., Fig. 3, which presents quite a realistic picture of such a contact), the seal is probably much better. 12 This represents the equilibrium level as modified by forces due to capillary action in the upper chamber; thus, no strain is placed on the seal. 13O. P. Hamill, A Marry, E. Neher, B. Sakmann, and F. J. Sigworth, Pfluegers Arch. 391, 85 (1981). 14 B. Sakmann and E. Neher, eds., "Single-Channel Recording." Plenum, New York, 1983.
352
EXPRESSIONOF ION CHANNELS
[21]
then relaxes again as the perfusion stops. Note that excessive pressure may break the seal or the membrane. To exchange the internally perfused solution, perform the following steps. Withdraw the perfusion tube from the oocyte and empty its contents, replace the syringe with one containing the new solution, fill the perfusion tube with the new solution, and insert the tube again into the oocyte and wash in the new solution. If a simple addition of a drug to the interior of the oocyte is desired, it is not necessary to exchange the solution in the entire internal solution chamber; ifa more drastic change (such as replacement of an ion by another) takes place, the solution in the lower chamber should be replaced with the new one. The resistance of the oocyte "runs down" (decreases) during the experiment 4 (Fig. 5), although currents through C1- and Ca 2+ channels can still be recorded for many minutes. Our preliminary results (Ref. 4 and Fig. 5) indicate that addition of 1.5 m M MgATP to the interior of the oocyte may prevent and even reverse this rundown. In the experiment shown in Fig. 5, after the addition of ATP, Ba 2+ current through the voltage-dependent Ca 2+ channels was recorded without decrement for 1 hr after the addition of ATP (see Ref. 15). Possible Drawbacks A major problem in the internal perfusion method is the lack of an accurate estimate of the seal resistance. Current flowing through the shunt resistance of the seal may influence or even distort the recording of the current flowing through the membrane, if seal resistance is insufficiently high.l.3 However, as noted above, the seal between devitellinized Xenopus oocytes and glass seems to be very good, and the errors in current amplitude estimation should be less than 10%.4 If there is only a loose seal at the edges of the contact of the glass with the membrane, the voltage control there will be poor, and this will distort the recorded current. A rigorous test of the validity of the method for measuring fast currents has not yet been performed.
Acknowledgments We are grateful to Dr. I. Lotan for critical reading of the manuscript. During preparation of this chapter, the authors were supported in part by grants from the Muscular Dystrophy Association (N.D.), the IsraelAcademy of Sciences and Humanities (N.D.), the USA-Israel Binational Fund (N.D. and H.A.L.), National Institutesof Health Grant G M 29836 (H.A.L.), and a grant from the Markcy Charitable Trust (H.A.L.). 15j. E. Chad and R. Eekert, J. Physiol. (London) 378, 31 (1986).
[21 ]
345
be useful for expression of neurotransmitter receptors coupled to phospholipase C, for such receptors are often monitored by activation of the C1current. The injections of RNA are technically more difficult in stage II-III oocytes. This can, however, be overcome with some simple modifications of the injection apparatus, mainly inclusion of a pump or similar device for actual injections.
[21] I n t r a c e l l u l a r P e r f u s i o n o f Xenopus O o c y t e s .By N A T H A N DASCAL, G A V I N CHILCOTT,
and HENRY A. LESTER
Introduction This chapter describes a method for recording macroscopic voltage clamp currents through the membrane of intracellularly perfused Xenopus oocytes. The method is similar to other internal perfusion methods described for ascidian eggs,~-3 with several modifications, notably the addition of a perfusion tube that allows exchange of the intracellular solution within a few seconds. The extracellular solution can also be rapidly exchanged. Thus, this method allows full control of the solutions bathing the membrane. Moreover, sequential addition of transmitters, second messengers, regulatory proteins, and other agents becomes possible. The internal perfusion of large volumes of solution assures homogeneous distribution of the intracellular environment, in contrast to the spatial gradients that occur when the substances are injected (by pressure or iontophoresis) into intact oocytes. The technique has been applied in a limited number of experiments,4 but it will presumably be exploited in the future for detailed studies of (1) ion channel permeation and (2) modulation of ion channels by second messengers and intracellular proteins. The potential importance of the internal perfusion method is enhanced by the widespread use of Xenopus oocytes for the heterologous expression of wild-type and mutated ion channels.5-7 K. Takahashi and M. Yoshii, J. Physiol. (London) 279, 519 (1978). 2 S. Hagiwara and M. Yoshii, J. Physiol. (London) 279, 251 (1979). 3 M. Yoshii and K. Takahashi, in "Intracellular Perfusion of Excitable Cells" (P. G. Kostyuk and O. A. Krishtal, eels.), p. 77. Wiley, New York, 1984. 4 N. Dascal, G. Chilcott, and H. A. Lester, J. Neurosci. Meth. 39, 29 (1991). 5 N. Dascal, Crit. Rev. Biochem. 22, 317 (1987). 6 T. P. Snutch, Trends Neurosci. 11, 250 (1988). 7 H. A. Lester, Science 241, 1057 (1988). METHODS IN ENZYMOLOGY, VOL. 207
Copyright© 1992by AcademicPrtm,Inc. All rightsof reproductionin any formreserved.
346
EXPRESSION OF ION CHANNELS
[21 ]
Intracellular Perfusion of Oocytes
General Description of Method The setup is presented schematically in Fig. I. The oocyte is placed in a fire-polished Pasteur pipette (upper chamber), filled with the extracellular solution. The upper chamber is lowered into the "internal solution" chamber. The recording employs a two-electrode voltage clamp configuration. The ground electrode is in the extracellular solution; current and voltage electrodes are in the intracellular solution. Not shown in Fig. 1 are the oocyte internal perfusion tube and the perfusion apparatus of the upper chamber (see Fig. 3).
Internal Perfusion Setup The internal solution chamber is made of transparent polystyrene (e.g., the lower third of a 25-cm 2 tissue culture flask) and should be large enough to accommodate the upper chamber, voltage and current electrodes, the perfusion tubes used to change the solution in this chamber (Fig. 1), and the internal perfusion tube (Fig. 3). The experiment is performed under visual observation using a standard stereomicroscope. If the microscope is on a vertical mount, a mirror is placed behind the chamber to redirect the line of sight (Fig. 2); if the microscope can be mounted horizontally, the mirror is unnecessary. The upper chamber, current and voltage electrodes, and the perfusion tubes are permanently mounted on steady rods with crocodile holders. The upper chamber holder should allow adjustment along the vertical axis. The upper chamber is made of a Pasteur pipette cut at both ends. The
ground electrode\
voltage
electro
upper chamber .(Pasteurpipette)
current trode P
oocyte Internal solution chamber / Fie. 1. Schematic presentation of the experimental arrangement for internal perfusion of an oocyte.
[21 ]
INTRACELLULAR PERFUSION OF Xenopus OOCYTES
EYEPIECJE~ ~ .
~
347
/SCOPE
LINE OF SIG~ REFLECTEr THROUGH MIR
CHAMBER
( FIG. 2. Side view of the microscope and the lower chamber.
wide part is cut to a length of about 4 cm; the narrow part is cut where the outer diameter tapers to 1.2-1.8 mm. The narrow edge is then fire polished until the desired inner diameter (300-400/~m) is obtained (the oocyte passes through larger openings). The pipette serving as upper chamber can be used for only one oocyte. Both soft and borosilicate glass provides a good seal. The perfusion tubes of the upper chamber and the ground electrode are arranged as shown in Fig. 3. The perfusion tubes must be provided with vertical movement, allowing change of the extracellular solution level. Note that the edge of the infusion tube protrudes 2 - 3 m m below the level of the meniscus, assuring that the perfusate will not drop on the meniscus and agitate the oocyte. The actual position of the ground electrode is of little importance as long as it comes in contact with the solution; therefore, it can be mounted separately from the perfusion tubes. All perfusion tubes, as well as the voltage and current electrodes, are made of 1.2-2 m m (outer diameter) glass tubes. The internal perfusion tube is made as follows (Fig. 3). First, the tip is pulled manually or with a microelectrode puller, then broken to an outer diameter of 50- 100/zm. Then the tube is bent, in a Bunsen burner, to give the shape shown in Fig. 3. The tube is mounted on a micromanipulator, to allow precise position-
348
EXPRESSION OF ION CHANNELS
[21 ]
wire or agar.
Ag
. ~ IILOOCVTE ~ !jl Pn'ETTE
/ ~
OOCYTE
PEP~-USION PIPETTE
INTERNAL.I- I--PERFUSATE /,
FIG. 3. Upper chamber perfusion devices and the internal perfusion pipette.
ing under the oocyte and upward-downward movement afterward. The wide end of the perfusion pipette is connected through polyethylene tubing to a syringe containing the desired internal perfusion solution. The electrodes are filled with 3 M KCI in 1.5% agar. The ground electrode is made in the same way, or a simple Ag/AgCI wire can be used if no changes of C1- concentration in the extracellular solution are planned. The electrical resistance of the KC1/agar electrodes is a few tens of kiloohms (at most). The electrodes are connected to a negative feedback amplifier in the usual two-electrode voltage clamp configuration. Note that most commercially available voltage clamp amplifiers are designed for stability with electrodes of megohm resistances and may not perform optimally with low-resistance electrodes.
Preparation of Oocytes Frogs are maintained and dissected as described) The oocytes are defolliculated by treatment with 1.5-2 mg/ml collagenase in a Ca2+-free s A. L. Goldin, this volume [15].
[21 ]
INTRACELLULAR PERFUSION OF Xenopus OOCYTES 1
2
349
3
_J
350 ~m OOCYTE INTRODUCED INTO PIPETTE
MEMBRANE SEALS AGAINST GLASS
LOWER MEMBRANE IS RUPTURED
FIG. 4. Xenopus oocyte in a pipette.
solution, 9 transferred to a normal physiological solution (e.g., ND 969), and treated as desired (injected with RNA, incubated under different conditions, etc.). Just before the internal perfusion, the vitelline membrane is stripped, essentially as described. L° In brief, the oocyte is placed for 2 0 30 min into a high-osmolarity solution (e.g., ND 96 with the addition of 50 m M NaC1 or 100 m M sucrose). The oocyte shrinks, and the viteUine membrane detaches from the surface at some points and can be mechanically removed with a pair of fine forceps. Do not expose the deviteUinized oocyte to air as it will explode immediately; transfer it inside a horizontally positioned Pasteur pipette (to prevent sliding to the tip and exposure to air).
Succession of Events in Internal Perfusion The succession of events in internal perfusion is shown schematically in Fig. 4. The lower and upper chambers are filled with a normal physiological solution (all solutions used in the study should be filtered through 0.29 N. Dascal, T. P. Snutch, H. Lubbert, N. Davidson, and H. A. Lester, Science 231, 1147 (1986). to C. Methfessel, V. Witzemann, B. Sakmann, T. Takahashi, M. Mishina, and S. Numa, PfluegersArch. 407, 577 (1986).
350
EXPRESSION OF ION CHANNELS
[21 ]
or 0.45-~m filters). Solutions containing C a 2+ appear to promote the formation of a seal between the glass and the membrane and are therefore recommended. The oocyte is dropped into the upper chamber and settles over the bottom opening. From this moment on, the oocyte should not be moved or mechanically disturbed until the seal has formed completely, for movements would permanently destroy the seal. The solution level in the upper chamber is set 4 - 8 m m higher than that in the lower chamber; this positive pressure on the oocyte helps the formation of the seal. At this stage, there is no potential difference between the two chambers. The resistance of the oocyte and the seal (connected in parallel) should be continuously monitored. To do so, the feedback loop is closed, the voltage between the chambers is clamped at 0 mV, and rectangular pulses of 10 to 20-mV amplitude are applied periodically at intervals of several seconds. 4 The development of the seal is indicated by a steady increase of the measured resistance from 50-200 k.Q to a constant level of 2 - 1 0 Mr2 over 15- 30 min (Fig. 5). Resistances significantly lower than 2 M ~ usu"extemar'-> ND 96 L . . . . . . . B..a acetate "internal"-> ND 96 ]High K+, EGTA,I High K +, EGTA, Cs +, ATP Cs +
E2 Ilmembrane broken
o-o
.1.
o.-~
--o-o
~oo____o.~O-----..o..~" 0
0
A
i
20
40
i
i
60 80 time, min
i
1oo
•
120
FIG. 5. Time course of changes in ~ as a function of time, membrane rupture, solution changes, and the addition of 1.5 m M ATP in an oocyte injected with cardiac RNA 3 days before the recording (see Ref. 9 for details). The two rows at the top indicate the solutions used: "external," the solution in the Pasteur pipene; "internal," the solution in the internal solution chamber. The vertical bars show the times of solution changes. The composition of the solutions was as follows: ND 96, 96 m M NaCI, 2 m M KCI, 1 m M MgCI2, 1.8 m M CaC12, 5 m M HEPES (pH 7.5); Ba acetate solution, 40 m M barium acetate, 2 m M potassium acetate, 60 m M N-methyl-D-glucamine (acetate salt), 5 m M HEPES (pH 7.5); high K + solution, 86 m M KC1, 12 m M NaCI, 5 m M HEPES (pH 7.4), 1 m M EGTA, and 5 m M CsC1.
[21 ]
INTRACELLULAR PERFUSIONOF Xenopus OOCYTES
351
ally suggest low seal quality; such cells should be abandoned. The formation of a stable seal can be verified by the fact that changes in the upper chamber solution level do not affect the measured resistance. The actual resistance of the seal cannot be measured directly, but calculations show that it is at least an order of magnitude or more higher than the resistance of half of the oocyte membrane.4,H; the latter is usually between 0.5 and 2 Mfl in normal physiological solution. Following the formation of a seal, the upper chamber solution level is reduced until it exceeds that of the lower chamber by no more than 1-3 m m n (excessive positive pressure may cause collapse of the oocyte after the lower membrane is ruptured). The lower membrane (facing the internal solution chamber) is ruptured by the tip of the internal perfusion pipette, which can be inserted a few hundred microns into the oocyte and moved in and out, to promote efflux of yolk. The yolk, however, still leaks out of the oocyte for tens of minutes even after extensive internal washings. At this time, the internal chamber solution can be changed to the desired intracellular solution. The solutions used are designed in the same way as for experiments done in the whole-cell configuration of the patch clamp, 13,~4for example, containing a high concentration of K ÷, low Na ÷, Cs ÷, and EGTA (Fig. 5). The voltage protocol is changed to one designed to measure the ionic currents under study, and the extracellular solution can be changed to one specially designed to measure currents through a particular class of channel; for example, currents through Ca 2+ channels can be recorded with Ba 2÷ as the charge carrier (see Fig. 5). Constituents of the internal chamber solution diffuse into the oocyte extremely slowly. ~-4 However, the internal perfusion tube allows one to introduce various substances and, apparently, change the actual ionic composition of the intracellular solution in just tens of seconds. The perfusion is accomplished by applying positive pressure from the syringe to the oocyte perfusion tube and blowing the solution into the oocyte. The oocyte expands visibly under the pressure of the inflowing solution and 11 Assuming an average resistance of the seal formed between a normal patch pipette of 1 #m diameter with a cell membrane to be 3 G~, the seal of a 300-/zm pipette with the oocyte membrane will be approximately 10 Mfl, assuming a similar contact between the cell surface and the glass. However, because the surface area of the contact per unit diameter is much larger than in the usual patch (see, e.g., Fig. 3, which presents quite a realistic picture of such a contact), the seal is probably much better. 12 This represents the equilibrium level as modified by forces due to capillary action in the upper chamber; thus, no strain is placed on the seal. 13O. P. Hamill, A Marry, E. Neher, B. Sakmann, and F. J. Sigworth, Pfluegers Arch. 391, 85 (1981). 14 B. Sakmann and E. Neher, eds., "Single-Channel Recording." Plenum, New York, 1983.
352
EXPRESSIONOF ION CHANNELS
[21]
then relaxes again as the perfusion stops. Note that excessive pressure may break the seal or the membrane. To exchange the internally perfused solution, perform the following steps. Withdraw the perfusion tube from the oocyte and empty its contents, replace the syringe with one containing the new solution, fill the perfusion tube with the new solution, and insert the tube again into the oocyte and wash in the new solution. If a simple addition of a drug to the interior of the oocyte is desired, it is not necessary to exchange the solution in the entire internal solution chamber; ifa more drastic change (such as replacement of an ion by another) takes place, the solution in the lower chamber should be replaced with the new one. The resistance of the oocyte "runs down" (decreases) during the experiment 4 (Fig. 5), although currents through C1- and Ca 2+ channels can still be recorded for many minutes. Our preliminary results (Ref. 4 and Fig. 5) indicate that addition of 1.5 m M MgATP to the interior of the oocyte may prevent and even reverse this rundown. In the experiment shown in Fig. 5, after the addition of ATP, Ba 2+ current through the voltage-dependent Ca 2+ channels was recorded without decrement for 1 hr after the addition of ATP (see Ref. 15). Possible Drawbacks A major problem in the internal perfusion method is the lack of an accurate estimate of the seal resistance. Current flowing through the shunt resistance of the seal may influence or even distort the recording of the current flowing through the membrane, if seal resistance is insufficiently high.l.3 However, as noted above, the seal between devitellinized Xenopus oocytes and glass seems to be very good, and the errors in current amplitude estimation should be less than 10%.4 If there is only a loose seal at the edges of the contact of the glass with the membrane, the voltage control there will be poor, and this will distort the recorded current. A rigorous test of the validity of the method for measuring fast currents has not yet been performed.
Acknowledgments We are grateful to Dr. I. Lotan for critical reading of the manuscript. During preparation of this chapter, the authors were supported in part by grants from the Muscular Dystrophy Association (N.D.), the IsraelAcademy of Sciences and Humanities (N.D.), the USA-Israel Binational Fund (N.D. and H.A.L.), National Institutesof Health Grant G M 29836 (H.A.L.), and a grant from the Markcy Charitable Trust (H.A.L.). 15j. E. Chad and R. Eekert, J. Physiol. (London) 378, 31 (1986).
