- Email: [email protected]
[8] Prevention of rundown in electrophysiological recording
[8]
INHIBITION OF RUNDOWN
[8] P r e v e n t i o n
149
o f R u n d o w n in E l e c t r o p h y s i o l o g i c a l Recording
By R I C H A R D
H O R N a n d STEPHEN J. K O R N
Introduction The invention of the gigaseal patch clamp technique I has led to stupendous strides in the study of electrophysiological phenomena. Of the several variants of this methodology, whole-cell recording is the most popular, largely because of the ease and speed of obtaining and analyzing macroscopic currents. Nonetheless, standard whole-cell recording suffers from several problems related to the need to break the membrane at the interface between the cytoplasm and the interior of the patch pipette. The exchange of material between the interior of the cell and the pipette solution results in the dilution, or "washout," of cytoplasmic constituents that may affect the functional properties of ion channels. Common examples of the deleterious consequences of the diluted cytoplasm include the decline (i.e., rundown) of calcium (Ca2+) channel function and the disappearance of receptor-activated second-messenger responses shortly after breaking into a cell. Other, perhaps more subtle, changes accompanying whole-cell recording include alterations in the activity of cytoplasmic buffers and enzymes. We shall discuss and evaluate several alternative methods that reduce or avoid the problems presented by whole-ceU recording. We do not, however, consider methods used for single-channel recording, which include the cytoplasm-preserving cell-attached patch configuration. Microelectrodes Patch clamp recording has largely supplanted the use of microelectrodes, especially in experiments with small mammalian cells that are difficult to penetrate benignly with a microelectrode. Microelectrode recording nonetheless retains several advantages, including (1) washout and dialysis of cytoplasm are minimized, (2) it is possible to impale cells that are inaccessible to a patch electrode, for example, cells deeply embedded in tissue; and (3) for large currents in very large cells, such as Xenopus oocytes, a two-microelectrode voltage clamp avoids the series resistance O. P. Hamill, A. Marry, E. Neher, B. Sakmann, and F. J. Sigworth, PluegersArch. 391, 85 (1981).
METHODS IN ENZYMOLOGY,VOL. 207
Copyright© 1992by AcademicPress,Inc. Allfightsofreproductionin any formreserved.
150
ELECTROPHYSIOLOGICAL TECHNIQUES
[8]
errors arising from passing current and recording voltage through the same electrode, as done in whole-cell recording. Disadvantages in the use of microelectrodes include (I) the electrical resistance between the cytoplasm and interior of the recording pipette is usually higher in microelectrode recording than in whole-cell recording. A high electrode resistance reduces the quality of the recording, especially in voltage clamp experiments; (2) microelectrodes are frequently more noxious than a patch pipette and may damage a cell2; and (3) there is some exchange between the pipette and cytoplasm. For example, microelectrodes filled with CsC1, instead of KC1, are effective in reducing potassium currents in impaled cells. Also, because large molecules (e.g., messenger RNA) may be pressure injected through microelectrodes, other substances may exchange at unknown rates between the cytoplasm and the electrode. Technical details for microelectrode recording have been described elsewhere:
Big Patches: Loose and Tight Cell-attached patch recording usually implies that cytoplasm is maintained on the intracellular surface of the plasma membrane. With this configuration one usually observes tingle-channel currents. However, if channel density is very high (e.g., see Ref:) or if very large pipettes are used, macroscopic currents may be observed in cell-attached patches. Presumably such currents are modulable by second messengers and do not wash out. One of the technical difficulties with large pipettes is that the probability of obtaining a gigohm seal is inversely related to pipette size. The current jargon thus differentiates macropatch recording (with a seal) and loose patch recording (without a seal). The seal improves the signal-tonoise ratio and increases the dynamic range of the recording amplifier. In certain experiments, however, loose patch recording may be preferable, for example, when multiple recordings are obtained from different locations on the same cell. Because the macroscopic currents through a large patch may be substantial, compared with the input impedance of the cell under study, it may be necessary to voltage clamp the cell with separate elec2 E. M. Fenwick, A. Marty, and E. Neher, J. Physiol. (London) 331, 557 (1982). 3 T. G. Smith, H. Lecar, S. J. Redman, and P. W. Gage, eds., "Voltage and Patch Clamping with Microelectrodes." American Physiological Society, Bethesda, Maryland, 1985. 4 j, Patlak and M. Oa'~, J. Gen. Physiol. 86, 89 (1985).
[8]
INHIBITION OF RUNDOWN
151
trodes, in order to maintain the membrane potential during current flow through the patch. (For further details see Refs. 5 and 6).
Whole-Cell Recording with S u p p l e m e n t e d Pipette Solutions The most direct method to prevent washout in whole-cell recording is to supplement the pipette solution with ingredients that will maintain the normal function of the ionic currents under study. This approach has been used successfully with Ca 2÷ currents, which may be maintained with a soup containing substances that inhibit proteases and support phosphorylation by cyclic AMP (e.g., ATP, Mg 2+, catalytic subunit of cAMP-dependent protein kinase, leupeptin, and Ca 2+ buffersT). This method may not, however, prevent the loss of hormone or transmitter responses, either because the essential ingredients are unknown or because they are added in the wrong concentrations. Furthermore, a second-messenger response to an extracellular stimulus involves transient changes in the concentration of intracellular substances; such transients are expected to be suppressed in whole-cell recording because the pipette solution tends to clamp cytoplasmic concentrations. The extent of suppression will depend on the time course of the transient and the rates of diffusion of relevant substances between the cell and the pipette. In one report a cytoplasmic extract, added to the pipette solution in whole-cell recording, inhibited the rundown of a hormone response in pituitary cells, s
Whole-Cell Recording with Small Pipettes The speed of washout depends on the resistance of a whole-cell pipette and the size of the cell? Therefore small pipettes have been favored in some experimentsJ ° The obvious penalty in such an approach is the concomitant increase in series resistance between the pipette and the cell
5W. M. Robertsand W. Almers,this volume[9]. 6W. Sttihmer,this volume[19]. 7j. Chad, D. Kalman,and D. Armstrong,in "CellCalciumand the Controlof Membrane Transport" (D. C. Eaton and L. J. Mandel, eds.), Vol. 42, p. 167. Societyof General PhysiologistsSeres, RockefellerUniv. Press, New York, 1987. 8B. Dufy,A. MacDermott,and J. L. Barker,Biochem. Biophys. Res. Commun. 137(1), 388 (1986). 9A. Manyand J. Zimmerberg,Cell. Signalling 1, 259 (1989). ~oL. O. Trusselland M. B. Jackson,J. Neurosci. 7, 3306 (1987).
152
ELECTROPHYSIOLOGICAL TECHNIQUES
[8]
interior. Another problem with this approach is that the exchange is slowed, but not prevented. This means that the concentrations of some cytoplasmic substances are changing slowly during the course of an experiment, and that the rate of exchange will depend on the molecular weight of each constituent.9,~ A final problem is that the series resistance often increases spontaneously when using small pipettes (i.e., they tend to "seal over").
Permeabilized Patch Recording The prevention of rundown in whole-cell recording clearly requires a diminution of the exchange of material between the recording pipette and the cytoplasm. The methods in this section rely on the plasma membrane itself as a dialysis membrane. Ordinarily the membrane under a patch electrode during cell-attached patch recording is far too resistive either to measure the membrane potential of a cell or to control the cell's voltage while recording current. Nonetheless we have occasionally measured a fraction of the membrane potential through a cell-attached patch during spontaneous action potentials (Fig. 1). Of course this method can only yield qualitative results. Peering through a patch into the cell was finally accomplished with the inclusion in the pipette solution of ionophores that permeabilized the patch, z2 Lindau and Fernandez 13used ATP, which induces a conductance on the external surface of mast cells. When placed into the patch pipette in the cell-attached configuration, the series resistance is decreased from unmeasurably high to 100-5000 Mf~. This is, however, approximately 100200 times greater than observed in whole-cell recording, resulting in slow capacitative currents in response to voltage steps, hence the name slow whole-cell recording. The large series resistance and the specificity of ATP for a selected range of cell types limits the use of slow whole-cell recording. Both of these problems were obviated by the introduction of the polyene antibiotic nystatin as the ionophore?4 This method is called perforated patch recording. Recently another polyene antibiotic, amphotericin B, has been introduced for perforated patch recordings? 5 The theory and methods for per-
11 M. Pusch and E. Neher, PfluegersArch. 411,204 (1988). 12B. D. Gomperts and J. M. Fernandez, Trends Biochem. Sci. 10, 414 (1985). 13M. Lindau and J. M. Fernandez, Nature (London) 319, 150 (1986). 14 R. Horn and A. Many, J. Gen. Physiol. 92, 145 (1988). 15 j. Rae, K. Cooper, P. Gates, and M. Watsky, J. Neurosci. Methods 37, 15 (1991).
[8]
INHIBITION OF RUNDOWN
153
iliiiiii!iii-lii!iiii~ii~ii~ii~i-~-~i!-~i~i~.:~!~iii~i~iiiT~L~--~-.-~-~i~-~i~:-~ii~iii~ii!i!~iii~ii!!~ii~ iiii~ii~iil¸ ~ i FIG. 1. Picoleak recording of fluorescence and membrane potential from an AtT-20 pituitary cell [see Ref. 17 and S. J. Korn, A. Bolden, and R. Horn, J. Physiol. (London) 439, 423 (1991) for technical details]. The cell was loaded with the membrane-permcant form of Fura-2. The Fluorescence (top) shows a continuous measurement at an excitation wavelength of 380 nm; at this wavelength a decrease (downward trend) in fluorescence indicates an increase in intracellular Ca 2+ concentration. The voltage (bottom), which was recorded from a cell-attached patch using a pipette filled with the normal Ringer's bath solution, shows a series of spontaneous long duration action potentials accompanied by a prolonged Ca 2+ influx. The membrane potential is an unknown fraction of that inside the cell, and it depends on the resistance of the seal and the membrane patch under the patch pipette.
forated patch recording have been described extensively, t4-17 We present only a brief overview here. In lipid bilayers and cell membranes nystatin and amphotericin B form channels that are selective to monovalent or uncharged molecules smaller than about 0.8 nm. Because these antibiotics cannot pass through a bilayer membrane, they do not enter the cell in perforated patch experiments. This is consistent with the longevity and high resistance of cells examined with this method. Recent experiments 18also suggest that nystatin is incapable of diffusing laterally through the plasma membrane from the perforated
~6S. J. Korn and R. Horn, J. Gen. PhysioL 94, 789 (1989). ~7 S. J. Korn, A. Marty, J. A. Connor, and R. Horn, in "Methods in Neuroseiences" (P. M. Conn. ed.), Vol. 4, p. 364. Academic Press, San Diego, California, 1991. ~s R. Horn, Biophys. J. 60, 329 (1991).
154
ELECTROPHYSIOLOGICAL TECHNIQUES
[8]
patch, under the glass-membrane seal, and into the surrounding plasma membrane. The series resistance achieved in perforated patch recordings is not much larger than observed for whole-cell recordings under similar conditions, and the polyene antibiotics are apparently promiscuous in their ability to form channels in a wide variety of membranes. A detailed description of the methods used for perforated patch recording may be found in Refs. 15 and 17. A few new technical details15 are worth mentioning: (1) the stock solution of antibiotic in dimethyl sulfoxide (DMSO) may lose its potency in the freezer and should be made up fresh every time the pipette solution is prepared, and (2) blunt-shaped pipettes are preferable to thin pipettes, presumably because of the membrane areas of patches. An abbreviated protocol follows. 1. Dissolve either nystatin or amphotericin B in DMSO (60 mg/ml). Add 20/xl of this stock to 5 ml of pipette solution and sonicate briefly ( --- 20 sec). This solution retains potency for around 2 hr. 2. Fill the electrode tip (e.g., by dipping it for a few seconds) with antibiotic-free solution. Increase the amount of this solution in the electrode tip only if seals are a problem. Backfill the rest of the pipette with the solution containing nystatin or amphotericin B. 3. Get the patch pipette sealed onto a cell as quickly as possible, to prevent the movement of the antibiotic to the pipette tip, where it may inhibit seals. 4. Set a holding potential and observe capacitative transients in response to 10-mV pulses. Usually a slow transient appears within a few minutes. The time course of this transient becomes increasingly rapid as the patch becomes perforated with antibiotic. The series resistance reaches steady state within about 30 min. The final value of series resistance depends on the size and shape of the pipette and on the amount of antitiotic-free solution in the tip. Values below 3 Mf~ have been reported for both nystatin and amphotericin B. It is not yet clear if either of these antibiotics has an advantage over the other, although it is possible that some membranes may be able to differentiate them. Perforated patch recording preserves many of the components of the cytoplasm, including buffers, enzymes, and multivalent ions, while permitting the exchange of small monovalent ions, such as K +, Na +, Cs +, and C1-. Initial reports indicate that the rundown of both ionic currents and second-messenger responses is reduced or eliminated. It is even possible to excise vesicles and study the modulation of single channels in the nonperforated membrane facing the bath. ~5,19 19 E. S. Levitan and R. K. Kramer, Nature (London) 348, 545 (1990).
[9]
15 5
LOOSE-SEAL VOLTAGE CLAMP
Improvements in perforated patch recording are possible. For example, it would be useful to have a battery of ionophores with different selectivities, so that it would be possible to choose the ingredients filtered by the pores. Many ionophores are presently available, of course, but few have all the advantages of nystatin and amphotericin B, namely, fixed pore size, inability to pass through the patch into the cell, high enough conductance (and/or density of channels incorporated into the membrane) to allow reasonable voltage clamp of whole-cell currents, and weak dependence of the conductance on transmembrane voltage. Conclusion We have evaluated several methods that have been used to prevent rundown in electrophysiological experiments. These techniques preserve, to varying extents, the integrity of the cytoplasm. Each has its own virtues and pitfalls, and it is unlikely that one method will suffice for all purposes. In our laboratory, for example, we use most of the above methods, depending on the particular experiment. Usually the choice of technique involves a compromise between the ability to determine the composition of the intracellular solution and the preservation of the cytoplasm. Acknowledgments We thank Dr. JamesRae for sendingus a preprintof his paper)5 20 S. J. Korn, A. Bolden, and R. Horn, J.
Physiol. (London) 439,
423 (1991).
[9] P a t c h V o l t a g e C l a m p i n g w i t h L o w - R e s i s t a n c e Loose Patch Clamp
Seals:
By WILLIAM M. ROBERTSand WOLFHARD ALMERS Introduction Patch voltage clamp methods employ smooth-tipped glass micropipettes to record electric currents across small regions of cell membrane. Unlike sharp pipettes, used to penetrate the lipid bilayer of the plasma membrane for intracellular recording, the smooth rim of a patch pipette nestles against the extracellular surface of the membrane, forming a seal that impedes the flow of electric current between the lumen of the pipette METHODS IN ENZYMOLOGY, VOL. 207
Copyright© 1992by AcademicPrem, Inc. All rightsof rL'productionin any form reserved.
INHIBITION OF RUNDOWN
[8] P r e v e n t i o n
149
o f R u n d o w n in E l e c t r o p h y s i o l o g i c a l Recording
By R I C H A R D
H O R N a n d STEPHEN J. K O R N
Introduction The invention of the gigaseal patch clamp technique I has led to stupendous strides in the study of electrophysiological phenomena. Of the several variants of this methodology, whole-cell recording is the most popular, largely because of the ease and speed of obtaining and analyzing macroscopic currents. Nonetheless, standard whole-cell recording suffers from several problems related to the need to break the membrane at the interface between the cytoplasm and the interior of the patch pipette. The exchange of material between the interior of the cell and the pipette solution results in the dilution, or "washout," of cytoplasmic constituents that may affect the functional properties of ion channels. Common examples of the deleterious consequences of the diluted cytoplasm include the decline (i.e., rundown) of calcium (Ca2+) channel function and the disappearance of receptor-activated second-messenger responses shortly after breaking into a cell. Other, perhaps more subtle, changes accompanying whole-cell recording include alterations in the activity of cytoplasmic buffers and enzymes. We shall discuss and evaluate several alternative methods that reduce or avoid the problems presented by whole-ceU recording. We do not, however, consider methods used for single-channel recording, which include the cytoplasm-preserving cell-attached patch configuration. Microelectrodes Patch clamp recording has largely supplanted the use of microelectrodes, especially in experiments with small mammalian cells that are difficult to penetrate benignly with a microelectrode. Microelectrode recording nonetheless retains several advantages, including (1) washout and dialysis of cytoplasm are minimized, (2) it is possible to impale cells that are inaccessible to a patch electrode, for example, cells deeply embedded in tissue; and (3) for large currents in very large cells, such as Xenopus oocytes, a two-microelectrode voltage clamp avoids the series resistance O. P. Hamill, A. Marry, E. Neher, B. Sakmann, and F. J. Sigworth, PluegersArch. 391, 85 (1981).
METHODS IN ENZYMOLOGY,VOL. 207
Copyright© 1992by AcademicPress,Inc. Allfightsofreproductionin any formreserved.
150
ELECTROPHYSIOLOGICAL TECHNIQUES
[8]
errors arising from passing current and recording voltage through the same electrode, as done in whole-cell recording. Disadvantages in the use of microelectrodes include (I) the electrical resistance between the cytoplasm and interior of the recording pipette is usually higher in microelectrode recording than in whole-cell recording. A high electrode resistance reduces the quality of the recording, especially in voltage clamp experiments; (2) microelectrodes are frequently more noxious than a patch pipette and may damage a cell2; and (3) there is some exchange between the pipette and cytoplasm. For example, microelectrodes filled with CsC1, instead of KC1, are effective in reducing potassium currents in impaled cells. Also, because large molecules (e.g., messenger RNA) may be pressure injected through microelectrodes, other substances may exchange at unknown rates between the cytoplasm and the electrode. Technical details for microelectrode recording have been described elsewhere:
Big Patches: Loose and Tight Cell-attached patch recording usually implies that cytoplasm is maintained on the intracellular surface of the plasma membrane. With this configuration one usually observes tingle-channel currents. However, if channel density is very high (e.g., see Ref:) or if very large pipettes are used, macroscopic currents may be observed in cell-attached patches. Presumably such currents are modulable by second messengers and do not wash out. One of the technical difficulties with large pipettes is that the probability of obtaining a gigohm seal is inversely related to pipette size. The current jargon thus differentiates macropatch recording (with a seal) and loose patch recording (without a seal). The seal improves the signal-tonoise ratio and increases the dynamic range of the recording amplifier. In certain experiments, however, loose patch recording may be preferable, for example, when multiple recordings are obtained from different locations on the same cell. Because the macroscopic currents through a large patch may be substantial, compared with the input impedance of the cell under study, it may be necessary to voltage clamp the cell with separate elec2 E. M. Fenwick, A. Marty, and E. Neher, J. Physiol. (London) 331, 557 (1982). 3 T. G. Smith, H. Lecar, S. J. Redman, and P. W. Gage, eds., "Voltage and Patch Clamping with Microelectrodes." American Physiological Society, Bethesda, Maryland, 1985. 4 j, Patlak and M. Oa'~, J. Gen. Physiol. 86, 89 (1985).
[8]
INHIBITION OF RUNDOWN
151
trodes, in order to maintain the membrane potential during current flow through the patch. (For further details see Refs. 5 and 6).
Whole-Cell Recording with S u p p l e m e n t e d Pipette Solutions The most direct method to prevent washout in whole-cell recording is to supplement the pipette solution with ingredients that will maintain the normal function of the ionic currents under study. This approach has been used successfully with Ca 2÷ currents, which may be maintained with a soup containing substances that inhibit proteases and support phosphorylation by cyclic AMP (e.g., ATP, Mg 2+, catalytic subunit of cAMP-dependent protein kinase, leupeptin, and Ca 2+ buffersT). This method may not, however, prevent the loss of hormone or transmitter responses, either because the essential ingredients are unknown or because they are added in the wrong concentrations. Furthermore, a second-messenger response to an extracellular stimulus involves transient changes in the concentration of intracellular substances; such transients are expected to be suppressed in whole-cell recording because the pipette solution tends to clamp cytoplasmic concentrations. The extent of suppression will depend on the time course of the transient and the rates of diffusion of relevant substances between the cell and the pipette. In one report a cytoplasmic extract, added to the pipette solution in whole-cell recording, inhibited the rundown of a hormone response in pituitary cells, s
Whole-Cell Recording with Small Pipettes The speed of washout depends on the resistance of a whole-cell pipette and the size of the cell? Therefore small pipettes have been favored in some experimentsJ ° The obvious penalty in such an approach is the concomitant increase in series resistance between the pipette and the cell
5W. M. Robertsand W. Almers,this volume[9]. 6W. Sttihmer,this volume[19]. 7j. Chad, D. Kalman,and D. Armstrong,in "CellCalciumand the Controlof Membrane Transport" (D. C. Eaton and L. J. Mandel, eds.), Vol. 42, p. 167. Societyof General PhysiologistsSeres, RockefellerUniv. Press, New York, 1987. 8B. Dufy,A. MacDermott,and J. L. Barker,Biochem. Biophys. Res. Commun. 137(1), 388 (1986). 9A. Manyand J. Zimmerberg,Cell. Signalling 1, 259 (1989). ~oL. O. Trusselland M. B. Jackson,J. Neurosci. 7, 3306 (1987).
152
ELECTROPHYSIOLOGICAL TECHNIQUES
[8]
interior. Another problem with this approach is that the exchange is slowed, but not prevented. This means that the concentrations of some cytoplasmic substances are changing slowly during the course of an experiment, and that the rate of exchange will depend on the molecular weight of each constituent.9,~ A final problem is that the series resistance often increases spontaneously when using small pipettes (i.e., they tend to "seal over").
Permeabilized Patch Recording The prevention of rundown in whole-cell recording clearly requires a diminution of the exchange of material between the recording pipette and the cytoplasm. The methods in this section rely on the plasma membrane itself as a dialysis membrane. Ordinarily the membrane under a patch electrode during cell-attached patch recording is far too resistive either to measure the membrane potential of a cell or to control the cell's voltage while recording current. Nonetheless we have occasionally measured a fraction of the membrane potential through a cell-attached patch during spontaneous action potentials (Fig. 1). Of course this method can only yield qualitative results. Peering through a patch into the cell was finally accomplished with the inclusion in the pipette solution of ionophores that permeabilized the patch, z2 Lindau and Fernandez 13used ATP, which induces a conductance on the external surface of mast cells. When placed into the patch pipette in the cell-attached configuration, the series resistance is decreased from unmeasurably high to 100-5000 Mf~. This is, however, approximately 100200 times greater than observed in whole-cell recording, resulting in slow capacitative currents in response to voltage steps, hence the name slow whole-cell recording. The large series resistance and the specificity of ATP for a selected range of cell types limits the use of slow whole-cell recording. Both of these problems were obviated by the introduction of the polyene antibiotic nystatin as the ionophore?4 This method is called perforated patch recording. Recently another polyene antibiotic, amphotericin B, has been introduced for perforated patch recordings? 5 The theory and methods for per-
11 M. Pusch and E. Neher, PfluegersArch. 411,204 (1988). 12B. D. Gomperts and J. M. Fernandez, Trends Biochem. Sci. 10, 414 (1985). 13M. Lindau and J. M. Fernandez, Nature (London) 319, 150 (1986). 14 R. Horn and A. Many, J. Gen. Physiol. 92, 145 (1988). 15 j. Rae, K. Cooper, P. Gates, and M. Watsky, J. Neurosci. Methods 37, 15 (1991).
[8]
INHIBITION OF RUNDOWN
153
iliiiiii!iii-lii!iiii~ii~ii~ii~i-~-~i!-~i~i~.:~!~iii~i~iiiT~L~--~-.-~-~i~-~i~:-~ii~iii~ii!i!~iii~ii!!~ii~ iiii~ii~iil¸ ~ i FIG. 1. Picoleak recording of fluorescence and membrane potential from an AtT-20 pituitary cell [see Ref. 17 and S. J. Korn, A. Bolden, and R. Horn, J. Physiol. (London) 439, 423 (1991) for technical details]. The cell was loaded with the membrane-permcant form of Fura-2. The Fluorescence (top) shows a continuous measurement at an excitation wavelength of 380 nm; at this wavelength a decrease (downward trend) in fluorescence indicates an increase in intracellular Ca 2+ concentration. The voltage (bottom), which was recorded from a cell-attached patch using a pipette filled with the normal Ringer's bath solution, shows a series of spontaneous long duration action potentials accompanied by a prolonged Ca 2+ influx. The membrane potential is an unknown fraction of that inside the cell, and it depends on the resistance of the seal and the membrane patch under the patch pipette.
forated patch recording have been described extensively, t4-17 We present only a brief overview here. In lipid bilayers and cell membranes nystatin and amphotericin B form channels that are selective to monovalent or uncharged molecules smaller than about 0.8 nm. Because these antibiotics cannot pass through a bilayer membrane, they do not enter the cell in perforated patch experiments. This is consistent with the longevity and high resistance of cells examined with this method. Recent experiments 18also suggest that nystatin is incapable of diffusing laterally through the plasma membrane from the perforated
~6S. J. Korn and R. Horn, J. Gen. PhysioL 94, 789 (1989). ~7 S. J. Korn, A. Marty, J. A. Connor, and R. Horn, in "Methods in Neuroseiences" (P. M. Conn. ed.), Vol. 4, p. 364. Academic Press, San Diego, California, 1991. ~s R. Horn, Biophys. J. 60, 329 (1991).
154
ELECTROPHYSIOLOGICAL TECHNIQUES
[8]
patch, under the glass-membrane seal, and into the surrounding plasma membrane. The series resistance achieved in perforated patch recordings is not much larger than observed for whole-cell recordings under similar conditions, and the polyene antibiotics are apparently promiscuous in their ability to form channels in a wide variety of membranes. A detailed description of the methods used for perforated patch recording may be found in Refs. 15 and 17. A few new technical details15 are worth mentioning: (1) the stock solution of antibiotic in dimethyl sulfoxide (DMSO) may lose its potency in the freezer and should be made up fresh every time the pipette solution is prepared, and (2) blunt-shaped pipettes are preferable to thin pipettes, presumably because of the membrane areas of patches. An abbreviated protocol follows. 1. Dissolve either nystatin or amphotericin B in DMSO (60 mg/ml). Add 20/xl of this stock to 5 ml of pipette solution and sonicate briefly ( --- 20 sec). This solution retains potency for around 2 hr. 2. Fill the electrode tip (e.g., by dipping it for a few seconds) with antibiotic-free solution. Increase the amount of this solution in the electrode tip only if seals are a problem. Backfill the rest of the pipette with the solution containing nystatin or amphotericin B. 3. Get the patch pipette sealed onto a cell as quickly as possible, to prevent the movement of the antibiotic to the pipette tip, where it may inhibit seals. 4. Set a holding potential and observe capacitative transients in response to 10-mV pulses. Usually a slow transient appears within a few minutes. The time course of this transient becomes increasingly rapid as the patch becomes perforated with antibiotic. The series resistance reaches steady state within about 30 min. The final value of series resistance depends on the size and shape of the pipette and on the amount of antitiotic-free solution in the tip. Values below 3 Mf~ have been reported for both nystatin and amphotericin B. It is not yet clear if either of these antibiotics has an advantage over the other, although it is possible that some membranes may be able to differentiate them. Perforated patch recording preserves many of the components of the cytoplasm, including buffers, enzymes, and multivalent ions, while permitting the exchange of small monovalent ions, such as K +, Na +, Cs +, and C1-. Initial reports indicate that the rundown of both ionic currents and second-messenger responses is reduced or eliminated. It is even possible to excise vesicles and study the modulation of single channels in the nonperforated membrane facing the bath. ~5,19 19 E. S. Levitan and R. K. Kramer, Nature (London) 348, 545 (1990).
[9]
15 5
LOOSE-SEAL VOLTAGE CLAMP
Improvements in perforated patch recording are possible. For example, it would be useful to have a battery of ionophores with different selectivities, so that it would be possible to choose the ingredients filtered by the pores. Many ionophores are presently available, of course, but few have all the advantages of nystatin and amphotericin B, namely, fixed pore size, inability to pass through the patch into the cell, high enough conductance (and/or density of channels incorporated into the membrane) to allow reasonable voltage clamp of whole-cell currents, and weak dependence of the conductance on transmembrane voltage. Conclusion We have evaluated several methods that have been used to prevent rundown in electrophysiological experiments. These techniques preserve, to varying extents, the integrity of the cytoplasm. Each has its own virtues and pitfalls, and it is unlikely that one method will suffice for all purposes. In our laboratory, for example, we use most of the above methods, depending on the particular experiment. Usually the choice of technique involves a compromise between the ability to determine the composition of the intracellular solution and the preservation of the cytoplasm. Acknowledgments We thank Dr. JamesRae for sendingus a preprintof his paper)5 20 S. J. Korn, A. Bolden, and R. Horn, J.
Physiol. (London) 439,
423 (1991).
[9] P a t c h V o l t a g e C l a m p i n g w i t h L o w - R e s i s t a n c e Loose Patch Clamp
Seals:
By WILLIAM M. ROBERTSand WOLFHARD ALMERS Introduction Patch voltage clamp methods employ smooth-tipped glass micropipettes to record electric currents across small regions of cell membrane. Unlike sharp pipettes, used to penetrate the lipid bilayer of the plasma membrane for intracellular recording, the smooth rim of a patch pipette nestles against the extracellular surface of the membrane, forming a seal that impedes the flow of electric current between the lumen of the pipette METHODS IN ENZYMOLOGY, VOL. 207
Copyright© 1992by AcademicPrem, Inc. All rightsof rL'productionin any form reserved.