[46]
PATCH CLAMP TECHNIQUES TO STUDY ORGANELLES
673
[46] P a t c h C l a m p T e c h n i q u e s t o S t u d y I o n C h a n n e l s from OrganeUes
By BERNHARDU. KELLERand RAI~ER HEDRICH Introduction Ionic transport across intracellular membranes is thought to be involved in a variety of cellular responses, such as synaptic transmission, stimulus-secretion coupling, or muscle contraction. To study ionic processes involved in the various aspects ofintracellular signal transduction, it is desirable to utilize the molecular resolution provided by the patch clamp technique. Until recently, however, it has been a considerable problem to investigate ionic signals across intracellular membranes on the basis of single channels, mainly because organeUes are too small and difficult to access within the living cell. Here, several recently developed strategies are presented to isolate intracellular membranes and yield organelles large enough for patch clamp experiments. In this chapter, we focus on the methods utilized for patch clamping membranes of mitochondria, endoplasmic reticulum, and plant vacuoles. In addition, the potential application of these techniques for other intracellular membranes is discussed. Osmotic Swelling Techniques: Mitochondria and Chloroplasts
Patch Clamp of Mitochondrial Membranes The most important function of mitochondria is to supply the cell with adenosine triphosphate (ATP). ATP synthesis is driven by the protonmorive force, which is maintained across the inner mitochondrial membrane by the activity of the respiratory chain. It was thought to be unlikely that the inner mitochondrial membrane would contain ion channels like those present in the plasma membrane, because the high rates of ion transport characteristic of open channels would dissipate the protonmotive force. By patch damping the inner mitochondrial membrane, it is possible to test this concept directly and, moreover, investigate in more general terms the molecular mechanisms of ion transport across the inner and outer mitochondrial membranes. To patch clamp the mitochondrial membranes, two problems need to be solved. First, the mitochondria have to be enlarged to reach a suitable size, and second, the outer and inner mitochondrial membranes have to be separated. Both problems can be solved by osmotic swelling of mitochonMETHODS IN ENZYMOLOGY, VOL. 207
~ t © 1992 by Ae~lemie Pre~ Inc. All rights of reproduefioa in any form reserved.
674
CHANNEL RECORDING IN ORGANELLES A N D MICROBES
[46] J
....i ~ i ( i ~ ~i~i~i~ ~
~i~i~!~!~ ~i¸
~'~/~ii~!~i?~~i:,!~i~ ~ , ~ ~
i
~, ~ , ~ ~ii~i~iii~i~~iiiL~iiiii~/i
/
Fro. 1. Micrographs of intracellular membranes utilized for patch clamp measurements. (A) Electron micrograph of a liver cell showing the different intracellular membranes (ER, endoplasmic reticulum; N, nucleus; M, mitochondria). (B) Patch clamped vacuole isolated from sugar beet as described by R. Hedrich and E. Neher, Nature (London) 329, 833 (1987). (C) Giant vesicles obtained by a dehydration/rehydration cycle, forming at the edge of a lipid film. (D) Giant vesicle, obtained with the hydration technique, of approximately 30#m diameter in the attached-patch conliguration. (E) Parent mitochondria as obtained after isolation from liver cells. Bar, 10 gin. (F) Swollen mitoplasts obtained after osmotic swelling of parent mitochondria. Note the residual fraction of outer mitochondrial membrane at the "cap" region of a mitoplast (arrow). Bar, l0 gin.
drial membranes. The first experiments to patch clamp the inner membrane of mitochondria were performed on mitochondria obtained from liver cells of mice fed with cuprizone, a compound which is known to induce the formation of larger liver mitochondria. In a later series of experiments, however, cuprizone was found to be unnecessary for successful experiments, mainly because osmotic swelling itself yielded mitochondria which were large enough for patch clamp measurements.l-3 In a typical experiment, mitochondria were isolated from liver cells by standard centrifugation procedures as described by Sorgato et al. 1 and references therein. Before swelling, isolated mitochondria (Fig. 1E) were loaded with a solution containing 150 mM KC1, 20 mM HEPES- KOH at pH 7.2. Osmotic shock was performed by exposing mitochondria to a 5- to 10-fold KC1 gradient for 2 - 5 rain. After that, inner mitochondrial memM. C. Sorgato, B. U. Keller, and W. StOhmer, Nature (London) 330, 498 (1987). 2 M. C. Sorgato, O. Moran, V. De Pinto, B. U. Keller, and W. Stfihmer, £ Bioenerg. Biomembr. 21, 485 (1989). a W. Stfihmer, B. U. Keller, G. Lippe, and M. C. Sorgato, in "Hormones and Cell Regulation" (J. Nunes, J. E. Dumont, and E. Carafoli, eds.), Vol. 165, p. 89. Libbey Eurotext, Pads, 1988.
[46]
PATCH CLAMP TECHNIQUES TO STUDY ORGANELLES
675
branes had usually unfolded, yielding large vesicles (mitoplasts) of 3- 6 #m in diameter. Moreover, fractions of outer mitochondrial membranes were visible as dark "cap" regions on top of the swollen mitoplasts (Fig. IF, arrow). For patch clamp experiments, aliquots containing 5-10 #1 of mitochondria or mitoplasts were layered on the glass bottom of the recording chamber and then diluted with 200- 300/zl of bath solution, which usually contained 150 m M KCI, 0.1 m M CaC12, 20 m M HEPES- KOH at pH 7.2. After allowing the mitoplasts to adhere to the bottom of the chamber for a minimum of 15 rain, the chamber was mounted on the patch clamp setup and extensively perfused with the bath solution. The pipette was usually filled with the same solution as in the bath. Usually, pipettes with resistances of 5-18 Mf~ were used for patch clamp experiments. Seals with resistances greater than 10 Gf~ formed relatively easily, provided the chamber had been extensively perfused. In such recordings, voltage-dependent ion channels could be identified in the inner mitochondrial membrane (IMM). At membrane voltages negative to - 20 mV (mitoplast inside negative), IMM channels were completely closed (Fig. 2A). For depolarizations positive to 0 mV, continuous openings and closings of single channel molecules could be observed (Fig. 2B). From reversal potential measurements, IMM channels were found to be anion selective, with a conductance of 107 pS in 150 m M KCI. Since the first findings of these channels in the inner mitochondrial membrane, several other electrophysiological studies have confirmed the existence of such entities.4,5 Although the physiological role of this channel in vivo is not completely understood, it may represent a suitable pathway for the transport of small molecules or metabolites into the mitochondria.6
Patch Clamp of Photosynthetic Membranes of Plants Bulychev et al. 7 reported small membrane potentials (10- 15 mV relative to the cytoplasm) across the chloroplast membranes when giant chloroplasts from leaves of Peperomia metallica were impaled by microelectrodes. Because the magnitude of these potentials depended on fight, the tip of the electrode was likely inside the thylakoid. Measurements of pH demonstrated that photosynthetic electron transport of the thylakoid generated a proton gradient which in turn drove the ATP synthase. Surpris4 M. Thieffry, J. Chich, D. Goldschmidt, and J. Henry, EMBO J. 7, 1449 (1988). s K. W. Kinnally, M. L. Campo, and H. Tedeschi, J. Bioenerg, Biomembr. 21, 497 (1989). 6 S. J. Singer, P. A. Maher, and M. P. Yalfe, Proc. Natl. Acad. Sci. U.S.A. 84, 1015 (1987). 7 A. A. Bulychev, V. K. Andrianov, G. A. KureUa, and F. F. Litvin, Biochim. Biophys. Acta 420, 336 (1976).
676
CHANNEL R E C O R D I N G
IN O R G A N E L L E S
IlnAl
A
AND
C
1,0
[46]
MICROBES
-
-
~
.8O 0 mV -20
5 nA
O2
=
6O
VlmVl
80
V{mV)
B
D -7/.
o c .
.
.
.
.
". . . . .
---
I° . . . . . . . . . . .
: -
-
t:u
IlOpA
lOOms I
:
0
-3o
Is°A , 250ms
I
FIG. 2. (A) Whole-mitoplastcurrent I flowingthrough the mitoplast membrane at different applied voltages V. Note the increase in membrane current for applied voltages more positive than 0 inV. (B) Openings and closingsof a single ion channel in the inner mitochondrlal membrane in the attached-patch configuration. The applied voltage is indicated in millivolts at fight. Depolarizing voltages evoked voltage-dependent ion channels with a conductance of 107 pS. Note the close correlation between single channel openings and the increase in whole-mitoplast currents shown in (A). (C) Whole-vacuolecurrents in response to a series of voltage steps from a holding potential with the vacuoleexposed to 10--4MCa 2+ on the cytoplasmic side of the membrane. (D) Single channel openings and closings recorded from an outside-out patch excised form the vacuole shown in (C) using the same voltage protocol. Hyperpolarizing voltages evoked voltage-dependent ion channels with a conductance of 60 pS. The slow activation and voltage dependence of the 60 pS channel reflect the macroscopic currents shown in (C).
ingly, the m e m b r a n e potential only transiently exceeded 20 mY. Thus, it was proposed that fluxes o f counterion across the thylakoid such as chloride a n d magnesium m a y have short-circuited photosynthetic H + transport.
To patch clamp the photosynthetic membrane, leafslicesof Peperomia metallica were incubated in 2% cellulase O n o z u k a R-10 and 1% Mazerozyme R-10 (Yakalt Honsha, Tokyo, Japan), 0.5% bovine serum albumin (BSA), 0.27 M sorbitol, 1 m M CaC12 for about 1 hr. Resulting protoplasts
[46]
PATCH CLAMP TECHNIQUES TO STUDY ORGANELLES
677
were washed with 0.35 M sorbitol, 1 m M CaC12, and separated from debris by filtration through 200- and 25-#m nylon nets followed by centrifugation (7 min at 100 g). The protoplasts were suspended in wash medium, stored on ice, and used within 4 hr. Protoplast suspensions (5-10 #1) were rapidly mixed with a 100-fold volume of bath solution (20 m M KC1, 5 m M MgC12, 2 m M MOPS [3-(N-morpholino)propanesulfonic acid)]-KOH, pH 6.9) in the recording chamber. After 15 rain of osmotic swelling in the dark, plasma membranes, vacuoles, and thylakoid envelopes had ruptured and thylakoids formed large blebs. The chamber was perfused with bath solution, and blebs adhering to the bottom of the chamber were used for patch-clamp studies. Experiments were performed with 20 m M KC1, 5 m M MgC12, 2 m M MOPS-KOH, pH 6.9, inside the pipette. By applying this protocol to Peperomia metallica chloroplasts, Sch6nknecht et al. 8 gained access to the thylakoid membrane with patch pipettes. In such experiments, voltage-dependent chloride-selective channels with a conductance of 80-100 pS in 100 m M KCI were identified. As has been discussed in the literature, these anion channels may be essential to balance the transthylakoid potential and to establish a pH gradient across the thylakoid membrane.7 Hydration Technique: Intracellular Membranes Intracellular membranes like the endoplasmic reticulum (ER) are thought to be the central store for intraceUular Ca2+, which is released in response to intracellular second messengers. Also, the modulation of calcium release by monovalent ion concentrations has suggested the existence of transport pathways for monovalent cations and anions in ER membranes. 9 These transport pathways can be investigated by using a fusion strategy based on a dehydration/rehydration cycle t° of isolated membrane preparations. In a typical experiment, a lysed membrane preparation was isolated by standard centrifugation techniques for endoplasmic or sarcoplasmic reticulum membranes." Isolated membranes were suspended in 5/tl fusion buffer containing 10 m M MOPS adjusted to pH 7.4. To protect the protein maximally against damage by water loss, an additional 10/~l of fusion
s G. Sch6nknecht, R. Hedrich, W. Junge, and K. Raschke, Nature (London) 336, 589 (1988). 9 S. K. Joseph and J. R. Williamson, J. BioL Chem. 261, 14658 (1986). zo B. U. Keller, R. Hedrich, W. L. C. Vaz, and M. Criado, PfluegersArch 411, 94 (1988). it M. Criado and B. U. Keller, FEBS Lett. 224, 172 (1987).
678
CHANNEL RECORDING IN ORGANELLES AND MICROBES
[46]
buffer containing 10% ethylene glycol v/v was added to the suspension. Subsequently, membranes were deposited on a glass slide to form a circle about 8 mm in diameter. The membrane-containing drop was dehydrated at 4 ° in a desiccator containing CaC12 granules. After 3 hr, a partially dehydrated lipid film could be observed, which was subsequently hydrated by covering it with 20#1 of 100 m M KCI. Complete rehydration of the lipid film was performed by incubating it for several hours at 4 ° in a closed petri dish. A wet filter paper pad was placed under the glass slide to ensure full rehydration. Usually, cell size vesicles could be observed after 2 - 3 hr at the edge of the lipid film (Fig. IC). To either reduce the density of ion channels or facilitate the fusion process, it was often desirable to dilute the isolated membranes by exogenous lipids. In this case, crude L-a-lecithin from soybean was obtained from Sigma (St. Louis, MO; type II-S) and suspended in water using a Branson sonifier at 40 W for 5 rain. The stock solution was prepared with 100 mg/ml lecithin. For the formation of lipid vesicles, 10 mg/ml lecithin was dissolved in 1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonic acid), 100 m M NaC1, and 20 m M MOPS adjusted to pH 7.4. Subsequently, dialysis was performed for 24 and 48 hr against 500 volumes of dialysis buffer containing 100 m M NaCI and 20 m M Tris-C1 adjusted to pH 7.4. The resulting small lipid vesicles were stored at - 8 0 ° until use. In a typical experiment, isolated membranes were diluted with lipid vesicles at the desired concentration and centrifuged for 40 min at 15,000 rpm. After adding the fusion buffer, giant vesicles of diluted membranes were performed as previously described. Figure 1C,D display giant vesicles formed by fusing ER membranes using the hydration technique. Vesicles up to 100 g m in diameter could be readily observed at the edge of the dehydrated film. For patch clamp experiments, a few microliters of fused vesicles were removed with a pipette and diluted in 300 gl filtered buffer solution. Usually, the buffer contained 50 m M KC1, 0.1 m M CaC12, and 5 m M HEPES-KOH adjusted to pH 7.2. Large vesicles 10-20/~m in diameter were preferably used for patch clamp experiments. Before starting electrophysiological experiments, vesicles were allowed to settle firmly on the glass surface of the recording chamber. Single-channel recordings were carded out using standard patch clamp equipment) 2 Channel-free membrane patches usually displayed ohmic resistances in the range Rp > 10 G~. In a typical ER experiment, more than 40% of all patches showed steplike current activities of one or several ion channels. ~20. P. Hamill, A. Malty, E. Neher, B. Sakmann, and F. J. Sigworth, Pfluegers Arch. 391, 85 (1981).
[46]
PATCH CLAMP TECHNIQUES TO STUDY ORGANELLES
679
Mechanical Isolation of Large Organelles: Plant Vacuoles
Patch Clamp of Vacuolar Membrane Mature plant cells are characterized by the presence of large central vacuoles. The storage of solutes in vacuoles and their subsequent release are important in cell metabolism and play a fundamental role in the balance of the osmotic pressure and the control of the electrical potential difference across the vacuolar membrane. The intracellular location of this organelle has, until recently, complicated the study of the electrical properties of the vacuolar membrane from higher plant cells by standard electrophysiological techniques. Improvement of methods for the isolation of stable vacuoles and the application of the patch clamp technique made possible much more incisive studies on the electrophysiology of the vacuolar membrane. The fastest method for the isolation of small numbers of intact vacuoles directly from intact tissue is the one described by Coyaud et al. 13. In short, the surface of a freshly cut tissue slice is rinsed with buffer solution14 to wash the liberated vacuoles directly into the recording chamber. Thus, fresh vacuoles can be isolated for each experiment and vacuole isolation and seal formation can be performed within 2 - 5 min. The whole-cell configuration of the patch clamp technique was established after patch pipettes were sealed against an isolated vacuole, and the underlying membrane was broken by alternate _+0.6 V pulses 1 - 3 msec in duration. After access to the lumen of the vacuoles was gained by patch pipettes, the pipette solution equilibrated with the vacuolar sap. Using solutions with symmetric ion compositions on both sides of the membrane, steady-state conditions were indicated by a resting potential of 0 mV (which was reached within 1 -5 rain for a 20-pF vacuole. Vacuoles were exposed to solutions containing either 200 m M KC1 or KNO3. Both bathing media included 5 m M MgC12,0 or 0.1 m M CaC12, and 5 m M Tris- MES (4-morpholinoethanesulfonic acid) or citrate - KOH buffered to pH 7.5. The vacuole was equilibrated with either 200 m M KC1 including 5 m M MgC12, 1 mMCaC12, and 5 m M MES-Tris, pH 7.5 and 5.5, or citrate-KOH, pH 3.5 and 4.5. A patch clamp survey of the electrical properties of the vacuolar membrane from a large variety of plant materials has demonstrated the presence of voltage-dependent ion channels and electrogenic pumps as general features of ion transport in higher plant vacuoles. t3 L. Coyaud, A. Kurkdjian, R. Kado, and R. Hedrich, Biochim. Biophys. Acta 902, 263 (1987). 14 R. Hedrieh and E. Neher, Nature (London), 329, 833 (1987).
680
CHANNEL RECORDING IN ORGANELLES AND MICROBES
[46]
At high cytoplasmic Ca 2+ (>0.3 pM), the ionic conductance of the vacuolar membrane was found to be entirely accounted for by currents directed into the vacuole. These currents are activated at negative voltages (negative inside the vacuole), as well as at slightly positive potentials (
PATCH CLAMP TECHNIQUES TO STUDY ORGANELLES
673
[46] P a t c h C l a m p T e c h n i q u e s t o S t u d y I o n C h a n n e l s from OrganeUes
By BERNHARDU. KELLERand RAI~ER HEDRICH Introduction Ionic transport across intracellular membranes is thought to be involved in a variety of cellular responses, such as synaptic transmission, stimulus-secretion coupling, or muscle contraction. To study ionic processes involved in the various aspects ofintracellular signal transduction, it is desirable to utilize the molecular resolution provided by the patch clamp technique. Until recently, however, it has been a considerable problem to investigate ionic signals across intracellular membranes on the basis of single channels, mainly because organeUes are too small and difficult to access within the living cell. Here, several recently developed strategies are presented to isolate intracellular membranes and yield organelles large enough for patch clamp experiments. In this chapter, we focus on the methods utilized for patch clamping membranes of mitochondria, endoplasmic reticulum, and plant vacuoles. In addition, the potential application of these techniques for other intracellular membranes is discussed. Osmotic Swelling Techniques: Mitochondria and Chloroplasts
Patch Clamp of Mitochondrial Membranes The most important function of mitochondria is to supply the cell with adenosine triphosphate (ATP). ATP synthesis is driven by the protonmorive force, which is maintained across the inner mitochondrial membrane by the activity of the respiratory chain. It was thought to be unlikely that the inner mitochondrial membrane would contain ion channels like those present in the plasma membrane, because the high rates of ion transport characteristic of open channels would dissipate the protonmotive force. By patch damping the inner mitochondrial membrane, it is possible to test this concept directly and, moreover, investigate in more general terms the molecular mechanisms of ion transport across the inner and outer mitochondrial membranes. To patch clamp the mitochondrial membranes, two problems need to be solved. First, the mitochondria have to be enlarged to reach a suitable size, and second, the outer and inner mitochondrial membranes have to be separated. Both problems can be solved by osmotic swelling of mitochonMETHODS IN ENZYMOLOGY, VOL. 207
~ t © 1992 by Ae~lemie Pre~ Inc. All rights of reproduefioa in any form reserved.
674
CHANNEL RECORDING IN ORGANELLES A N D MICROBES
[46] J
....i ~ i ( i ~ ~i~i~i~ ~
~i~i~!~!~ ~i¸
~'~/~ii~!~i?~~i:,!~i~ ~ , ~ ~
i
~, ~ , ~ ~ii~i~iii~i~~iiiL~iiiii~/i
/
Fro. 1. Micrographs of intracellular membranes utilized for patch clamp measurements. (A) Electron micrograph of a liver cell showing the different intracellular membranes (ER, endoplasmic reticulum; N, nucleus; M, mitochondria). (B) Patch clamped vacuole isolated from sugar beet as described by R. Hedrich and E. Neher, Nature (London) 329, 833 (1987). (C) Giant vesicles obtained by a dehydration/rehydration cycle, forming at the edge of a lipid film. (D) Giant vesicle, obtained with the hydration technique, of approximately 30#m diameter in the attached-patch conliguration. (E) Parent mitochondria as obtained after isolation from liver cells. Bar, 10 gin. (F) Swollen mitoplasts obtained after osmotic swelling of parent mitochondria. Note the residual fraction of outer mitochondrial membrane at the "cap" region of a mitoplast (arrow). Bar, l0 gin.
drial membranes. The first experiments to patch clamp the inner membrane of mitochondria were performed on mitochondria obtained from liver cells of mice fed with cuprizone, a compound which is known to induce the formation of larger liver mitochondria. In a later series of experiments, however, cuprizone was found to be unnecessary for successful experiments, mainly because osmotic swelling itself yielded mitochondria which were large enough for patch clamp measurements.l-3 In a typical experiment, mitochondria were isolated from liver cells by standard centrifugation procedures as described by Sorgato et al. 1 and references therein. Before swelling, isolated mitochondria (Fig. 1E) were loaded with a solution containing 150 mM KC1, 20 mM HEPES- KOH at pH 7.2. Osmotic shock was performed by exposing mitochondria to a 5- to 10-fold KC1 gradient for 2 - 5 rain. After that, inner mitochondrial memM. C. Sorgato, B. U. Keller, and W. StOhmer, Nature (London) 330, 498 (1987). 2 M. C. Sorgato, O. Moran, V. De Pinto, B. U. Keller, and W. Stfihmer, £ Bioenerg. Biomembr. 21, 485 (1989). a W. Stfihmer, B. U. Keller, G. Lippe, and M. C. Sorgato, in "Hormones and Cell Regulation" (J. Nunes, J. E. Dumont, and E. Carafoli, eds.), Vol. 165, p. 89. Libbey Eurotext, Pads, 1988.
[46]
PATCH CLAMP TECHNIQUES TO STUDY ORGANELLES
675
branes had usually unfolded, yielding large vesicles (mitoplasts) of 3- 6 #m in diameter. Moreover, fractions of outer mitochondrial membranes were visible as dark "cap" regions on top of the swollen mitoplasts (Fig. IF, arrow). For patch clamp experiments, aliquots containing 5-10 #1 of mitochondria or mitoplasts were layered on the glass bottom of the recording chamber and then diluted with 200- 300/zl of bath solution, which usually contained 150 m M KCI, 0.1 m M CaC12, 20 m M HEPES- KOH at pH 7.2. After allowing the mitoplasts to adhere to the bottom of the chamber for a minimum of 15 rain, the chamber was mounted on the patch clamp setup and extensively perfused with the bath solution. The pipette was usually filled with the same solution as in the bath. Usually, pipettes with resistances of 5-18 Mf~ were used for patch clamp experiments. Seals with resistances greater than 10 Gf~ formed relatively easily, provided the chamber had been extensively perfused. In such recordings, voltage-dependent ion channels could be identified in the inner mitochondrial membrane (IMM). At membrane voltages negative to - 20 mV (mitoplast inside negative), IMM channels were completely closed (Fig. 2A). For depolarizations positive to 0 mV, continuous openings and closings of single channel molecules could be observed (Fig. 2B). From reversal potential measurements, IMM channels were found to be anion selective, with a conductance of 107 pS in 150 m M KCI. Since the first findings of these channels in the inner mitochondrial membrane, several other electrophysiological studies have confirmed the existence of such entities.4,5 Although the physiological role of this channel in vivo is not completely understood, it may represent a suitable pathway for the transport of small molecules or metabolites into the mitochondria.6
Patch Clamp of Photosynthetic Membranes of Plants Bulychev et al. 7 reported small membrane potentials (10- 15 mV relative to the cytoplasm) across the chloroplast membranes when giant chloroplasts from leaves of Peperomia metallica were impaled by microelectrodes. Because the magnitude of these potentials depended on fight, the tip of the electrode was likely inside the thylakoid. Measurements of pH demonstrated that photosynthetic electron transport of the thylakoid generated a proton gradient which in turn drove the ATP synthase. Surpris4 M. Thieffry, J. Chich, D. Goldschmidt, and J. Henry, EMBO J. 7, 1449 (1988). s K. W. Kinnally, M. L. Campo, and H. Tedeschi, J. Bioenerg, Biomembr. 21, 497 (1989). 6 S. J. Singer, P. A. Maher, and M. P. Yalfe, Proc. Natl. Acad. Sci. U.S.A. 84, 1015 (1987). 7 A. A. Bulychev, V. K. Andrianov, G. A. KureUa, and F. F. Litvin, Biochim. Biophys. Acta 420, 336 (1976).
676
CHANNEL R E C O R D I N G
IN O R G A N E L L E S
IlnAl
A
AND
C
1,0
[46]
MICROBES
-
-
~
.8O 0 mV -20
5 nA
O2
=
6O
VlmVl
80
V{mV)
B
D -7/.
o c .
.
.
.
.
". . . . .
---
I° . . . . . . . . . . .
: -
-
t:u
IlOpA
lOOms I
:
0
-3o
Is°A , 250ms
I
FIG. 2. (A) Whole-mitoplastcurrent I flowingthrough the mitoplast membrane at different applied voltages V. Note the increase in membrane current for applied voltages more positive than 0 inV. (B) Openings and closingsof a single ion channel in the inner mitochondrlal membrane in the attached-patch configuration. The applied voltage is indicated in millivolts at fight. Depolarizing voltages evoked voltage-dependent ion channels with a conductance of 107 pS. Note the close correlation between single channel openings and the increase in whole-mitoplast currents shown in (A). (C) Whole-vacuolecurrents in response to a series of voltage steps from a holding potential with the vacuoleexposed to 10--4MCa 2+ on the cytoplasmic side of the membrane. (D) Single channel openings and closings recorded from an outside-out patch excised form the vacuole shown in (C) using the same voltage protocol. Hyperpolarizing voltages evoked voltage-dependent ion channels with a conductance of 60 pS. The slow activation and voltage dependence of the 60 pS channel reflect the macroscopic currents shown in (C).
ingly, the m e m b r a n e potential only transiently exceeded 20 mY. Thus, it was proposed that fluxes o f counterion across the thylakoid such as chloride a n d magnesium m a y have short-circuited photosynthetic H + transport.
To patch clamp the photosynthetic membrane, leafslicesof Peperomia metallica were incubated in 2% cellulase O n o z u k a R-10 and 1% Mazerozyme R-10 (Yakalt Honsha, Tokyo, Japan), 0.5% bovine serum albumin (BSA), 0.27 M sorbitol, 1 m M CaC12 for about 1 hr. Resulting protoplasts
[46]
PATCH CLAMP TECHNIQUES TO STUDY ORGANELLES
677
were washed with 0.35 M sorbitol, 1 m M CaC12, and separated from debris by filtration through 200- and 25-#m nylon nets followed by centrifugation (7 min at 100 g). The protoplasts were suspended in wash medium, stored on ice, and used within 4 hr. Protoplast suspensions (5-10 #1) were rapidly mixed with a 100-fold volume of bath solution (20 m M KC1, 5 m M MgC12, 2 m M MOPS [3-(N-morpholino)propanesulfonic acid)]-KOH, pH 6.9) in the recording chamber. After 15 rain of osmotic swelling in the dark, plasma membranes, vacuoles, and thylakoid envelopes had ruptured and thylakoids formed large blebs. The chamber was perfused with bath solution, and blebs adhering to the bottom of the chamber were used for patch-clamp studies. Experiments were performed with 20 m M KC1, 5 m M MgC12, 2 m M MOPS-KOH, pH 6.9, inside the pipette. By applying this protocol to Peperomia metallica chloroplasts, Sch6nknecht et al. 8 gained access to the thylakoid membrane with patch pipettes. In such experiments, voltage-dependent chloride-selective channels with a conductance of 80-100 pS in 100 m M KCI were identified. As has been discussed in the literature, these anion channels may be essential to balance the transthylakoid potential and to establish a pH gradient across the thylakoid membrane.7 Hydration Technique: Intracellular Membranes Intracellular membranes like the endoplasmic reticulum (ER) are thought to be the central store for intraceUular Ca2+, which is released in response to intracellular second messengers. Also, the modulation of calcium release by monovalent ion concentrations has suggested the existence of transport pathways for monovalent cations and anions in ER membranes. 9 These transport pathways can be investigated by using a fusion strategy based on a dehydration/rehydration cycle t° of isolated membrane preparations. In a typical experiment, a lysed membrane preparation was isolated by standard centrifugation techniques for endoplasmic or sarcoplasmic reticulum membranes." Isolated membranes were suspended in 5/tl fusion buffer containing 10 m M MOPS adjusted to pH 7.4. To protect the protein maximally against damage by water loss, an additional 10/~l of fusion
s G. Sch6nknecht, R. Hedrich, W. Junge, and K. Raschke, Nature (London) 336, 589 (1988). 9 S. K. Joseph and J. R. Williamson, J. BioL Chem. 261, 14658 (1986). zo B. U. Keller, R. Hedrich, W. L. C. Vaz, and M. Criado, PfluegersArch 411, 94 (1988). it M. Criado and B. U. Keller, FEBS Lett. 224, 172 (1987).
678
CHANNEL RECORDING IN ORGANELLES AND MICROBES
[46]
buffer containing 10% ethylene glycol v/v was added to the suspension. Subsequently, membranes were deposited on a glass slide to form a circle about 8 mm in diameter. The membrane-containing drop was dehydrated at 4 ° in a desiccator containing CaC12 granules. After 3 hr, a partially dehydrated lipid film could be observed, which was subsequently hydrated by covering it with 20#1 of 100 m M KCI. Complete rehydration of the lipid film was performed by incubating it for several hours at 4 ° in a closed petri dish. A wet filter paper pad was placed under the glass slide to ensure full rehydration. Usually, cell size vesicles could be observed after 2 - 3 hr at the edge of the lipid film (Fig. IC). To either reduce the density of ion channels or facilitate the fusion process, it was often desirable to dilute the isolated membranes by exogenous lipids. In this case, crude L-a-lecithin from soybean was obtained from Sigma (St. Louis, MO; type II-S) and suspended in water using a Branson sonifier at 40 W for 5 rain. The stock solution was prepared with 100 mg/ml lecithin. For the formation of lipid vesicles, 10 mg/ml lecithin was dissolved in 1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonic acid), 100 m M NaC1, and 20 m M MOPS adjusted to pH 7.4. Subsequently, dialysis was performed for 24 and 48 hr against 500 volumes of dialysis buffer containing 100 m M NaCI and 20 m M Tris-C1 adjusted to pH 7.4. The resulting small lipid vesicles were stored at - 8 0 ° until use. In a typical experiment, isolated membranes were diluted with lipid vesicles at the desired concentration and centrifuged for 40 min at 15,000 rpm. After adding the fusion buffer, giant vesicles of diluted membranes were performed as previously described. Figure 1C,D display giant vesicles formed by fusing ER membranes using the hydration technique. Vesicles up to 100 g m in diameter could be readily observed at the edge of the dehydrated film. For patch clamp experiments, a few microliters of fused vesicles were removed with a pipette and diluted in 300 gl filtered buffer solution. Usually, the buffer contained 50 m M KC1, 0.1 m M CaC12, and 5 m M HEPES-KOH adjusted to pH 7.2. Large vesicles 10-20/~m in diameter were preferably used for patch clamp experiments. Before starting electrophysiological experiments, vesicles were allowed to settle firmly on the glass surface of the recording chamber. Single-channel recordings were carded out using standard patch clamp equipment) 2 Channel-free membrane patches usually displayed ohmic resistances in the range Rp > 10 G~. In a typical ER experiment, more than 40% of all patches showed steplike current activities of one or several ion channels. ~20. P. Hamill, A. Malty, E. Neher, B. Sakmann, and F. J. Sigworth, Pfluegers Arch. 391, 85 (1981).
[46]
PATCH CLAMP TECHNIQUES TO STUDY ORGANELLES
679
Mechanical Isolation of Large Organelles: Plant Vacuoles
Patch Clamp of Vacuolar Membrane Mature plant cells are characterized by the presence of large central vacuoles. The storage of solutes in vacuoles and their subsequent release are important in cell metabolism and play a fundamental role in the balance of the osmotic pressure and the control of the electrical potential difference across the vacuolar membrane. The intracellular location of this organelle has, until recently, complicated the study of the electrical properties of the vacuolar membrane from higher plant cells by standard electrophysiological techniques. Improvement of methods for the isolation of stable vacuoles and the application of the patch clamp technique made possible much more incisive studies on the electrophysiology of the vacuolar membrane. The fastest method for the isolation of small numbers of intact vacuoles directly from intact tissue is the one described by Coyaud et al. 13. In short, the surface of a freshly cut tissue slice is rinsed with buffer solution14 to wash the liberated vacuoles directly into the recording chamber. Thus, fresh vacuoles can be isolated for each experiment and vacuole isolation and seal formation can be performed within 2 - 5 min. The whole-cell configuration of the patch clamp technique was established after patch pipettes were sealed against an isolated vacuole, and the underlying membrane was broken by alternate _+0.6 V pulses 1 - 3 msec in duration. After access to the lumen of the vacuoles was gained by patch pipettes, the pipette solution equilibrated with the vacuolar sap. Using solutions with symmetric ion compositions on both sides of the membrane, steady-state conditions were indicated by a resting potential of 0 mV (which was reached within 1 -5 rain for a 20-pF vacuole. Vacuoles were exposed to solutions containing either 200 m M KC1 or KNO3. Both bathing media included 5 m M MgC12,0 or 0.1 m M CaC12, and 5 m M Tris- MES (4-morpholinoethanesulfonic acid) or citrate - KOH buffered to pH 7.5. The vacuole was equilibrated with either 200 m M KC1 including 5 m M MgC12, 1 mMCaC12, and 5 m M MES-Tris, pH 7.5 and 5.5, or citrate-KOH, pH 3.5 and 4.5. A patch clamp survey of the electrical properties of the vacuolar membrane from a large variety of plant materials has demonstrated the presence of voltage-dependent ion channels and electrogenic pumps as general features of ion transport in higher plant vacuoles. t3 L. Coyaud, A. Kurkdjian, R. Kado, and R. Hedrich, Biochim. Biophys. Acta 902, 263 (1987). 14 R. Hedrieh and E. Neher, Nature (London), 329, 833 (1987).
680
CHANNEL RECORDING IN ORGANELLES AND MICROBES
[46]
At high cytoplasmic Ca 2+ (>0.3 pM), the ionic conductance of the vacuolar membrane was found to be entirely accounted for by currents directed into the vacuole. These currents are activated at negative voltages (negative inside the vacuole), as well as at slightly positive potentials (
