[47] Patch clamp studies of microbial ion channels

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vantage of the hydration technique is demonstrated by the fact that the preparation of interest can be biochemically modified. With this additional tool, channel analysis may range from the study of ion channels in a purely synthetic environment to channels in an environment which largely resembles the physiological situation. The hydration technique may, therefore, provide a basis to investigate ion channel structure and function in its original environment. In some cases, when the organdies are large enough, they can be patch damped directly without any manipulation to enlarge their size. In the case of plant vacuoles mechanically isolated from plant tissue, they can be patch clamped in an osmotically adapted bathing solution. Owing to the large size of vacuoles (10- 15 #m), the classic patch clamp configurations can be employed directly in that system. In a similar approach, Mazzanti et al.lS studied ionic channels in the nuclear membrane. In their experiments, standard microelectrodes were inserted into the nucleus so that the nucleus adhered to the micropipette when it was withdrawn from the cell. After removal of the nucleus, the nuclear membranes could be directly accessed by patch pipettes. Taken together, these experiments demonstrate that there is no general strategy for patch damping different organdies. Instead, the specific structure of each organeUe has to be considered before choosing an appropriate patch clamp strategy. However, the methods presented here promise to serve as versatile tools for a better understanding of intracellular ion transport and signal transduction. ~8M. Mazzanti, L. J. De Felice, J. Cohen, and H. Malter, Nature (London)343, 764 (1990).

[47] P a t c h C l a m p S t u d i e s o f M i c r o b i a l I o n C h a n n e l s By YOSHIRO SAIMI, BORIS MARTINAC, ANNE H. DELCOUR, PETER V. MINORSKY, MICHAEL C. GUSTIN, MICHAEL R. CULBERTSON, JULIUS ADLER, a n d CHING KUNG

Introduction Interest in microbial ion channels stems from three sources: (1) microbial biology, (2) channel evolution, and (3) the use of microbes to study the principles of channel structures, functions, and regulations. We have pioneered patch clamp studies of Paramecium (a dilated protozoan), budding yeast (a fungus), and Escherichia coli (a gram-negative bacterium). MethMETHODSIN ENZYMOLOGY,VOL.207

Copyright© 1992byAcademicPre~ Inc. Allrightsofretn~uctionin anyformreserved.

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C H A N N E L R E C O R D I N G IN O R G A N E L L E S A N D M I C R O B E S

STARTING MATERIAL

PROCEDURES

OBJECTS FOR PATCH CLAMP

I ~

[47]

vesicles

he'~'~or s

~

10 O ~ 1 - - ~ 't ~"

cilia

--"~-~~

O spher°plasts

"~ow osmo.

Q mitochondria

~0

highosmo.

0 oCreu~

a

I~o./EOTA

~

A

vacuoles

i nt sphi~lasts

de-

.-'~

c:

"~

~%: ." q

+

azolectin

'i';':i:'." + azolectin

> deMg2+ ( ~ re-hyd. ~11 "----->

>

blisters liposome blisters

FIG. I. Outline of procedures. The starting material (left) Paramecium, yeast, E. coli, membrane vesicles, and yeast killer toxin have dimensions of the order of 100, 10, l, 0. l and 0.01 gin, respectively. The procedures (center), detailed in the text, convert them to objects used successfully in patch clamp experiments. These objects (fight) are all about 5 to 15/zm in diameter except detached cilia from Paramecium (-2/zm) and yeast mitochondria and vacuoles (-3/zm). AW405 is a wild-type E. coli strain. Lpp- OmpA- and AW693 are mutants. Osmo., osmolarity; ceph., cephalexin; lyso./EDTA, lysozyme and EDTA; de- rehyd., dehydration/rehydration. Objects not drawn to scale.

ods we developed are diagrammed in Fig. I and described below. Activities of channel types we found have been reviewed frequently l-s and are summarized in Table I.9-2° I y. Saimi, B. Martinac, M. C. Gustin, M. R. Culbertson, J. Adler, and C. Kung, Trends Biochem. Sci. 13, 304 (1988). 2 B. Martinac, Y. Saimi, M. C. Gustin, and C. Kung, in "Calcium and Ion Channel Modulation" (A. D. Grinnell, D. Armstrong, and M. B. Jackson, eds.), p. 415. Plenum, New York, 1988.

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ION CHANNELS

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TABLE I MICROBIAL CHANNELS RECORDED USING PATCH CLAMP METHODS

Organism

Channel

Paramecium

K + channel K + channel Na + channel CI- channel Cation channel K + channel MSehannel a Mitoehondrial VDAC b Vacuolar channel I c Vacuolar channel 2~ Killer toxin MS channel ° Cation channel I' Cation channel 2/ MS channel °

Yeast

E. coil

B. subtilis

Conductance (approx.) (pS) 150 70 19 <430 150 20 40 400 465 160 120 900 90 30 100

Ion specificity K K Na CI K ~ Na K K ~ Na

: ~ Na :~ Na :~ K :~ K :I~ C1 :~- Na : ~ C1

C] 3~..K K > Ca ~. C1 K ~ Na : ~ CI K ~ Na : ~ CI CI > K K>CI K > CI ?

Gating principle

Refs.

Caz+, depolarization Ca 2+, hyperpolarization Ca 2+ ? ATP, depolatization Depolarization Tensions

3,4 9 10 3,4 3,4 4,11 4,12

Voltage Ca2+, voltage Ca 2+, voltage None Tension.g depolarization Depolarization Voltage Tensions

13 13,14 13,14 15 4,7,16-18 4,17-20 18 7

* MS channel, meehanosensitive channel. b Mitoehondrial VDAC, mitochond_~al volt.e-dependent anion channel. Vacuolar channel 1, vacuolar Ca2+-release channel, which has a 465-pS conductance for K +, 100 pS for Ba~+ and Ca 2÷, and is activated by millimolar vacuolar Ca 2+, micromolar cyloplasmic Ca 2+, and cytoplasmic depolarization. d Vacuolar channel 2, vacuolar cation channel. ' Cation channel 1, outer membrane channel, most likely a porin, which shows cooperative closing and reopening on depolarization. fCation channel 2, location unknown. s Tension, membrane tension.

3 B. Martinac, Y. Saimi, M. C. Gustin, M. R. Culbertson, J. Adler, and C. Kung, Period. Biol. 90, 375 (1988). 4 y. Saimi, B. Martinac, M. C. Gustin, M. R. Culbertson, J. Adler, and C. Kung, Cold Spring Harbor Syrup. Quant. Biol. 53, 667 (1988). 5 C. Kung, Y. Saimi, and B. Martinac, Curr. Top. Membr. Transp. 26, 145 (1990). 6 C. Kung, in "The Evolution of the First Nervous Systems" (P. A. V. Anderson, ed.), p. 203. Plenum, New York, 1990. 7 B. Martinac, A. H. Delcour, M. Buechner, J. Adler, and C. Kung, in "Comparative Aspects of Mechanoreceptor Systems" (F. Ito, ed.), Springer-Verlag, in press. 8 R. R. Preston, J. A. Kink, R. D. Hinrichsen, Y. Saimi, and C. Kung, Annu. Rev. Physiol. 53, 309 (1991). 9 y. Saimi and B. Martinac, J. Membr. Biol. 112, 79 (1989). to y. Saimi and K.-Y. Ling, Science 249, 1441 (1990). H M. C. Gustin, B. Martinac, Y. Saimi, M. R. Culbertson, and C. Kung, Science 233, 1195 (1986). ~2M. C. Gustin, X.-L. Zhou, B. Martinac, and C. Kung, Science 242, 762 (1988). ~3p. V. Minorsky, X.-L. Zhou, M. R. Culbertson, and C. Kung, unpublished observations (1989). ~4p. V. Minorsky, X.-L. Zhou, M. R. Culbertson, and C. Kung, Plant Physiol. 89, 148 (Abstr.) (1989). ~5B. Martinac, H. Zhu, A. Kubalski, X.-L. Zhou, M. Culbertson, H. Bussey, and C. Kung, Proc. Natl. Acad. Sci. U.S.A. 87, 6228 (1990).

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Paramecium Blister Membranes Paramecium tetraurelia is a large cell, over 100 a m long and 50 a m in diameter. Macroscopic currents can be measured readily with a conventional two-electrode voltage clamp. 2~ Attempts to patch clamp live paramecia, ciliated or deciliated, have failed. Isolated membrane vesicles in the form of detached surface blisters have been patch-clamped successfully.9,~ Paramecium can be grown in several types of medium, such as 0.1 g/liter glucose, 0.1 g/liter casamino acid, 0.5 m M K2HPO4, 0.2 m M MgSO4, 0.2 m M CaC12, 5 mg/liter stigmasterol, 7.5 mg/liter phenol red, 5 m M HEPES (pH adjusted to 7.0-7.5 with NaOH). The medium (5 ml in a test tube) is bacterized with a tiny scoop ( - 10/zl) of Aerobacter aerogenes (food bacterium) from a plate with a platinum loop 1 day before the Paramecium inoculation and 2 days before experiments. Add as aseptically as possible 0.5-1 ml of Paramecium culture or 500-1000 cells to the bacterized medium. Higher growth temperatures (around 31 °) and stigmasterol supplement (3- 10 mg/liter) in the medium appear crucial. These factors may affect the rigidity of the membrane and therefore the stability of the patch. Tens of cells are transferred to a chamber where blisters are induced in 100-150 m M sodium glutamate or NaCI, 10- ~ M free Ca 2+ (buffered with EGTA), 5 m M HEPES (pH ~ 7.0). The blistering process, which takes place in 5 min, can be monitored under a phase-contrast microscope with a magnification of ×300. If the free Ca 2+ level is below 10-6 M or above 10-4 M, the cells do not blister consistently. Also avoid cells in the stationary growth phase, which is marked by the absence of dividing cells in the culture. Cells in the late logarithmic to early stationary phase, where unperturbed cells start to descend and spread in the culture tube, are preferred for blistering. The blisters can be detached from the actively swimming cell by passing the cell in and out of a glass suction pipette whose bore is slightly smaller than a paramecium. Several detached vesicles 5 - 20 a m in diameter are then picked up with the same suction pipette and laid down on the bottom of a separate

16B. Martinac, M. Buechner, A. H. Delcour, J. Adler, and C. K u n ~ Proc. Natl. Acad. Sci. U.S.A. 84, 2297 (1987). 17M. Buechner, A. H. Delcour, B. Martinac, J. Adler, and C. Kunf, Biochim. Biophys. Acta 1024, 111 (1990). ~s A. H. Delcour, B. Martinac, J. Adler, and C. Kung, Biophys. J. 56, 631 (1989). 19A. H. Delcour, B. Martinac, J. Adler, and C. Kung, J. Membr. Biol. 112, 267 (1989). 2o A. H. Delcour, J. Adler, and C. Kung, J. Membr. Biol. 119, 267 (1991). 2t D. O. Oertel, S. J. Schein, and C. Kung, Nature (London) 268, 120 (1977). 22A. Kubalski, B. Martinac, and Y. Saimi, J. Membr. Biol. 112, 91 (1989).

[47]

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chamber containing the seal-forming solution, which may have (in mM) IOO K + or Na +, 10 MgC12, 5 HEPES (pH - 7.0), an appropriate amount of anion, and 10-8 to 10-4 M free Ca 2+, depending on the type of experiments. The patch pipette often contains a similar solution. The blisters are double layered. Depending on the osmolarity difference between the blistering and gigohm seal-forming solutions, the blisters may stay round or start to peel off to give rise to a vesicle (with the inner, probably the alveolar membrane) capped with hemispherical membrane (outer, the plasma membrane). Gigohm seals can be formed on either membrane. The inner membrane appears electrically silent in our survey; the outer membrane contains channels (Table I). After gigohm seal formation, the membrane patches can be excised by air exposure or shaking the vesicle off by tapping the pipette holder. If the free Ca 2+ level in the gigaseal forming solution is higher than 10-6 M, the excised membrane may be closed into vesicles. Fire-polished, coated pipettes of Boralex glass (Dynalabs, Rochester, NY, Cat. No. 5068) are favored although other types of pipettes have been used successfully.

Paramecium Cilia Each Paramecium is covered with some 5000 cilia, whose membranes are continuous with the body membrane. Forcing live paramecia through a narrow-bore pipette as above can shear off cilia. Each detached cilium retains its microtubular axoneme inside, and its membrane apparently reseals, since it appears osmotically active. These ciliary vesicles can be transferred to the recording chamber, form seals with recording pipettes, and show activities of several channel types. 2 Yeast Spheroplasts Cells of budding yeast (Saccharomyces cerevisiae) are about 7 p m in diameter and have external cell walls. For preparation of yeast spheroplasts,11,12 haploid or diploid yeast is cultured in liquid YEPD media [I % yeast extract, 2% Bacto-peptone (Difco, Detroit, MI), 2% dextrose] at 30 ° overnight. A second culture is prepared the next morning by a 1:20 dilution into flesh YEPD. After incubation at 30 ° for 90 to 120 min, the cells are washed twice with distilled water by centrifugation (2000 rpm, for 5 min in a tabletop centrifuge), and the final peUct is resuspended in water at an A~oo of 1.5. This suspension is diluted 1 : 1 (final volume 0.75 ml) with a solution containing 0.8 M sorbitol plus 0.133 mg/ml Zymolyase (100T, ICN, Costa Mesa, CA) to remove the cell wall. After incubation at 30 ° for 18 rain, 3 ml of solution A (in mM, 120 KC1, 50 MgC12, 5 HEPES,

686

CHANNEL RECORDING IN ORGANELLES AND MICROBES

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pH 7.2) is added. The spheroplasts are then spun down and resuspended in 0.5 ml of solution A. A small aliquot of this suspension is diluted into a chamber containing the bath solution (solution A plus 2 mM CaC12). Spheroplasts are used only on the day of preparation. Various recording configurations (on-cell, excised inside-out and outside-out, and whole-cell modes) can be and have been used. Seals are usually obtained on freefloating spheroplasts; a spheroplast is moved off the bottom by slight positive pressure through the recording pipette and then captured in the mouth of the pipette by suction. High gigohm seals (> 10 Gf2) will form but take longer and probably require more suction than with animal cells. A 3-ml syringe with a two-way valve connected by tubing to the port on the pipette holder is used to apply the strong and prolonged suction necessary. A pressure transducer (PX 143, Omega Engineering, Stamford, CT) in line is used for monitoring the applied pressure. At least 5-20 cm Hg appears to be necessary for seal formation. Often during maintained application of suction the resistance will abruptly decrease (probably owing to the pinching off of surface membrane blebs), only to start slowly increasing again. Gigohm seals will still usually develop on such spheroplasts. Recording at seal resistances less than 10 GK2 is not recommended. Patch pipettes are made from Boralex glass, pulled to a bubble number23 around 3.5 (8- I0 Mfl). Two sets of solutions have been used most often: pipette hypotonic [bath, solution A; pipette, (in raM) 150 KC1, 5 MgCI2, 0.1 EGTA, 5 HEPES, pH 7.2] and pipette hypertonic24 (bath, 10 potassium glutamate, 390 mannitol, 2 MgC12, 1 CaCI2, 10 MES, pH 5.5; pipette, 100 potassium glutamate, 2 EGTA, 2 ATP, 4 MgC12, 250 mannitol, 10 HEPES, pH 7.2). Seals form more easily with the former, but whole-cell recordings are more stable with the latter. Yeast Mitochondria and Vacuoles Each yeast cell has a giant mitochondrion and a large vacuole. When yeast spheroplasts are lysed under the microscope in a hypoosmotic solution of (in mM) 50 sorbitol, 5 MgC12, 50 KC1, 5 HEPES, 0.1 EGTA, pH 7.2, two types of vacuolelike structures are evident. The less abundant type appears colorless in plain light optics, has a more rigid membrane, and encloses many particles in Brownian motion. When reshrunk in the sealforming solution (see below) and patch clamped, these organelles are found t4 to possess an ion channel with a conductance (-390 pS in sym23 D. P. Corey and C. F. Stevens, in "Single Channel Recording" (B. Sakmann and E. Neher, eds.), p. 53. Plenum, New York, 1983. 24 j. I. Schroeder, J. Gen. Physiol. 92, 667 (1988).

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PATCH CLAMP STUDIES OF MICROBIAL ION CHANNELS

687

metrical 100 m M KC1) and voltage dependency virtually identical to those reported for the voltage-dependent anion channel (VDAC) found in the outer membrane of all eukaryotic mitochondria so far examined.25 The more abundant organelle released following lysis is extremely elastic, contains few or no particles, appears lavender, and has a complement of channel activities entirely different from those of the mitochondrial or plasma membrane. The kinds of channels encountered indicate that these organelles are most likely vacuoles. Following shrinkage of the isolated vacuoles by perfusion of the gigohm-forming solution (in mM; 300 potassium glutamate, 5 MgC12, 5 HEPES, adjusted to pH 7.2 with KOH), excised, vacuolar-side-out patches can be attained simply by touching the membrane with a fire-polished Boralex pipette filled with (in raM) 300 potassium glutamate, 0.1 MgC12, 5 EGTA, variable CaC12, 5 HEPES, adjusted to pH 7.2 with KOH. We have succeeded in establishing reliably the on-vacuole or vacuole-side-out excised recording configuration but not other modes. Our studies indicate that the vacuolar membrane possesses several distinct channel types, but we have only examined two conductances in detail.~3,~4

Escherichia coli Giant Cells or Giant Spheroplasts Escherichia coli is rod shaped, 0.5 #m in diameter and 2/zm long. Patch clamping such cells directly was unsuccessful. We have developed five methods t6a~ to generate giant cells or giant spheroplasts 5 to 10 p m in diameter for patch clamp experiments. Giant Spheroplasts from Cephalexin- Treated Cells Wild-type E, coli, such as strain AW405, grown in modified LuriaBertani medium (MLB) [1% Bacto-tryptone (Difco), 0.5% yeast extract, 0.5% NaCI] at 35 ° (up to an A~9oof 0.5-0.6) is used to inoculate a culture (1 : 10 dilution of bacteria) in MLB plus 60 #g/ml ofcephalexin at 42 ° until unseptated filaments of 50- 150 g m observed under a microscope (× 400) are formed (in 2.5-3 hr). The harvested filaments are resuspended in 0.8 M sucrose and digested with lysozyme (200 #g/ml) in the presence of (in mM) 50 Tris buffer and 6 NaEDTA, pH 7.2, to hydrolyze the peptidoglycan layer (cell wall) at room temperature for 7 - 10 min. The progress of spheroplast formation is followed under a microscope (× 400). 26 This treatment apparently does not digest the peptidoglycan completely, but 25 M. Colombini, J. Membr. Biol. 111, 103 (1989). 26 H.-J. Ruthe and J. Adler, Biochim. Biophys. Acta 819, 105 (1985).

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CHANNEL RECORDING IN ORGANELLES AND MICROBES

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clips the polymers and weakens the cell wall, thereby allowing the swelling ofE. coli spheroplasts. A similar lysozyme treatment followed by overnight growth and activation of autolysin can also generate giant spheroplasts of Bacillus subtilis, a Gram-positive bacterium. 7 Bacillus subtilis is grown overnight in 10 ml of 2% Bacto-peptone (Difco) in the presence (crucial) of 0.5% (w/v) NaCI and 25 m M KC1. A 1 : 100 dilution of the culture is made into 80 ml of the same fresh medium as above, and the bacteria are grown at 35 ° to an A59o of 0.8-0.9. Cells are harvested by centrifugation in a Sorvall SS-34 rotor at 5000 rpm for 15 rain. The cell pellet is resuspended in 70 ml of 0.5 M sucrose, 16 m M MgSO4, and 0.5 M potassium phosphate buffer (pH 7.0) in a l-liter flask. The cell suspension is incubated at 35 ° for 1 hr in the presence of 2 mg/ml lysozyme. Spheroplasts are harvested by centrifugation in a Sorvall SS-34 rotor at 5000 rpm for 15 rain. The spheroplasts are resuspended in 1.6 ml of the same solution as above. The spheroplasts must be grown further to larger sizes in order to be patch-clamped. A 1 : 25 dilution (4- I 0 ml total) of the spheroplast suspension is made with the succinate medium (0.5 Msodium succinate, 0. I MKCI, 1 mMMgSO4, 10 mg/ml casamino acid, 0.6 mg/ml fructose bisphosphate) and then added (per ml) with 40/tl of 5% casamino acid and 10/tl of 10% yeast extract. The culture is shaken slowly (50-75 rpm) at 33 ° for 24 hr before the giant spheroplasts are harvested by centrifugation in a Sorvall SS-34 rotor at 5000 rpm for I0 min. The spheroplast pellet is resuspended gently into 10 ml of the activating solution (1 M sucrose, 6 m M KC1, 20 m M NaHCO3, and 10 mMMgSO4, pH 7.5), and incubated at 35 ° for 90 min. This treatment activates the autolysin. RNase and DNase (0.8 mg/ml each) can be added to the suspension during this time to reduce viscosity, but this is optional. Spheroplasts are diluted directly into the chamber, where gigohm seals are formed on them with patch pipettes.

Giant Spheroplasts from Cells Treated with Ultraviolet Light Escherichia coli cells irradiated with UV light also form unseptate filaments. Bacterial cultures (A59o0.1-0.2, 5 m m in depth) in plastic petri dishes are shaken at 40 rpm under UV light (254 rim, 124 erg m m -2 see-~) for 3 min, pooled into 9 volumes of a growth medium (MLB), and cultured at 42 ° until filaments 80-100/tin long developed (2.5-3 hr). Spheroplasts are prepared with lysozyme as above. Magnesium-Induced Giant Cell Ceils are grown in LB (1% Bacto-tryptone, 0.5% yeast extract, 1% NaC1) plus 50 m M MgC12 at 35* to an A59o of 0.5, then diluted 1 : 10 into same medium with the addition of 60/tg/ml cephalexin, and grown for 2 hr by

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shaking at 42 °. This culture yields a mixture of giant round cells (5 - 10 # m in diameter) and long filaments, some with bulges on the sides. These bulges and giant cells can be used directly for patch clamp studies.

lpp- ompA- Giant Cells Cells of a mutant E. coli strain which lacks both Lpp and OmpA proteins, two major components of the outer membrane, round up in the presence of 30 m M MgC12. Mutant ceils are cultured in LB plus 30 m M MgClz at 35 ° to ariA59o of 0.5, then diluted 1 : 10 in LB plus 30 m M MgC12 containing 60 #g/ml cephalexin, and grown for 4 hr to form giant round cells 5 - 1 0 #m in diameter. They are usually washed twice with 0.8 M NaC1 by centrifugation in a Sorvall SS-34 rotor at 5000 rpm for 3 min. We found an increase in seal resistance after this salt wash.

Giant Cells of an Osmotic-Sensitive Mutant Escherichia coli AW693 is selected for its failure to grow at high osmolarity, t3 AW693 cells are grown in LB plus 400 m M KC1 to an A59o of about 0.6 and incubated overnight at 4* without shaking. A few of the cells are large, some large enough ( 4 - 8 # m in diameter) to be patch clamped directly. The number of these large ceils increases to about 10% of the total by the inclusion of 10 m M MgC12 in the growth medium. Most recordings are conducted in the on-cell or excised inside-out mode. As with yeast spheroplasts, seals form more slowly with these bacterial preparations than with most animal ceils. Seals of several gigohms can nonetheless be formed routinely. We found that giant spheroplasts from cephalexin- or UV-treated cells and from lpp- ompA- cells gave higher seal resistances. A mechanosensitive channel is most commonly encountered in all five preparations. A voltage-gated channel, which tends to close cooperatively, is encountered less frequently. Note that E. coli has an outer and an inner membrane. We have provided evidence that the mechanosensitive and voltage-gated channels are located in the outer membrane. ~7 We also found a mechanosensitive channel in B. subtilis. 7 Reconstitution of Microbial Channels in Liposome Blisters We reconstitute channels in liposomes by a fusion procedure ~a similar to that of Criado and Keller27 except that azolectin is prepared without the use of detergent. High gigohm seals are formed more reproducibly with this preparation than when azolectin is prepared with a detergent.

27 M. Criado and U. B. Keller, FEBSLett. 224, 172 (1987).

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CHANNEL RECORDING IN ORGANELLES AND MICROBES

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Azolectin (Sigma) of 10 mg/ml is sonicated to clarity by a Branson probe sonicator for 5 min in the presence of 5 m M Tris-HC1 (pH - 7.2). Aliquots of 1 ml are twice frozen in dry ice-acetone (5 rain each) and thawed (15 rain each). This process yields multilamellar liposomes. Aliquots of bacterial native membrane vesicles 18-2° are mixed with 0.5 ml of freeze-thawed azolectin at a desired protein-to-lipid ratio (w/w; 1 : 600 for the outer membrane protein, 1 : 6 for the inner membrane protein), and the mixture is pelleted for 1 hr at 95000 g at 4 ° (add 5 m M Tris-HCl, pH 7.2, as necessary). Large membrane vesicles, such as those obtained by a freeze-thaw procedure, pellet better and are needed to obtain a reproducible recovery of lipid. To form giant liposomes, the pelleted membranes are resuspended into 25/tl of buffer containing l0 m M M O P S and 5% (w/v) ethylene glycol (pH 7.2). Aliquots of the suspension are placed onto a clean glass slide and subjected to a 4-hr dehydration in a desiccator at 10°C. The dehydrated lipid film on the slide glass is then rehydrated at 4 ° overnight with a solution of 1 5 0 r a m KC1, 0.1 m M EDTA, 10-5M CaCI2, and 5 m M HEPES (pH 7.2). We found that a lipid concentration of at least 90 mg/ml is needed during the rehydration step to produce giant liposomes. The liposomes are not suited for gigohm seal formation, but we have developed a protocol whereby unilamellar blisters are induced to grow out of these liposomes. The blisters form high-resistance seals readily and very reproducibly with patch pipettes. Blisters are formed by placing a few microliters of the rehydrated suspension in the patch clamp chamber, which contains the experimental buffer plus 20 m M MgC12. The presence of MgC12 causes the liposomes to collapse. Within a few minutes, faint and most likely unilamellar blisters emerge from the sides of the collapsed liposomes. Blisters form rapidly (3- l0 rain) at low protein-to-lipid ratios, but slowly (30-60 min) at high protein-to-lipid ratios. Once formed, the blisters are stable for hours, as long as MgC12 is in the buffer. We found that blisters made form pure synthetic lipids alone tended to yield artifactual channellike currents. However, experiments done with azolectin (a mixture of lipids extracted from soybeans), in the absence of fused native membranes or toxins, yield high-resistance seals and quiet background. Care must be taken to use very clean, sterile solutions, especially during the rehydration procedure, to avoid contamination by exogenous microbial membrane fragments. Bacterial membrane vesicles can be prepared mostly inside-out from French-pressed cells or mostly outside-out from sonicated spheroplasts. Inner and outer bacterial membranes fractions can be separated via sucrose gradient centrifugation. All membrane fractions can be stored at - 80 ° for 2 to 3 months without loss of channel activity. Using this method, detailed

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elsewhere, ts,t9 we have reconstituted three types of E. coli channels: a mechanosensitive channel, a voltage-sensitive channel, and a Small cationselective channel. The first two have also been observed during recordings of outer membrane activity using live cells and spheroplasts, and they have retained their native properties. The third type has been seen only in reconstituted membranes so far and is, most likely, an inner membrane channel. This method can be used for reconstitution of channels from other sources. Killer strains of yeast harbor a double-stranded RNA virus, which produces a dimeric toxin capable of kiUing the virus-free strains. This toxin forms channels. We showed this, in part, by incorporating it into liposome blisters. ~5 Partially purified toxin or a toxin-containing concentrated filtrate of a killer-yeast culture is added to azolectin (toxin: lipid, 1 : 1000, molar ratio) in 100 m M KC1 plus 1X Halvorson salts. 2s The rest of the procedure is as above. Conclusions It appears that all plasma membranes and organelle membranes, including those of microbes, are equipped with ion channels. Thus, ion channels apparently evolved early. Patch clamp experiments have revealed many types of ion channels in different microbial preparations. These channels appear to underlie large sectors of interesting biology yet to be explored. The methods described here have been developed for this exploration. Acknowledgments We thank M. Buectmer, C. Hirscher, A. Kubalski, X.-L. Zhou, and H. Zhu for their technical or other assistance in developing the methods described here. The work in our laboratories described here was supported by National Institutes of Health Grants GM22714, GM32386, GM37925, DK93121, and a grant from the Lucille P. Markey Trust. zs Composition of Halvorson salts [H. O. Halvorson, Biochim. Biophys. Acta 27, 267 (1958)]: in raM, 30 (NH4)2SO4, 50 K2HPO4, 50 succinic acid, 2.71 CaC12, 4.14 MgSO4,plus trace metals [in gM, 7.7 Fez (SO4)3, 16.6 MnSO4, 15.5 ZnSO4, 15.7 CuSO4].