Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments Derived from Eukaryotic Microorganisms

CHAPTER 4

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments Derived from Eukaryotic Microorganisms Yoshinori Muto1,* and Kiyoshi Kawai2 1

Department of Basic Health Science and Fundamental Nursing, Gifu University School of Medicine, 1-1, Yanagido, Gifu 501-1193, Japan 2 Department of Nutrition, Faculty of Wellness, Chukyo Women’s University, Ohbu 474-0011, Aichi, Japan

Contents 1. Introduction 2. Structures and properties of quinones studied 2.1. Versicolorin A and averufin 2.2. Emodin and skyrin 2.3. Blepharismin 3. Procedures for analyzing quinone effects 3.1. Isolation and purification of quinone compounds 3.1.1. Isolation of fungal quinones 3.1.2. Extraction and purification of blepharismins 3.2. Planar bilayer techniques 3.2.1. General considerations 3.2.2. Setup for membrane formation 3.2.3. Membrane formation using the folding method 3.2.4. Electrical equipment and recording 3.3. Mitochondrial techniques 3.3.1. General considerations 3.3.2. Preparation of rat liver mitochondria 3.3.3. Measurement of mitochondrial respiration 3.3.4. Measurement of mitochondrial swelling 4. Effects of quinones on mitochondrial function 4.1. Fungal quinones 4.1.1. Effects of versicolorin A and averufin on mitochondrial respiration 4.1.2. Effects of emodin and skyrin on mitochondrial respiration 4.2. Blepharismin 4.2.1. Effects of blepharismin on mitochondrial respiration 4.2.2. Induction of mitochondrial swelling by blepharismin

123 124 124 124 126 126 126 126 126 127 127 128 128 130 130 130 131 132 133 134 134 134 135 136 136 138

* Corresponding author. Tel./Fax: C81-58-293-3241; E-mail: [email protected] ADVANCES IN PLANAR LIPID BILAYERS AND LIPOSOMES, VOLUME 1 ISSN 1554-4516 DOI: 10.1016/S1554-4516(05)01004-5

q 2005 Elsevier Inc. All rights reserved

122

Y. Muto and K. Kawai

5. Effects of quinones on planar bilayer membranes 5.1. Fungal quinones 5.1.1. Effects of versicolorin A on conductance of planar bilayer membranes 5.1.2. Characteristics of planar bilayer membranes in the presence of versicolorin A 5.1.3. Effects of averufin on conductance of planar bilayer membranes 5.1.4. Effects of emodin and skyrin on conductance of planar bilayer membranes 5.2. Blepharismin 5.2.1. Effects of blepharismin on conductance of planar bilayer membranes 5.2.2. Characteristics of the channels formed with blepharismin 6. Concluding remarks Acknowledgements References

138 139 139 141 144 145 146 146 147 151 151 152

Abstract Numerous quinone and quinoid pigments have been isolated from a variety of eukaryotic microorganisms, including fungi and protozoa. Structural information on many of these quinone compounds suggests that these molecules are amphiphilic in nature, which is indicative of a strong membrane association potential. Therefore, we have extensively investigated the interaction of various quinone pigments with typical biological membrane models, planar lipid bilayer membranes and mitochondrial membranes. Anthraquinone mycotoxins, versicolorin A and averufin are metabolic precursors of aflatoxin B1, and have been found to exhibit genotoxic effects in the hepatocyte/DNA repair test. Using planar lipid bilayer membranes, we demonstrated that versicolorin A and averufin greatly increased the proton conductance of bilayer membranes. The concentrations employed are comparable to those used in mitochondrial experiments and thus the effects of both toxins on mitochondrial oxidative phosphorylation might be mediated by increases in the proton permeability of the mitochondrial membrane. On the other hand, it is interesting that averufin increased ionic permeability in addition to proton permeability in planar bilayer membranes. This observation suggests that the mechanism of averufin-induced uncoupling may differ somewhat from that of versicolorin A. Blepharismins are polycyclic quinones found in the pigment granules of the ciliated protozoan, Blepharisma. At cytotoxic concentrations, blepharismins formed cation-selective channels in planar phospholipid bilayer membranes. The channels formed in a diphytanoylphosphatidylcholine bilayer had a KC/ClK permeability ratio of 6.6:1. Single-channel recordings revealed the conductance to be quite heterogeneous, ranging from 0.2 to 2.8 nS in solutions containing 0.1 M KCl, possibly reflecting different states of aggregation of blepharismin. We also studied the effects of blepharismins on membrane permeability in rat liver mitochondria. The results further substantiated the channel formation by blepharismins in biological membranes. The analyses presented here, in conjunction with other biochemical studies described in this chapter, indicate that planar lipid bilayer membranes offer powerful tools for answering important questions regarding the structure and function of a diverse range of naturally occurring compounds.

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

123

1. INTRODUCTION A chemically diverse group of quinone and quinoid compounds have been isolated from various microorganisms [1]. Fungi is the most prominent taxonomical class of organisms that produce biologically active quinones, which are categorized into three structural subsets: benzoquinone, naphthoquinone and anthraquinone derivatives [2]. Certain members of these fungal quinone compounds, particularly anthraquinone derivatives, have medical and toxicological importance due to their significant bioactivity [3]. For example, genotoxicity, including mutagenicity, carcinogenicity and anti-carcinogenicity of various anthraquinone compounds have been reported [4–9]. Toxicity to cellular and mitochondrial functions has also been studied in several anthraquinones from fungi [10–13]. In particular, investigations using isolated mitochondria revealed that several anthraquinones, such as emodin and versicolorin A, impede mitochondrial function by uncoupling oxidative phosphorylation [14–17]. Besides being abundant in fungi, some quinone pigments are known to be produced by ciliated protozoa, such as Blepharisma [18–22] and Stentor [23–27]. The quinone compounds in these protozoa are polycyclic quinones, and are believed to function as photoreceptors modulating the photobehavior of the cell [28–34]. Moreover, blepharismins produced by Blepharisma have been extensively characterized and shown to exert cytotoxicity against certain other protozoa [35–38]. The molecular bases of the various biological activities exhibited by microbial quinone pigments are of great interest from a medical point of view [39,40]. However, the precise mechanisms of action are not fully understood. Many of the quinone pigments whose structures have been clarified possess a hydrophobic skeleton with several peri-hydroxyl groups [41–43]. This suggests that these compounds are polarized and amphipathic in nature, which is indicative of a strong membrane association potential [44–46]. It is therefore of interest to investigate the interaction of these quinone compounds with membranes in order to gain further insight into the mechanism of quinone action. Planar lipid bilayer membranes are widely used to characterize the interaction of various agents with cellular membranes [47,48]. Many natural products of microbial origin, such as linear and cyclic peptides, polyethers and polyene macrolides, facilitate ionic transport through lipid bilayer membranes via the formation of pores or carriers, thus enabling passive ion flux [49,50]. Detailed information about the ionic permeability and particularly about the ionic selectivity induced by pores or carriers can be obtained from measurements in lipid bilayer membranes [51,52]. Because the method is sufficiently sensitive to detect permeability changes caused by various agents, studies of quinone compounds in planar lipid bilayers seem particularly attractive. On the other hand, isolated mitochondria provide another convenient experimental system for screening membrane-active compounds and for analyzing transport mechanisms [41,53]. Because isolated

124

Y. Muto and K. Kawai

mitochondrial membranes contain a fully active respiratory chain, they are well suited for investigating uncoupling activity and oxidative phosphorylation [14,15]. In addition, mitochondrial preparations permit direct spectrophotometric swelling measurements, thus facilitating detection of various cation permeabilities [54,55], which is essential for assessing quinone-induced membrane permeability. Through the use of planar bilayers and isolated mitochondria, we have investigated the interactions between membranes and various quinone pigments derived from eukaryotic microorganisms [56–58]. In this chapter we focus on anthraquinone derivatives and blepharismins and describe results from our laboratory that indicate the mechanism of quinone-induced ion transport and correlation between quinone structure and ionophoric properties.

2. STRUCTURES AND PROPERTIES OF QUINONES STUDIED 2.1. Versicolorin A and averufin Aflatoxin B1, a potent hepatotoxic and carcinogenic mycotoxin, was isolated from Aspergillus flavus and A. parasiticus and was shown to cause turkey X-disease [59,60]. In the course of studies on the biosynthesis of aflatoxin B1, a series of anthraquinone compounds, such as norsolorinic acid, averantin, averufin and versicolorins A and B, was identified as intermediates in aflatoxin B1 biosynthesis [61,62]. Of these anthraquinone intermediates, versicolorin A (Fig. 1(1)) is strongly suspected of being carcinogenic as a result of screening tests and because its chemical structure contains a dihydrobisfuran ring, like aflatoxin B1 and sterigmatocystin [5,63]. Moreover, averufin (Fig. 1(2)), norsolorinic acid and versicolorin A were found to be potent uncouplers of oxidative phosphorylation [14,64]. Details are provided in Section 4.1.1.

2.2. Emodin and skyrin More than 20 quinone and quinoid compounds have been isolated from Penicillium islandicum, which is responsible for production of yellow rice toxins [8,65]. Of these quinoid metabolites, luteoskyrin and rugulosin are hepatotoxic and carcinogenic [9,66]. Flavoskyrin, rubroskyrin and several anthraquinone metabolites of P. islandicum exhibit various degrees of toxicity in HeLa cells [12]. It is characteristic that this fungus produces both anthraquinones and their corresponding bis derivatives, such as emodin (Fig. 1(3)) and its dimer, skyrin (Fig. 1(4)). Emodin is a polyhydroxyanthraquinone with a relatively simple structure, and is found not only in fungi but also in higher plants as a secondary metabolite [1]. This pigment exhibits strong mutagenicity as well as anti-carcinogenicity, but the mechanism has not been elucidated [67].

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

125

Fig. 1. Chemical structures of versicolorin A, averufin, emodin, skyrin and blepharismins. Blepharismin-1: R1ZR2Zethyl, R3ZH; blepharismin-2: R1Z ethyl, R2Zisopropyl, R3ZH; blepharismin-3: R1ZR2Zisopropyl, R3ZH; blepharismin-4: R1Zethyl, R2Zisopropyl, R3Zmethyl or R1Zisopropyl, R2Z ethyl, R3Zmethyl; blepharismin-5: R1ZR2Zisopropyl, R3Zmethyl. Mitochondrial experiments demonstrated that emodin and skyrin also uncouple the oxidative phosphorylation and that the hydroxyl group at the b position of the anthraquinone nucleus is important for this uncoupling effect [13,16] (see Section 4.1.2).

126

Y. Muto and K. Kawai

2.3. Blepharismin The ciliated protozoan, Blepharisma japonicum [19], has numerous pigment granules containing quinone pigments, called blepharismins (Fig. 1(5)). These granules are located just beneath the plasma membrane [68–70]. The pigments are believed to function as photoreceptors modulating the photobehavior of the cell [29,31]. Blepharismins have also been reported to be toxic to certain other kinds of protozoa [35,36] and to kill a variety of protozoa, including predators which actively feed on Blepharisma [37,38]. The molecular structures of blepharismins (Fig. 1(5)) possess a naphthodianthrone skeleton with four peri-hydroxyl groups as a common structural component [42,43]. This structural information suggests the polarized amphipathic nature of blepharismin molecules, which is indicative of a strong membrane association potential.

3. PROCEDURES FOR ANALYZING QUINONE EFFECTS 3.1. Isolation and purification of quinone compounds 3.1.1. Isolation of fungal quinones Isolation of averufin and versicolorin A from P. parasiticus is essentially based on the method described previously [71]. Here, we describe the procedure briefly. Pigments were obtained by chloroform–methanol (1:1 v/v) extraction of the mycelium in a Soxhlet apparatus, followed by solvent partition with hexane–90% methanol. The 90% methanol layer containing averufin and versicolorin A was separated by chromatography on silica gel H under pressure (1 kg/cm2). The column was developed with chloroform–methanol (97:3 v/v). Crystallization from acetone and chloroform–methanol gave pure averufin and versicolorin A, respectively. Isolation of emodin and skyrin from P. islandicum was achieved by chromatography on an active charcoal column to absorb mono- and bianthraquinones, which were recovered by elution with 1 M sodium hydroxide solution followed by acidification. Details are as described previously by Betina [72].

3.1.2. Extraction and purification of blepharismins The procedure described here is essentially based on the method of Matsuoka et al. [70]. Blepharisma japonicum was cultured at 23 8C in the dark in an infusion of 0.1% cereal leaves containing Enterobacter aerogenes as food. The cells, collected by centrifugation at 150g for 5 min at room temperature, were suspended and extracted in acetone for 1 min at room temperature. After centrifugation at 800g for 10 min, the supernatant fluid was concentrated and dried

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

127

on a rotary evaporator (Rotavapor, Shibata, Tokyo, Japan). Blepharismin-1, blepharismin-2, blepharismin-3, blepharismin-4 and blepharismin-5 were then purified by thin-layer chromatography (TLC) using silica gel plates (60 F254, Merck, Germany) or an HPLC column (Hitachi 655 liquid chromatography system). Dried pigments were dissolved in 30% acetonitrile solution for application to the column (COSMOSIL 5C18-300, 4.6!150 mm, Nakarai) for reverse-phase HPLC with an acetonitrile gradient. In the case of TLC, pigments were dissolved in acetone for application to normal-phase TLC plates, which were developed with a solvent system consisting of ethyl acetate and acetone (4:1, 3:1 or 2:1 v/v). All pigment preparation and TLC procedures were carried out under dim light conditions (below 0.05 W/m2). The purified blepharismins were dissolved in ethanol and were stored and protected from light at K20 8C until use.

3.2. Planar bilayer techniques 3.2.1. General considerations There are two main techniques for forming planar bilayer membranes: a painting method and a folding method [73,74]. In the painting method originally described by Mueller et al. [73], bilayer membranes are formed by placing a small amount of a lipid solution, usually dissolved in an organic solvent such as decane, onto an aperture in a septum separating two aqueous solutions. After spreading the lipid solution across the aperture, most of the solvent drains away and a bilayer membrane is formed spontaneously. The folding method of Montal and Mueller [74], on the other hand, employs monolayers spontaneously formed at the air– water interface of aqueous compartments. By raising the water levels in both compartments, the two monolayers are apposed within an aperture in a thin Teflon septum. Although both methods efficiently produce relatively stable bilayer membranes, some differences are noted. Painted membranes contain a certain amount of organic solvent used in the membrane-forming lipid solution, but bilayers formed from monolayers are virtually free of solvent [75]. In addition, folded membranes can be formed asymmetrically by apposing two monolayers having different compositions. Because the presence of organic solvent in the painted membranes are more likely to affect the membrane actions of quinone compounds, we used the folding method for all the investigations presented here. The original technique for forming planar lipid bilayers from two monolayers is described by Montal and Mueller [74]. Further details and improvements in the basic procedure can be found elsewhere [76,77]. In the following we describe the planar bilayer technique used to investigate the membrane action of quinone pigments in more detail.

128

Y. Muto and K. Kawai

3.2.2. Setup for membrane formation The membrane chamber consists of two compartments, which can be constructed from Teflon blocks (Santsuri Kiko, Japan). The two compartments are separated by a 12.5 mm Teflon film (Yellow Spring Instruments, Yellow Springs, OH) and are mechanically clamped together using two stainless steel screw rods [78]. The use of high-vacuum silicone grease on the Teflon film adjoining the compartments prevents electrical leakage between the two aqueous phases in the chamber. Each compartment has two 1 mm holes from the top surface to the bottom for insertion of the polyethylene tubing, which is connected to a disposable syringe. The tubing and syringes are used to raise or lower the solution levels as well as to perfuse aqueous phase into each compartment (capacity of each compartment is 1.5 ml). The Teflon film separating the compartments contains an aperture (50–250 mm in diameter) across which the bilayer is formed. The formation of a smooth and circular aperture is of great importance for obtaining stable bilayer membranes. We used an electrical spark generated by an ignition coil to locally melt the Teflon film. With this method, smooth apertures of various sizes can easily be generated [79]. The diameter of the aperture is determined by the number of sparks passed through the film and the electrical voltage applied. An inverted microscope was used to inspect the smoothness of the aperture. The diameter of the aperture was also determined under a microscope with an objective micrometer. We used a relatively small aperture (ca. 100 mm) to measure single-channel fluctuations and a larger aperture to measure macroscopic currents. Figure 2 illustrates the entire Faraday cage and a block diagram of the electrical system. The chamber rests on a DC-powered magnetic stirrer, which is used to spin a small magnetic stir bar inside the compartment. Two Ag/AgCl electrodes are immersed in glass reservoirs containing 1 M KCl and connected on one end to the chamber compartments with salt bridges. The other end is connected directly to the BNC connector of an I–V converter (current-to-voltage converter). The whole chamber/stirrer/I–V converter assembly is placed in a box (Faraday cage) made from 3 mm thick aluminum plates (inside) and 15 mm thick wooden plates (outside). The syringes used to displace the water levels are located outside the box. The box serves as a shield from alternating current noise and acoustic vibration. To further attenuate floor vibrations, the box is mounted on a heavy platform composed of an 11 mm thick steel plate, which is supported by fresh tennis balls. This inexpensive configuration is particularly effective for damping high-frequency vibrations.

3.2.3. Membrane formation using the folding method Prior to membrane formation, the area of Teflon film around the aperture is coated with 0.5 ml of 0.5% (v/v) hexadecane in hexane [80]. The two compartments are

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

129

Fig. 2. Schematic diagram of the system used for forming planar lipid bilayers and for recording membrane currents. Details are described in the text. then filled to below the aperture with 0.5 ml of electrolyte solution. The monolayer can be generated by applying 10–20 ml of 10 mg/ml lipid solution in hexane to the surface of each water phase and allowing 5 min for the solvent to evaporate. To form a planar bilayer, more electrolyte solution is slowly injected under the monolayers using the syringes connected to the bottom of the compartments (Fig. 2). As the solution level rises, the bilayer membrane is formed by apposition of the monolayers at the aperture in the Teflon film. The formation of bilayer membranes from monolayers can be conveniently followed by continuous measurement of membrane capacitance. Capacitance measurements were achieved by applying rectangular pulses from a function generator (from 50 to 100 Hz at an output level of 1 mV) and directly recording the capacitative current, which occurs at the edge of the pulse. When the monolayers begin to form a bilayer, the capacitative current increases dramatically above the basal level of the Teflon film and reaches a final stable value once the bilayer is formed. These current changes are readily monitored with a storage oscilloscope. The lipid composition of the planar bilayer is critical to the stability and physical nature of the membranes [80]. We have used nonoxidizable phospholipid, diphytanoylphosphatidylcholine (Avanti Polar-Lipids, Alabaster, AL). This lipid forms a bilayer configuration more readily and the resultant bilayers are extremely stable. The conductance (G) of planar bilayers in symmetrical solutions is defined as GZI/V, where I is the transmembrane current flowing through the membranes

130

Y. Muto and K. Kawai

and V corresponds to the clamped membrane potential. Bilayer membranes whose basal conductance was less than 4 pS were used in our experiments.

3.2.4. Electrical equipment and recording The equipment for electrical measurements mainly consists of a voltage-clamp amplifier, a storage oscilloscope and a recording device. The voltage-clamp amplifier is made up of a current-to-voltage converter, a voltage amplifier, a lowpass filter and a high-frequency booster for step response corrections (Fig. 2). A summing amplifier circuit for introducing voltage-clamp commands and rectangular waves is also connected to an input stage of the amplifier. All the amplifier circuits were constructed in our laboratory from ordinary electronic components. Of prime importance to the amplifier design are the sensitivity and signal-to-noise ratio of the I–V converter [81]. We used the OPA104 operational amplifier (Burr-Brown, Tucson, AZ) with a 1 GU feedback resistor as an input circuit for the I–V converter. This combination of electrical components allows measurement of membrane currents at roughly 10K12 A levels, i.e., at a gain of 10K12 A/mV. Power for the I–V converter was supplied by two 9 V batteries, which were mounted on the magnetic stirrer inside the Faraday cage. Voltage outputs from the I–V converter were further amplified and displayed on an oscilloscope as well as a chart recorder [82]. Data were stored on a videotape recorder after A/D conversion using a digital audio processor (PCM-501ES, Sony) with a bandwidth extended to zero frequency by various modifications of the input stage [83,84]. To reduce extraneous noise, the outputs or the stored signals were filtered using Bessel low-pass filters at 5–500 Hz (NF Corporation, Yokohama, Japan). Upon measuring the quinone-induced currents, the compounds were added to one side of the membrane, defined as the cis side, with the other being the trans side. The potential was applied with agar salt bridges to the trans side, while the cis side was virtually grounded using the operational amplifier. The voltages of the trans side are reported in this study. Currents (cations) flowing from the trans to the cis compartment were considered positive and were plotted in an upward direction. The cis solution was stirred continuously with a magnetic stirrer under the applied membrane potential until the electric current reached steady-state levels.

3.3. Mitochondrial techniques 3.3.1. General considerations Mitochondria are intracellular organelles capable of synthesizing ATP by oxidative phosphorylation in eukaryotic organisms [85]. The major part of the machinery for

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

131

oxidative phosphorylation is composed of the electron transfer (respiratory chain) and the ADP phosphorylation (ATP synthase) systems. According to Peter Mitchell’s findings [86], the energy generated in the respiratory chain is used to move protons from the mitochondrial matrix to the mitochondrial intermembrane space in a process known as proton pumping. Because the inner mitochondrial membrane is normally impermeable to protons, respiratory chain-generated proton pumping produces a proton electrochemical potential across the inner mitochondrial membrane, which is used by mitochondrial ATP synthase to generate ATP from ADP and inorganic phosphate. ATP is thus produced in a highly elaborate fashion, a process essential for various cellular activities. The tight coupling of the electron transfer system with the ADP phosphorylation system is essential for continuous production of ATP, but is relatively easily released (uncoupled) by chemicals called uncouplers [87]. Numerous compounds have been demonstrated to be toxic to liver, kidney and heart as a result of inhibitory action on ATP synthesis in mitochondria, either depressing respiration or uncoupling oxidative phosphorylation. Thus, inhibition of mitochondrial function, particularly by uncoupling oxidative phosphorylation, may account for a substantial part of the in vivo toxicity of various compounds [88]. To efficiently assess the effects of quinone compounds on mitochondrial function, we employed isolated rat liver mitochondria as an in vitro assay system. Using isolated mitochondrial preparations, various parameters for respiratory chain inhibition and ionic permeabilities of mitochondrial membranes can easily be determined. In this section, we describe the techniques for mitochondria measurements that are used to elucidate the membrane action of quinone compounds.

3.3.2. Preparation of rat liver mitochondria Diverse types of modified procedures for mitochondrial preparation have been reported by many researchers since a fraction showing octanol oxidation was first isolated from liver homogenate by Schneider [89]. It should be emphasized that the electron-microscopically intact mitochondria are not always those showing tightly coupled respiration. Mitochondria exhibiting tightly coupled respiration can be isolated using a careful technique described here in detail. As a first step in our procedure, a liver is removed from Wistar albino male rat weighing 200–250 g. Mitochondria showing tightly coupled respiration are not prepared from livers congested with blood. It is desirable to eliminate blood from the liver by circulation with isolation medium before liver removal. The isolation medium contains 0.25 M sucrose, 1 mM EDTA and 10 mM Tris (pH 7.4) and should be cooled on ice. The liver is then immediately cooled in the ice-chilled isolation medium and is chopped into fine pieces using scissors while immersed in the medium. After washing with a small volume of the isolation medium (5–10 ml), the pieces are homogenized in a Potter–Elvehjem type Teflon–glass homogenizer at low speed. It is very important

132

Y. Muto and K. Kawai

to use a homogenizer with adequate clearance between the Teflon pestle and the glass tube. All procedures, including centrifugation, should be performed at temperatures below 4 8C. The homogenate is centrifuged at 850g for 10 min in order to remove unbroken cells, blood cells and nuclei as precipitates. The supernatant layer after the first centrifugation should be filtrated through glass fiber in order to remove floating serum lipids and the supernatant layer is again centrifuged at 3000g for 10 min. The precipitates (crude mitochondrial fraction) are thereafter washed three times in the isolation medium. The surface of the precipitate after each centrifugation is carefully washed with a few milliliters of isolation medium and is homogeneously suspended in isolation medium using a glass pipette. After the final centrifugation, the centrifuge tube is carefully shaken, during which the light mitochondrial fraction showing loosely coupled respiration is separated from the heavy mitochondrial precipitate showing tightly coupled respiration. After the fluffy layer is removed by pipetting, the surface of the heavy mitochondria is lightly washed with isolation medium. The precipitates are then suspended in 2–3 ml of reaction medium and kept on ice. It is desirable to keep mitochondria on ice in a small bottle with cotton cap. The isolated mitochondria should be used for experiments within a couple of hours. Freshly prepared mitochondria show respiration with a respiratory control (RC) index of 8–12.

3.3.3. Measurement of mitochondrial respiration Mitochondrial respiration is measured at 30 8C by means of a Galbani-type oxygen electrode (Iijima Electronics IMF Co. Ltd, Japan). Reaction medium is composed of 0.15 M KCl, 5 mM MgCl2, 5 mM inorganic phosphate, 0.5 mM EDTA, 20 mM Tris and 1 mg of mitochondrial protein in a final volume of 2.0 ml (pH 7.4). To equilibrate dissolved oxygen in this medium (237 mM oxygen in distilled water at 30 8C), it is kept at 30 8C for 30 min before starting the experiment. Mitochondrial respiration in vitro is discriminated by state 1–5 respirations according to substrate conditions. State 3 respiration is defined as ADP-driven respiration synthesizing ATP, while state 4 respiration is defined as restricted respiration due to lack of ADP. According to the method of Chance and Williams [90], RC index (a ratio of state 3 respiration to state 4 respiration) and P/O ratio (ratio of added ADP in nmol to consumed oxygen atoms in natom) are calculated from oxygraph data. Typical oxygraph data of mitochondrial respiration are depicted in Fig. 3. Curve 2 shows sample oxygraph data for tightly coupled mitochondrial respiration with a high RC index. The reaction is initiated by adding mitochondria (usually 1 mg protein) to a reaction chamber. The addition of a substrate (L-glutamate/malate in this reaction) gives state 4 respiration and subsequent addition of ADP gives state 3 respiration, which is full respiration that produces ATP. When added ADP is exhausted, state 3 respiration shifts to state 4 respiration. This cycle of state 3 and 4 respirations can be repeated until the dissolved oxygen is exhausted. Curve 1 shows the

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

133

Fig. 3. Typical oxygraph data of mitochondrial respiration. The following abbreviations are used: Glu/Mal, glutamate/malate (substrate); Mt, mitochondria. For details see text. respiration of unsuccessfully prepared mitochondria. In this preparation, mitochondria still show an explicitly accelerated oxygen uptake even at the state 4 respiration due to contamination with light mitochondria exhibiting uncoupled respiration. Uncoupling reagents (uncoupler) accelerate state 4 respiration, decreasing both RC index and P/O ratio. Chemicals that obstruct electron transport along respiratory chains inhibit both state 3 and 4 respirations.

3.3.4. Measurement of mitochondrial swelling The ion permeation of mitochondrial inner membrane is highly regulated in order to keep the membrane potential generated by electron transport along the respiratory chain. When membrane function is disturbed by chemicals or osmotic shock, electrolytes and water passively permeate into the matrix, which is accompanied by swift swelling of mitochondria. The permeability of mitochondrial membranes for ions was measured by following this energy-independent swelling in isosmotic KCl solutions, as described by Brierley [55]. According to this method, the permeability of mitochondrial membranes can be determined quantitatively and rather simply, based on the kinetics of their swelling in various saline solutions. Systematic variation of ionic constituents of the medium further permit one to study the permeability of inner mitochondrial membranes for specific ions under normal and experimental conditions. Mitochondrial swelling is accompanied by decreases in light scattering and light absorption of the mitochondrial suspension. The swelling process can therefore be photometrically monitored

134

Y. Muto and K. Kawai

by using a recording spectrophotometer according to the findings of Tedesch and Harris [91]. The standard reaction mixture contained 300 mmol of KCl, 40 mmol of Tris-chloride (pH 7.4) and 0.8 mg of mitochondrial protein in a final volume of 2.0 ml. Chloride salts of different cations, such as NaC, LiC and RbC, were used to study the relative permeability of inner membranes of mitochondria to these cations. The reaction was carried out at 24 8C in a glass cuvette set in a Hitachi 320S recording spectrophotometer (Hitachi Co. Ltd, Japan). Swelling is usually observed as a decrease in absorbance at around 500 nm. In the present study, the absorbance decrease was measured at 700 nm in order to avoid the interfering effects of light absorption by quinone pigments.

4. EFFECTS OF QUINONES ON MITOCHONDRIAL FUNCTION Here, we provide a simplified description of the effects of quinone pigments on mitochondrial function. Because mitochondria exhibit diverse functions, we mainly focus on the uncoupling activity of quinone compounds. The results described in this section are of considerable significance for the design of the planar bilayer experiments described later.

4.1. Fungal quinones 4.1.1. Effects of versicolorin A and averufin on mitochondrial respiration The effects of versicolorin A and averufin on mitochondrial respiration were studied using an oxygen electrode. Freshly prepared mitochondria exhibit tightly coupled respiration displaying high RC index and P/O ratio values. The addition of versicolorin A or averufin markedly accelerated state 4 respiration and caused significant decreases in the RC index and P/O ratio, which indicates the potent uncoupling effect of both pigments on oxidative phosphorylation in mitochondria. Versicolorin A and averufin were found to exhibit uncoupling effects on both NADand FAD-linked respiration, oxidizing L-glutamate and succinate, respectively [14]. The deteriorating effects of both compounds on FAD-linked respiration are shown in Fig. 4. RC index and P/O ratio were decreased dose dependently by both compounds, indicating a strong uncoupling effect. RC index was more sensitive than P/O ratio to inhibition by the pigments, which is consistent with repression of state 3 respiration. The uncoupling effect of averufin and versicolorin A was the strongest among the naturally occurring compounds reported to date. Versicolorin B, which has a tetrahydrobisfuran ring, also showed an uncoupling effect of the same potency as versicolorin A [64]. In contrast, the O-methylation of versicolorin A and averufin at the 6- and 8-hydroxyl groups completely eliminated

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

135

Fig. 4. Effects of averufin and versicolorin A on mitochondrial respiration. Reaction medium contained 0.15 M KCl, 5 mM MgCl2, 5 mM inorganic phosphate, 0.5 mM EDTA, 20 mM Tris and 1 mg of mitochondrial protein in a final volume of 2.0 ml (pH 7.4). Reaction was carried out at 30 8C. RC index (C) and P/O ratio (B) were calculated from oxygraph data. the uncoupling activity of both compounds, suggesting that the b-hydroxyl group is responsible for their uncoupling effects.

4.1.2. Effects of emodin and skyrin on mitochondrial respiration The effects of emodin and skyrin on mitochondrial respiration were also studied. Both compounds exhibited an uncoupling effect on oxidative phosphorylation, which was clearly substantiated by a decrease in both RC index and P/O ratio (Fig. 5). Emodin showed an uncoupling effect at similar concentrations as those observed for averufin and versicolorin A, whereas skyrin, a dimer of emodin, showed a markedly weaker uncoupling effect than emodin [13]. It is well understood that the strength of the uncoupling effect of chemicals is essentially dependent on lipophilicity and proton dissociation in physiological pH ranges [92,93]. Extremely high or low lipophilicity decreases the uncoupling potential. Therefore, the weaker uncoupling potency of skyrin might be the result of decreased lipophilicity due to an increased number of hydroxyl groups on the anthraquinone nucleus (Fig. 1(4)). In addition, pKa values of emodin and skyrin show some differences, and were determined to be 7.6 and 8.0, respectively. Because pKa value is a measure of proton dissociation, the higher uncoupling potency of emodin may be in part due to its different dissociation characteristics.

136

Y. Muto and K. Kawai

Fig. 5. Effects of emodin and skyrin on mitochondrial respiration. Reaction medium contained 0.15 M KCl, 5 mM MgCl2, 5 mM inorganic phosphate, 0.5 mM EDTA, 20 mM Tris and 1 mg of mitochondrial protein in a final volume of 2.0 ml (pH 7.4). Reaction was carried out at 30 8C. RC index (C) and P/O ratio (B) were calculated from oxygraph data. Physion, produced by Eurotium chevalieri, is the O-methyl ether of emodin but did not show a comparable uncoupling effect on mitochondrial respiration [13]. Parallel results were obtained for skyrin and dimethylskyrin. 1-Hydroxyanthraquinone and several dihydroxyanthraquinones, including some that have been synthesized, were further examined for uncoupling activity. 1-Hydroxyanthraquinone, 1,8-dihydroxyanthraquinone (chrysazin) and islandicin, which have no hydroxyl group at the b position of the anthraquinone nucleus, did not exhibit any uncoupling effect, whereas 1,2-dihydroxyanthraquinone (alizarin) induced uncoupling [94]. These observations again indicate that the hydroxyl group at the b position of the anthraquinone nucleus is closely associated with a potent uncoupling effect on oxidative phosphorylation.

4.2. Blepharismin 4.2.1. Effects of blepharismin on mitochondrial respiration The oxygraph data of mitochondrial respiration oxidizing L-glutamate are depicted in Fig. 6. Curve 4 shows the control experiment in the absence of blepharismin, in which distinct state 3 and 4 respirations were observed. Addition of blepharismin resulted in disappearance of state 3 and 4 respirations and dose-dependent repression of mitochondrial respiration. State 3 respiration was no longer induced by ADP in the presence of blepharismin at 15 nmol/mg protein (7 mM).

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

137

Fig. 6. Effects of blepharismin on mitochondrial respiration. Reaction medium contained 0.15 M KCl, 5 mM MgCl2, 5 mM inorganic phosphate, 0.5 mM EDTA, 20 mM Tris and 1 mg of mitochondrial protein in a final volume of 2.0 ml (pH 7.4). Reaction was initiated by adding substrate (L-glutamate/malate), and was carried out at 30 8C. Glu/Mal, glutamate/malate. Blepharismin released, but very weakly, state 4 respiration, indicating that the uncoupling effect of blepharismin is very weak and that the repressive effect is predominant over the uncoupling effect. Blepharismin is a type of bisanthraquinone compound and has several hydroxyl groups [43]. Spectrophotometric pH titration experiments revealed that pKa value(s) of these hydroxyl groups were at pH values higher than 10 (data not shown), suggesting that blepharismin is not a proton-conductive uncoupler.

138

Y. Muto and K. Kawai

Fig. 7. Induction of mitochondrial swelling by blepharismin. Reaction medium contained 0.15 M KCl, 20 mM Tris, 0.5 mM EDTA and 0.2 mg of mitochondrial protein in a final volume of 2.5 ml (pH 7.4). Reaction was carried out at 25 8C. Curves 1–3, blepharismin-induced swelling; curve 4, control experiment in the presence of BSA (bovine serum albumin).

4.2.2. Induction of mitochondrial swelling by blepharismin The effect of blepharismin on ion permeability of the mitochondrial inner membrane was examined by measuring the induction of mitochondrial swelling in isotonic KCl solution (pH 7.4). As shown in Fig. 7, addition of blepharismin to a mitochondrial suspension caused a rapid decrease in absorbance, indicating induction of mitochondrial swelling at similar concentrations to those for impairing mitochondrial respiration. Considering the results of the experiments using planar bilayer membranes described later, blepharismin might uncouple mitochondrial respiration not by the proton conductivity, but by induction of membrane permeability transition against potassium ions, which is accompanied by proton leak through the inner membrane [88]. Blepharismin induced mitochondrial swelling in isotonic LiCl, NaCl and RbCl as well as KCl mediums, showing no ion selectivity among these alkali metal cations.

5. EFFECTS OF QUINONES ON PLANAR BILAYER MEMBRANES In this section, we describe planar bilayer measurements and characterize the ionic conductance induced by several quinone pigments. The results show that anthraquinone pigments produced by fungi mainly behave as proton carriers (protonophores) in planar bilayer membranes. In contrast, blepharismin, derived

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

139

from ciliated protozoa, form ionic channels in planar bilayers, indicating that channel formation is a toxic mechanism of this quinone pigment.

5.1. Fungal quinones 5.1.1. Effects of versicolorin A on conductance of planar bilayer membranes In a typical experiment, a phospholipid bilayer membrane having very low ionic permeability (membrane conductance of less than 4.0 pS) was produced in the presence of 0.1 M KCl (pH 7.2 buffered with 10 mM Mops–Tris) in aqueous medium. When versicolorin A was added to the cis compartment of the chamber, the transmembrane current began to increase within 1 min. Figure 8 shows the relationship between steady-state current and applied voltage in the presence of versicolorin A. The range of the applied voltage was limited by the membrane electrical stability during steady-state measurement. The steady-state current– voltage characteristics of bilayers modified by versicolorin A were symmetrical and became nonlinear above applied potentials of approximately 50 mV. The specific membrane conductance calculated by regression of the linear portion of the current–voltage curve was 41.8 nS/cm2 in the presence of 27 mM versicolorin A [56]. Steady-state levels of bilayer conductance were usually reached within 5 min and the final conductance levels depended on the concentration of added versicolorin A. These observations indicate that versicolorin A has an ionophore action, which increased ionic permeability of planar bilayer membranes.

Fig. 8. Current–voltage relationship of a diphytanoylphosphatidylcholine bilayer membrane in the presence of versicolorin A. Bilayer membrane was formed in 100 mM KCl buffered at pH 7.2 with 10 mM Mops–Tris. Surface area of the membrane was 4.9!10K4 cm2 and versicolorin A concentration was 27 mM.

140

Y. Muto and K. Kawai

It has been shown that the conductance of bilayers is increased by uncouplers, such as 2,4-dinitrophenol (DNP), as a consequence of increased proton permeability [95]. The high proton permeability has been calculated based on proton diffusion potential, which was determined in a system with different proton concentrations in both compartments (proton concentration gradient) [92]. To investigate whether the conductance induced by versicolorin A was due to increased proton permeability, the same technique was applied to bilayer membranes in the presence of this compound. A planar bilayer membrane was prepared between the compartments, which contained a 10-fold gradient of proton concentrations across the planar membrane, and current was measured at various voltages. As shown by the current–voltage relationship in Fig. 9, reversal potential (voltage that brings the membrane current to zero) for versicolorin A had a positive sign and a value of approximately 54 mV. If the membrane is permeable to protons and to no other ions, the reversal potential is equal to the Nernst potential (EN): EN Z

RT C ln cis ZF Ctrans

where R is the gas constant (taken as 8.314 J/deg/mol), T the absolute temperature in degrees and F the Faraday constant (96,500 C/mol). Z is the

Fig. 9. Current–voltage relationship of a bilayer lipid membrane treated with versicolorin A under asymmetrical HC concentrations. Solutions separated by the bilayer membrane were 100 mM KCl buffered with 10 mM Mops–Tris. The pH of cis side was 7.0 and that of the trans side was 8.0. The reversal potential was estimated to be about 54 mV based on linear interpolation of the data between 40 and 60 mV. Surface area of the membrane was 1.3!10K4 cm2 and versicolorin A concentration was 20 mM.

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

141

valence of the ion and Ccis/Ctrans is the concentration ratio of the ion across the membrane [96]. For bilayers formed in the presence of a 10-fold proton concentration gradient, the Nernst potential is calculated to be 59 mV at 25 8C. Thus, the reversal potential in the presence of versicolorin A is very close to the theoretical value of 59 mV per pH unit for membranes exclusively permeable to protons. This finding suggests that in the presence of versicolorin A, planar bilayer membranes become selectively permeable to protons [56]. The concentrations of versicolorin A employed (Fig. 10, concentration dependency graph) are quite comparable to those used in mitochondrial experiments [64] and thus it may be reasonable to speculate that the uncoupling effect of versicolorin A on mitochondrial oxidative phosphorylation is mediated by an increase in the proton permeability of the mitochondrial membrane. Various compounds have been demonstrated to uncouple mitochondrial respiration [88], and the chemiosmotic hypothesis, in which the electrochemical potential generated by the proton gradient across the inner membranes is abolished by nonvectorial proton-carrying cycles of uncouplers, is generally accepted as the molecular mechanism of the uncoupling effect [97]. The protonophoretic action of versicolorin A demonstrated here agrees with this hypothesis of the mode of action of uncouplers on mitochondrial respiration.

5.1.2. Characteristics of planar bilayer membranes in the presence of versicolorin A In order to evaluate the carrier stoichiometry, bilayer conductance was measured as a function of versicolorin A concentration in the aqueous phase. Figure 10a shows plots of conductance versus versicolorin A concentration, and reveals that conductance is not linearly proportional to versicolorin A concentration. When the numerical values in Fig. 10a are plotted on logarithmic scales, an almost linear relationship is obtained (Fig. 10b). From the slope of the line, it is obvious that the conductance of a bilayer membrane varies quadratically with the total concentration of versicolorin A added to the aqueous phase. This suggests that the relative stoichiometry of versicolorin A and protons is not 1:1. Figure 11a illustrates the effects of pH on the relationship between steady-state current and applied voltage in the presence of versicolorin A. While the current and voltage curve showed similar patterns among different pH levels, the magnitude of conductance greatly varied depending on pH. In Fig. 11b, the dependence of bilayer conductance on pH at a fixed concentration of versicolorin A is shown. The maximum conductance is seen at around pH 8.0, which corresponds to the pKa of versicolorin A. Weak acids such as DNP act as carriers for protons, and the term proton ionophores or ‘protonophores’ is used [88]. These compounds efficiently increase proton permeability in mitochondrial membranes, thereby uncoupling

142

Y. Muto and K. Kawai

Fig. 10. Dependence of planar membrane conductance on versicolorin A concentration. (a) Values of conductance were plotted against versicolorin A concentrations in the cis compartment. Solutions separated by bilayer membrane contained 100 mM KCl buffered at pH 7.2 with 10 mM Mops–Tris. The membrane potential was clamped at C50 (C) or K50 mV (B). (b) The membrane conductance is a quadratic function of the aqueous concentration of versicolorin A. Abscissa: logarithm of the aqueous versicolorin A concentration. Ordinate: logarithm of the membrane conductance. Data are taken from the membrane depicted in (a). oxidation from phosphorylation (uncouplers). Dilger and McLaughlin [98,99] made a detailed study on the transport kinetics of weak acid uncouplers using artificial lipid bilayer membranes, and classified them into two groups. The first group is the AK class of weak acid protonophores for which the anionic form of

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

143

Fig. 11. Effect of aqueous phase pH on planar membrane conductance. (a) Current–voltage relationship of versicolorin A treated bilayers at several pH values. The current passing through the bilayer membrane was measured in 100 mM KCl solution whose pH was buffered at 7.0 (C), 7.5 (B) and 8.0 (-) with 10 mM Mops–Tris. The concentration of versicolorin A was 30 mM. (b) Dependence of conductance on pH at a fixed versicolorin A concentration of 27 mM. The conductances were determined at a membrane potential of C50 (C) or K50 mV (B). Other conditions were the same as in (a).

the weak acid, AK, acts as the charged permeant species in the membrane. These include the most potent protonophores, such as carbonylcyanide mchlorophenylhydrazone (CCCP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 5-chloro-3-tert-butyl-2 0 -chloro-4 0 -nitrosalicylanilide (S13). The second group is the HAK 2 class of weak acid protonophores. These compounds include DNP, 5,6-dichloro-2-trifluoromethylbenzimidazole (DTFB) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB). For this class of weak acids the charged permeant species is the HAK 2 dimer, a complex of the anionic (AK) and the protonated (HA) forms of the weak acid. According to their theoretical considerations [99], the model for HAK 2 protonophores predicts that the conductance should vary quadratically with the total concentration of uncoupler added to the aqueous phase and be maximal when pHZpKa. The data for versicolorin A described here agree strongly with the predicted properties of HAK 2 weak acid protonophores. Because versicolorin A has weakly dissociable hydroxyl group, quadratic relationship observed suggests that it has a similar transport mechanism to HAK 2 protonophores, such as DTFB and TTFB.

144

Y. Muto and K. Kawai

5.1.3. Effects of averufin on conductance of planar bilayer membranes The anthraquinone mycotoxin averufin, which is a precursor of versicolorin A in the aflatoxin B1 biosynthetic route, exerts a potent uncoupling effect on mitochondrial respiration that is comparable to that of versicolorin A [64]. Averufin was also tested for its effects on electrical conductance of planar bilayer membranes and was compared with versicolorin A. Figure 12a shows the relationship between steady-state current and applied voltage in the presence of averufin. Similar to versicolorin A, averufin increased the conductance of planar bilayer membranes, but exhibited its effects at lower concentrations than versicolorin A [58]. The steady-state current–voltage characteristics of bilayers modified by averufin were also symmetrical and became nonlinear at higher applied potentials (Fig. 12a). The specific membrane conductance calculated by regression of the linear portion of the current–voltage curve was 270 nS/cm2 in the presence of 28 mM averufin. The current–voltage relationship of the planar bilayer modified by averufin was then determined in the presence of a 10-fold proton gradient across the bilayer. As shown in Fig. 12b, the reversal potential for the bilayer in the presence of averufin

Fig. 12. Effect of averufin on planar bilayer conductance. (a) Current–voltage relationship under varying concentrations of averufin. Planar bilayer membrane was formed in 100 mM KCl buffered at pH 7.2 with 10 mM Mops–Tris. Surface area of the membrane was 2.3!10K4 cm2. Averufin concentrations were as follows: -, 28 mM; B, 21 mM; C, 14 mM. (b) Current–voltage relationship of bilayer lipid membrane treated with averufin under asymmetrical HC concentrations. Solutions separated by the bilayer membrane were 100 mM KCl buffered with 10 mM Mops–Tris. The pH of cis side was 7.0 and that of the trans side was 8.0. The reversal potential was estimated to be about 30 mV. Surface area of the membrane was 2.3!10K4 cm2 and averufin concentration was 27 mM.

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

145

was about 30 mV. Because this value is quite different from the theoretical value for membranes exclusively permeable to protons, it appears that averufin increased permeability to protons as well as other ions, such as KC. We have recently found evidence showing that averufin and versicolorin A do not always display identical biological activities. Averufin induced drastic mitochondrial swelling in an isotonic solution of alkali metal ions, but versicolorin A did not induce such swelling (unpublished data). These observations suggest that the averufin-induced permeability of ions other than protons might be implicated in the mitochondrial swelling and that the uncoupling mechanism of averufin may be somewhat different from that of versicolorin A.

5.1.4. Effects of emodin and skyrin on conductance of planar bilayer membranes As described in previous sections, emodin and its dimer, skyrin, are produced by yellow rice mold, P. islandicum, and uncouple oxidative phosphorylation in mitochondria [13]. For these compounds, we briefly investigated the effects on planar bilayer membranes. Figure 13 shows the current–voltage relationships of bilayer membranes in the presence of emodin or skyrin. The results indicate that both quinones increase the conductance of planar bilayer membranes, but the effects of emodin are stronger than those of skyrin. The specific membrane conductance for 25 mM emodin and skyrin were 51 and 10 nS/cm2, respectively.

Fig. 13. Current–voltage relationship of bilayer lipid membrane in the presence of emodin and skyrin. Bilayer membrane was formed in 100 mM KCl buffered at pH 7.2 with 10 mM Mops–Tris. Surface area of the membrane was 4.2!10K4 cm2. Membrane current was measured in the presence of 25 mM emodin (C) or 25 mM skyrin (B).

146

Y. Muto and K. Kawai

5.2. Blepharismin 5.2.1. Effects of blepharismin on conductance of planar bilayer membranes After the exposure of a planar lipid bilayer to blepharismin from Blepharisma japonicum, drastic increases of membrane conductance were observed. Figure 14 shows the effects of addition of blepharismin-2 (final concentration 5 mg/ml) to the cis side of a planar lipid bilayer formed by diphytanoylphosphatidylcholine. The trace shows the membrane current as a function of time, with a voltage of 40 mV imposed across the membrane. Initially, there was little current flow, due to the inherent impermeability of lipid membranes to ions, but after addition of blepharismin-2 (arrow), the current gradually increased. Following blepharismin addition, channel-like fluctuations of the bilayer conductance were initially seen,

Fig. 14. Induction of ionic current in a planar lipid bilayer membrane by blepharismin. The membrane current trace is shown as a function of time. The membrane voltage (voltage of the trans compartment with respect to the cis compartment to which blepharismin was added) was held constant at 40 mV, and the current through an unmodified lipid bilayer membrane was very low. Addition of 5 mg/ml of blepharismin-2 (arrow) to the aqueous phase induced current fluctuations and dramatically increased membrane current. The lipid bilayer membrane was composed of diphytanoylphosphatidylcholine and the aqueous salt solution in both compartments was 100 mM KCl, 10 mM Mops– Tris, pH 7.2. (Reprinted with the permission of the Federation of the European Biochemical Societies from Y. Muto, T. Matsuoka, A. Kida, Y. Okano, Y. Kirino, Blepharismins, produced by the protozoan, Blepharisma japonicum, form ionpermeable channels in planar lipid bilayer membranes, FEBS Lett. 508 (2001) 423–426.)

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

147

which, after several minutes, were replaced by large current fluctuations. Under the conditions described above, the membranes were significantly destabilized and the current increased continuously until the membrane broke. In the absence of blepharismin, no increase in, or fluctuation of, the membrane current was seen. Because membrane conductance reflects the permeability to ions in the bathing solution (for example KC or ClK), we concluded that blepharismin-2 increased the ionic permeability of lipid bilayer membranes [57]. For this effect, it was only necessary to add blepharismin-2 to one side of the bilayer. The other four homologous blepharismins (1, 3, 4 and 5) [43] induced similar conductance changes (data not shown). Blepharismin-2 is the only blepharismin distributed throughout the entire cell body in Blepharisma [70], and thus it was used as a model of this class of compounds in the following experiments. In order to characterize the conductance induced by blepharismin, the cis side of the membrane was perfused with bathing solution after addition of blepharismin and this resulted in cessation of the current increase and in steady-state conductance. All the results described below were obtained in experiments in which steady-state conductance was observed after perfusion. The ionic selectivity of bilayers modified with blepharismin-2 was determined by measuring the potential at zero current flow (reversal potential) in the presence of a KCl concentration gradient across the bilayer (200–70 mM). As shown in Fig. 15, a reversal potential of C19 mV (with reference to the side with lower KCl concentration) was observed. The positive sign of the potential indicates preferential cation selectivity. From the reversal potential Erev and the concentration gradient Ccis/Ctrans across the membrane, the ratio Pc/Pa of permeabilities (Pc for cations and Pa for anions) was calculated using the Goldman–Hodgkin– Katz equation [100,101]: Erev Z

RT Pc Ccis C Pa Ctrans ln F Pc Ctrans C Pa Ccis

where R, T and F have their usual thermodynamic meanings. For the numerical values described above, the relative ionic selectivity (Pc:Pa) of the channels for KC and ClK was estimated to be about 6.6:1. At this concentration difference, an ideally cation-selective channel would yield a potential of approximately C27 mV.

5.2.2. Characteristics of the channels formed with blepharismin Using a small aperture and a low blepharismin concentration, individual fluctuations, which might constitute macroscopic membrane current, could be easily resolved. As shown in Fig. 16a, discrete stepwise conductance fluctuations could be routinely observed after addition of a small amount of blepharismin. Because the current fluctuation consisted of unitary digital changes, it might reflect the opening and closing behaviors of single channels, suggesting the existence of

148

Y. Muto and K. Kawai

Fig. 15. Macroscopic current–voltage relationship for blepharismin-induced conductance in an asymmetrical solution of KCl. The solutions were 200 mM KCl, 10 mM Mops–Tris (pH 7.2) on the cis side and 70 mM KCl, 10 mM Mops–Tris (pH 7.2) on the trans side. Before the experiment shown, blepharismin-2 (5 mg/ml final concentration) was added to the cis compartment, then the cis side of the membrane was perfused with bathing solution. Current amplitudes were measured at the beginning of the voltage changes. The reversal potential was obtained from the intercept on the x-axis. (Reprinted with the permission of the Federation of the European Biochemical Societies from Y. Muto, T. Matsuoka, A. Kida, Y. Okano, Y. Kirino, Blepharismins, produced by the protozoan, Blepharisma japonicum, form ion-permeable channels in planar lipid bilayer membranes, FEBS Lett. 508 (2001) 423–426.) defined single pores. However, the amplitude of the various steps was highly variable. A histogram of the blepharismin-2 single-channel conductances seen under these conditions (Fig. 16b) shows a well-defined peak close to 0.8–1 nS, but the single-channel conductance was quite heterogeneous, ranging from 0.2 to 2.8 nS, possibly reflecting different states of aggregation of blepharismin and/or different conformations within the membrane [102,103]. We next studied the voltage sensitivity of blepharismin-induced conductivity using multichannel bilayers, the molecule being applied to the cis side. Figure 17 shows the current response of a blepharismin-treated membrane to a series of

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

149

Fig. 16. Single-channel currents induced by blepharismin. (a) The current due to single channels induced by blepharismin-2 at a final concentration of 0.5 mg/ml is shown. The aqueous salt solutions contained 100 mM KCl, 10 mM Mops–Tris, pH 7.2, and the membrane was formed from diphytanoylphosphatidylcholine. The membrane voltage was held constant at C40 mV throughout this recording. Note that current jumps of varying magnitude can be seen, reflecting the heterogeneity of channels induced by blepharismin. (b) Histogram of blepharismin single-channel conductances. Data are taken from the membrane depicted in (a). (Reprinted with the permission of the Federation of the European Biochemical Societies from Y. Muto, T. Matsuoka, A. Kida, Y. Okano, Y. Kirino, Blepharismins, produced by the protozoan, Blepharisma japonicum, form ion-permeable channels in planar lipid bilayer membranes, FEBS Lett. 508 (2001) 423–426.)

various voltage steps. The data show that subjecting the bilayer to positive and negative voltages had no measurable effect on its initial conductivity (instantaneous current), but the current frequently declined with time, accompanying closing events of the single channels, particularly when switching from zero to negative voltage (on the side of the blepharismin-free compartment). It should be noted that a brief reduction in the applied potential (to zero) brings about restoration of the conduction process, i.e., opening of the channels, as is clearly shown in the second and third responses in Fig. 17. The instantaneous current

150

Y. Muto and K. Kawai

Fig. 17. Multichannel conductance induced by blepharismin at different voltages. The currents in response to a series of voltage steps are shown for a membrane to which blepharismin-2 was added at a final concentration of 2 mg/ml; the other conditions are identical to those in Fig. 16. The inset illustrates the plots for instantaneous (,) and steady-state (C) currents as a function of voltage. (Reprinted with the permission of the Federation of the European Biochemical Societies from Y. Muto, T. Matsuoka, A. Kida, Y. Okano, Y. Kirino, Blepharismins, produced by the protozoan, Blepharisma japonicum, form ion-permeable channels in planar lipid bilayer membranes, FEBS Lett. 508 (2001) 423–426.) flowing through the membrane responded almost linearly to the applied voltage and was symmetrical with respect to the polarity of the electrical field (Fig. 17, inset). In the study described above, direct current measurement in planar lipid bilayers treated with blepharismin demonstrated the ability of this molecule to form time-variant cation-selective channels. Moreover, the results of the mitochondrial swelling experiments described above are quite compatible with channel formation by blepharismin. The discrete conductance fluctuations (Fig. 16a) and the large current fluctuations observed in the multichannel state (Fig. 14) clearly exclude the possibility of a carrier-mediated increase in bilayer conductance. However, the structure of the channels formed by blepharismin is unknown. The molecular structure of blepharismin (Fig. 1(5)) suggests that a single molecule does not form an ion channel in the membrane. In addition, the presence of polar OH groups in the peripheral region would probably prevent a single blepharismin molecule from being localized in the internal hydrophobic part of a lipid bilayer. Taking into account the fact that blepharismin can form channels of varying

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

151

conductance levels (Fig. 16b), we hypothesize that different sized clusters of blepharismin molecules are responsible for forming channels of different conductances. Similar single-channel heterogeneity has been observed for other small molecule channel formers, such as melittin [102], defensin [103], coumarin antibiotic [104] and beticolin [105].

6. CONCLUDING REMARKS Several lines of evidence have demonstrated that the induced permeabilization of cellular membranes is one of the most common injuring mechanisms of cytotoxic molecules. In the case of several peptidic or nonpeptidic toxins, the formation of transmembrane pores is considered to be responsible for membrane permeabilization and cell lysis [103,106,107]. The results described here further demonstrate that anthraquinone pigments, such as versicolorin A, averufin, emodin and skyrin, greatly increase the conductance of lipid bilayer membranes. The concentrations employed are quite comparable to those used in mitochondrial experiments [13,14] and thus the effects of these toxins on mitochondrial oxidative phosphorylation might be mediated by increases in proton permeability of mitochondrial membranes. On the other hand, the concentration range in which blepharismin induced the conductance increases is the same as that at which it is toxic for various free-swimming protozoa [36,38] and mammalian cells [35]. It is thus possible that channel formation, resulting in dissipation of the electrochemical gradient and subsequent cell lysis, might be the main physiological effect of blepharismin on the target cell membrane. Therefore, it follows that membrane permeabilization by a carrier or channel mechanism plays an important role in the cellular actions of toxic quinone compounds derived from various microorganisms. The experimental results presented in this article indicate that analysis using planar lipid bilayer membranes may assist in increasing our understanding of the membrane action of various quinone compounds [47,108]. Moreover, the approaches described in this chapter, in conjunction with other biochemical studies using biological membranes, offer powerful tools for answering important questions regarding the structure and function of a diverse range of naturally occurring compounds.

ACKNOWLEDGEMENTS We are grateful to Dr Tatsuomi Matsuoka and Dr Terue Harumoto for supplying blepharismins. Y. M. is indebted to Dr Yutaka Kirino for introducing him to the planar bilayer technique and for advice on amplifier construction. We also thank Mr. Fumiaki Muto for expert assistance in the preparation of illustrations in this chapter.

152

Y. Muto and K. Kawai

REFERENCES [1] R.H. Thomson, Naturally Occurring Quinones, Academic Press, London, 1971. [2] W.B. Turner, D.C. Aldridge, Fungal Metabolites II, Academic Press, London, 1983. [3] R.J. Cole, R.H. Cox, Handbook of Toxic Fungal Metabolites, Academic Press, New York, 1981. [4] H. Mori, K. Kawai, F. Ohbayashi, T. Kuniyasu, M. Yamazaki, T. Hamasaki, G.M. Williams, Genotoxicity of a variety of mycotoxins in the hepatocyte primary culture/DNA repair test using rat and mouse hepatocytes, Cancer Res. 44 (1984) 2918–2922. [5] H. Mori, J. Kitamura, S. Sugie, K. Kawai, T. Hamasaki, Genotoxicity of fungal metabolites related to aflatoxin B2 biosynthesis, Mutat. Res. 143 (1985) 121–125. [6] J.P. Brown, R.J. Brown, Mutagenesis by 9,10-anthraquinone derivatives and related compounds in Salmonella typhimurium, Mutat. Res. 40 (1976) 203–224. [7] J.P. Brown, P.S. Dietrich, Mutagenicity of anthraquinone and benzanthrone derivatives in the Salmonella/microsome test: activation of anthraquinone glycosides by enzymic extracts of rat cecal bacteria, Mutat. Res. 66 (1979) 9–24. [8] M. Enomoto, I. Ueno, Penicillium islandicum (toxic yellowed rice)–luteoskyrin– islanditoxin–cyclochlorotine, in: I.F.H. Purchase (Ed.), Mycotoxins, Elsevier, Amsterdam, 1974, pp. 303–326. [9] Y. Ueno, N. Sato, T. Ito, I. Ueno, M. Enomoto, H. Tsunoda, Chronic toxicity and hepatocarcinogenicity of (C)rugulosin, an anthraquinoid mycotoxin from penicillium species: preliminary surveys in mice, J. Toxicol. Sci. 5 (1980) 295–302. [10] H. Porumb, I. Petrescu, Interaction with mitochondria of the anthracycline cytostatics adriamycin and daunomycin, Prog. Biophys. Mol. Biol. 48 (1986) 103–125. [11] Y. Ueno, I. Ueno, N. Sato, Y. Iitoshi, M. Saito, M. Enomoto, H. Tsunoda, Toxicological approach to (K)rugulosin, an anthraquinoid mycotoxin of Penicillium rugulosum, Jpn. J. Exp. Med. 41 (1971) 177–188. [12] M. Umeda, M. Saito, S. Shibata, Comparison of cytotoxic effects of various anthraquinonoids on cultured mammalian cells, Jpn. J. Exp. Med. 44 (1974) 249–255. [13] K. Kawai, T. Kato, H. Mori, J. Kitamura, Y. Nozawa, A comparative study on cytotoxicities and biochemical properties of anthraquinone mycotoxins emodin and skyrin from Penicillium islandicum Sopp, Toxicol. Lett. 20 (1984) 155–160. [14] K. Kawai, Y. Nozawa, Y. Maebayashi, M. Yamazaki, T. Hamasaki, Averufin, an anthraquinone mycotoxin possessing a potent uncoupling effect on mitochondrial respiration, Appl. Environ. Microbiol. 47 (1984) 481–483. [15] K. Kawai, Y. Nozawa, H. Mori, Y. Ogihara, Inhibition of mitochondrial respiration by flavoskyrin, a toxic metabolite of Penicillium islandicum Sopp, Toxicol. Lett. 30 (1986) 105–111. [16] T. Ubbink-Kok, J.A. Anderson, W.N. Konings, Inhibition of electron transfer and uncoupling effects by emodin and emodinanthrone in Escherichia coli, Antimicrob. Agents Chemother. 30 (1986) 147–151. [17] V. Betina, S. Kuzela, Uncoupling effect of fungal hydroxyanthraquinones on mitochondrial oxidative phosphorylation, Chem. Biol. Interact. 62 (1987) 179–189. [18] M.R. Sevenants, Pigments of Blepharisma undulans compared with hypericin, J. Protozool. 12 (1965) 240–245. [19] A.C. Giese, Blepharisma, The Biology of a Light-Sensitive Protozoan, Stanford University Press, Stanford, 1973. [20] T. Matsuoka, D. Tokumori, H. Kotsuki, M. Ishida, M. Matsushita, S. Kimura, T. Itoh, G. Checcucci, Analyses of structure of photoreceptor organelle and blepharisminassociated protein in unicellular eukaryote Blepharisma, Photochem. Photobiol. 54 (2000) 709–713.

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

153

[21] T. Matsuoka, N. Moriyama, A. Kida, K. Okuda, T. Suzuki, H. Kotsuki, Immunochemical analysis of a photoreceptor protein using anti-IP3 receptor antibody in the unicellular organism, Blepharisma, J. Photochem. Photobiol. B 1143 (1993) 131–135. [22] T. Yamazaki, I. Yamazaki, Y. Nishimura, R. Dai, P.S. Song, Time-resolved fluorescence spectroscopy and photolysis of the photoreceptor blepharismin, Biochim. Biophys. Acta 1143 (1993) 319–326. [23] P.S. Song, I.H. Kim, S. Florell, N. Tamai, T. Yamazaki, I. Yamazaki, Structure and function of the photoreceptor stentorins in Stentor coeruleus. II. Primary photoprocess and picosecond time-resolved fluorescence, Biochim. Biophys. Acta 1040 (1990) 58–65. [24] I.H. Kim, J.S. Rhee, J.W. Huh, S. Florell, B. Faure, K.W. Lee, T. Kahsai, P.S. Song, N. Tamai, T. Yamazaki, Structure and function of the photoreceptor stentorins in Stentor coeruleus. I. Partial characterization of the photoreceptor organelle and stentorins, Biochim. Biophys. Acta 12 (1983) 43–57. [25] P.S. Song, Protozoan and related photoreceptors: molecular aspects, Annu. Rev. Biophys. Bioeng. 587 (1979) 35–68. [26] E.B. Walker, T.Y. Lee, P.S. Song, Spectroscopic characterization of the Stentor photoreceptor, Biochim. Biophys. Acta 32 (1962) 129–144. [27] K.M. Moller, On the nature of stentorin, C. R. Trav. Lab. Carlsberg 32 (1962) 471–498. [28] A. Kida, Y. Takada, H. Kotsuki, D. Tokumori, G. Checcucci, T. Matsuoka, Primary stages in photosignal transduction leading to step-up photophobic response in the unicellular eukaryote Blepharisma japonicum, Microbios 289 (2001) 189–201. [29] T. Matsuoka, H. Kotsuki, Photosensory transduction in unicellular eukaryote Blepharisma, J. Exp. Zool. 62 (1995) 467–471. [30] T. Matsuoka, Y. Watanabe, Y. Sagara, M. Takayanagi, Y. Kato, Additional evidence for blepharismin photoreceptor pigment mediating step-up photophobic response of unicellular organism, Blepharisma, Photochem. Photobiol. 3 (1989) 190–193. [31] F. Lenci, F. Ghetti, Photoreceptor pigments for photomovement of microorganisms: some spectroscopic and related studies, J. Photochem. Photobiol. B 3 (1989) 1–16. [32] H. Falk, From the photosensitizer hypericin to the photoreceptor stentorin – the chemistry of phenanthroperylene quinones, Angew. Chem. Int. Ed. Engl. 3 (1989) 3116–3136. [33] P.S. Song, K.J. Tapley Jr., J.D. Berlin, The photoreceptor in Stentor coeruleus, Symp. Soc. Exp. Biol. 12 (1983) 503–520. [34] P.S. Song, Protozoan and related photoreceptors: molecular aspects, Annu. Rev. Biophys. Bioeng. 12 (1983) 35–68. [35] Y. Kato, Y. Watanabe, Y. Sagara, Y. Murakami, M. Sugiyama, T. Matsuoka, The photoreceptor pigment of the unicellular organism Blepharisma generates hydroxy radicals, J. Photochem. Photobiol. B 34 (1996) 29–33. [36] M.N. Terazima, H. Iio, T. Harumoto, Toxic and phototoxic properties of the protozoan pigments blepharismin and oxyblepharismin, Photochem. Photobiol. 69 (1999) 47–54. [37] A. Miyake, T. Harumoto, B. Salvi, R. Rivola, Defensive function of pigment granules of Blepharisma japonicum, Eur. J. Protistol. 25 (1990) 310–315. [38] T. Harumoto, A. Miyake, N. Ishikawa, R. Sugibayashi, K. Zenfuku, H. Iio, Chemical defense by means of pigmented extrusomes in the Ciliate Blepharisma japonicum, Eur. J. Protistol. 34 (1998) 458–470. [39] K. Kawai, K. Hisada, H. Mori, Y. Nozawa, Molecular approach to the toxic action of quinone mycotoxins – chemical structure and biochemistry, in: M. Borgers, R. Hay, M.G. Rinaldi (Eds.), Current Topics in Medical Mycology, Springer, Heidelberg, 1992, pp. 207–230.

154

Y. Muto and K. Kawai

[40] A. Miyake, Cell–cell interaction by means of extrusomes in ciliates – particularly on the predator–prey interaction mediated by extrusomal toxins (in Japanese), Jpn. J. Protozool. 35 (2002) 97–117. [41] K. Kawai, J. Kitamura, T. Hamasaki, Y. Nozawa, The mode of action of anthraquinone mycotoxins on ATP synthesis in mitochondria, in: A.E. Pohland, V.R. Dowell, J.L. Richard (Eds.), Microbial Toxins in Foods and Feeds, Plenum Press, New York, 1990, pp. 369–375. [42] G. Checcucci, R.S. Shoemaker, E. Bini, R. Cerny, N. Tao, J.S. Hyon, F. Gioffre, F. Ghetti, F. Lenci, P.S. Song, Chemical structure of blepharismin, the photosensor pigment for Blepharisma japonicum, J. Am. Chem. Soc. 119 (1997) 5762–5763. [43] M. Maeda, H. Naoki, T. Matsuoka, Y. Kato, H. Kotsuki, K. Utsumi, T. Tanaka, Blepharismin 1–5, novel photoreceptor from the unicellular organism Blepharisma japonicum, Tetrahedron Lett. 38 (1997) 7411–7414. [44] B. de Kruijff, R.A. Demel, Polyene antibiotic–sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. 3. Molecular structure of the polyene antibiotic–cholesterol complexes, Biochim. Biophys. Acta 339 (1974) 57–70. [45] H. Vogel, Incorporation of melittin into phosphatidylcholine bilayers. Study of binding and conformational changes, FEBS Lett. 134 (1981) 37–42. [46] R. Chaloupka, T. Obsil, J. Plasek, F. Sureau, The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts, Biochim. Biophys. Acta 1418 (1999) 39–47. [47] H.T. Tien, A. Ottova, The lipid bilayer concept and its experimental realization: from soap bubbles, kitchen sink, to bilayer lipid membranes, J. Membr. Sci. 189 (2001) 83–117. [48] H.T. Tien, A. Ottova, Planar Lipid Bilayers (BLMs) and Their Applications, Elsevier, Amsterdam, 2003. [49] J.E. Hall, Channels and carriers in lipid bilayers, in: S.L. Bonting, J.J.H.H.M. de Pont (Eds.), Membrane Transport, Elsevier/North-Holland Biomedical Press, Amsterdam, 1981, pp. 107–121. [50] R. Latorre, O. Alvarez, Voltage-dependent channels in planar lipid bilayer membranes, Physiol. Rev. 61 (1981) 77–150. [51] O.S. Anderson, W.N. Green, B.W. Urban, Ion conduction through sodium channels in planar lipid bilayers, in: C. Miller (Ed.), Ion Channel Reconstitution, Plenum Press, New York, 1986, pp. 385–404. [52] B.L. Kagan, Y. Sokolov, Use of lipid bilayer membranes to detect pore formation by toxins, Methods Enzymol. 235 (1994) 691–705. [53] A.Y. Abramov, M.V. Zamaraeva, A.I. Hagelgans, R.R. Azimov, O.V. Krasilnikov, Influence of plant terpenoids on the permeability of mitochondria and lipid bilayers, Biochim. Biophys. Acta 1512 (2001) 98–110. [54] J.B. Chappel, A.R. Crofts, Ion transport and reversible volume changes of isolated mitochondria, in: J.M. Tager, S. Pappa, E. Quagliariello, E.C. Slator (Eds.), Regulation of Metabolic Processes in Mitochondria, Elsevier, Amsterdam, 1966, pp. 293–316. [55] G.P. Brierley, Passive permeability and energy-linked ion movements in isolated heart mitochondria, Ann. N. Y. Acad. Sci. 227 (1974) 398–411. [56] Y. Muto, M. Matsunami, K. Kawai, T. Hamasaki, The effect of versicolorin A on electrical conductance in planar lipid bilayer membranes, Mycotoxins 44 (1997) 45–48. [57] Y. Muto, T. Matsuoka, A. Kida, Y. Okano, Y. Kirino, Blepharismins, produced by the protozoan, Blepharisma japonicum, form ion-permeable channels in planar lipid bilayer membranes, FEBS Lett. 508 (2001) 423–426.

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

155

[58] Y. Muto and K. Kawai, The effects of polyhydroxyanthraquinones averufin and versicolorin A on the electric resistance of the artificial phospholipid planar membranes, Fourth International Conference on Biological Physics, Abstracts, 2001, p. 43. [59] J.I. Clifford, K.R. Rees, Aflatoxin: a site of action in the rat liver cell, Nature 209 (1966) 312–313. [60] P.M. Newberne, W.H. Butler, Acute and chronic effects of aflatoxin on the liver of domestic and laboratory animals: a review, Cancer Res. 29 (1969) 236–250. [61] M.T. Lin, D.P. Hsieh, Averufin in the biosynthesis of aflatoxin B, J. Am. Chem. Soc. 95 (1973) 1668–1669. [62] L.S. Lee, J.W. Bennett, A.F. Cucullu, J.B. Stanley, Synthesis of versicolorin A by a mutant strain of Aspergillus parasiticus deficient in aflatoxin production, J. Agric. Food Chem. 23 (1975) 1132–1134. [63] J.J. Wong, R. Singh, D.P. Hsieh, Mutagenicity of fungal metabolites related to aflatoxin biosynthesis, Mutat. Res. 44 (1977) 447–450. [64] K. Kawai, Y. Nozawa, Y. Maebayashi, M. Yamazaki, T. Hamasaki, The inhibition of mitochondrial respiration by anthraquinone mycotoxins, averufin and versicolorins A, B, Proc. Jpn. Assoc. Mycotoxicol. 18 (1983) 35–37. [65] A.C. Ghosh, A. Manmade, A.L. Demain, Toxins from Penicillium islandicum Sopp, in: J.V. Rodricks, C.W. Hesseltine, M.A. Mehlman (Eds.), Mycotoxins in Human and Animal Health, Pathotox Publishers, New York, 1977, pp. 625–638. [66] K. Uraguchi, M. Saito, Y. Noguchi, K. Takahashi, M. Enomoto, Chronic toxicity and carcinogenicity in mice of the purified mycotoxins, luteoskyrin and cyclochlorotine, Food Cosmet. Toxicol. 10 (1972) 193–207. [67] T. Masuda, Y. Ueno, Microsomal transformation of emodin into a direct mutagen, Mutat. Res. 125 (1984) 135–144. [68] F. Inaba, S. Nakamura, S. Yamaguchi, An electronmicroscopic study on the pigment granules of Blepharisma, Cytologia (Tokyo) 23 (1958) 72–79. [69] J.R. Kennedy, The morphology of Blepharisma undulans Stein, J. Protozool. 12 (1965) 542–561. [70] T. Matsuoka, M. Sato, H. Maeda, T. Naoki, H. Tanaka, H. Kotsuki, Localization of blepharismin photosensors and identification of a photoreceptor complex mediating the step-up photophobic response of the unicellular organism, Blepharisma, Photochem. Photobiol. 65 (1997) 915–921. [71] R.J. Cole, M.A. Schweikert, B.B. Jarvis, Handbook of Secondary Fungal Metabolites, Academic Press, London, 2003, Vol. I. [72] V. Betina, Mycotoxins: Chemical, Biological and Environmental Aspects, Elsevier, Amsterdam, 1989. [73] P. Mueller, D.O. Rudin, H.T. Tien, W.C. Wescott, Reconstitution of cell membrane structure in vitro and its transformation into an excitable system, Nature 194 (1962) 979–980. [74] M. Montal, P. Mueller, Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties, Proc. Natl Acad. Sci. USA 69 (1972) 3561–3566. [75] R. Benz, O. Fro¨hlich, P. La¨uger, M. Montal, Electrical capacity of black lipid films and of lipid bilayers made from monolayers, Biochim. Biophys. Acta 394 (1975) 323–334. [76] M. Montal, Formation of bimolecular membranes from lipid monolayers, Methods Enzymol. 32 (1974) 545–554. [77] M. Montal, R. Anholt, P. Labarca, The reconstituted acetylcholine receptor, in: C. Miller (Ed.), Ion Channel Reconstitution, Plenum Press, New York, 1986, pp. 157–204. [78] P. Labarca, J. Lindstrom, M. Montal, Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers, J. Gen. Physiol. 83 (1984) 473–496.

156

Y. Muto and K. Kawai

[79] R. Hartshorne, M. Tamkun, M. Montal, The reconstituted sodium channel from brain, in: C. Miller (Ed.), Ion Channel Reconstitution, Plenum Press, New York, 1986, pp. 337–362. [80] S.H. White, The physical nature of planar bilayer membranes, in: C. Miller (Ed.), Ion Channel Reconstitution, Plenum Press, New york, 1986, pp. 3–35. [81] O. Alvarez, How to set up a bilayer system, in: C. Miller (Ed.), Ion Channel Reconstitution, Plenum Press, New York, 1986, pp. 115–130. [82] K. Anzai, M. Hamasuna, H. Kadono, S. Lee, H. Aoyagi, Y. Kirino, Formation of ion channels in planar lipid bilayer membranes by synthetic basic peptides, Biochim. Biophys. Acta 1064 (1991) 256–266. [83] C. Fujiwara, K. Anzai, Y. Kirino, S. Nagao, Y. Nozawa, M. Takahashi, Cation channels from ciliary membrane of Tetrahymena reconstituted into planar lipid bilayer. Comparison between the channels from the wild T. thermophila and from its mutant which does not show ciliary reversal, J. Biochem. (Tokyo) 104 (1988) 344–348. [84] F. Bezanilla, A high capacity data recording device based on a digital audio processor and a video cassette recorder, Biophys. J. 47 (1985) 437–441. [85] A. Tzagoloff, Mitochondria, Plenum Press, New York, 1982. [86] P. Mitchell, J. Moyle, Stoichiometry of proton translocation through the respiratory chain and adenosine triphosphatase systems of rat liver mitochondria, Nature 208 (1965) 147–151. [87] Y. Hatefi, The mitochondrial electron transport and oxidative phosphorylation system, Annu. Rev. Biochem. 54 (1985) 1015–1069. [88] V.P. Skulachev, Uncoupling: new approaches to an old problem of bioenergetics, Biochim. Biophys. Acta 1363 (1998) 100–124. [89] W.C. Schneider, Intracellular distribution of enzymes III. The oxidation of octanoic acid by rat liver fraction, J. Biol. Chem. 176 (1948) 1287–1288. [90] B. Chance, G.R. Williams, The respiratory chain and oxidative phosphorylation, Adv. Enzymol. 17 (1956) 65–134. [91] H. Tedesch, D.L. Harris, The osmotic behavior and permeability to non-electrolytes of mitochondria, Arch. Biochem. Biophys. 58 (1955) 52–67. [92] U. Hopfer, A.L. Lehninger, W.J. Lennarz, The effect of polar moiety of lipids on bilayer conductance induced by uncouplers of oxidative phosphorylation, J. Membr. Biol. 3 (1970) 142–155. [93] B. Neumcke, The action of uncouplers on lipid bilayer membranes, Membranes 3 (1975) 215–253. [94] K. Kawai, H. Mori, S. Sugie, N. Yoshimi, T. Inoue, T. Nakamaru, Y. Nozawa, T. Matsushima, Genotoxicity in the hepatocyte/DNA repair test and toxicity to liver mitochondria of 1-hydroxyanthraquinone and several dihydroxyanthraquinones, Cell Biol. Toxicol. 2 (1986) 457–467. [95] U. Hopfer, A.L. Lehninger, T.E. Thompson, Protonic conductance across phospholipid bilayer membranes induced by uncoupling agents for oxidative phosphorylation, Proc. Natl Acad. Sci. USA 59 (1968) 484–490. [96] W.D. Stein, Physical basis of movement across cell membrane, Transport and Diffusion Across Cell Membranes, Academic Press, London, 1986. pp. 1–68. [97] P. Mitchell, Chemiosmotic coupling in oxidative and photosynthetic phosphorylation, Biol. Rev. Camb. Philos. Soc. 41 (1966) 445–502. [98] J. Dilger, S. McLaughlin, Proton transport through membranes induced by weak acids: a study of two substituted benzimidazoles, J. Membr. Biol. 46 (1979) 359–384. [99] S.G. McLaughlin, J.P. Dilger, Transport of protons across membranes by weak acids, Physiol. Rev. 60 (1980) 825–863. [100] D.E. Goldman, Potential, impedance and rectification in membranes, J. Gen. Physiol. 27 (1943) 37–60.

Ion Permeability Induced in Planar Lipid Bilayer Membranes by Quinone Pigments

157

[101] A.L. Hodgkin, B. Katz, The effect of sodium ions on the electrical activity of the giant axon of squid, J. Physiol. 108 (1949) 37–77. [102] M.T. Tosteson, D.C. Tosteson, The sting. Melittin forms channels in lipid bilayers, Biophys. J. 36 (1981) 109–116. [103] B.L. Kagan, M.E. Selsted, T. Ganz, R.I. Lehrer, Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes, Proc. Natl Acad. Sci. USA 87 (1990) 210–214. [104] A.M. Feigin, E.V. Aronov, J.H. Teeter, J.G. Brand, The properties of ion channels formed by the coumarin antibiotic, novobiocin, in lipid bilayers, Biochim. Biophys. Acta 1234 (1995) 43–51. [105] C. Goudet, J.P. Benitah, M.L. Milat, H. Sentenac, J.B. Thibaud, Cluster organization and pore structure of ion channels formed by beticolin 3, a nonpeptidic fungal toxin, Biophys. J. 77 (1999) 3052–3059. [106] A.W. Bernheimer, B. Rudy, Interactions between membranes and cytolytic peptides, Biochim. Biophys. Acta 864 (1986) 123–141. [107] J.I. Kourie, A.A. Shorthouse, Properties of cytotoxic peptide-formed ion channels, Am. J. Physiol. Cell Physiol. 278 (2000) C1063–C1087. [108] T.A. Mirzabekov, A.Y. Silberstein, B.L. Kagan, Use of planar lipid bilayer membranes for rapid screening of membrane active compounds, Methods Enzymol. 294 (1999) 661–674.