2021/07/12 更新

写真a

イデ トオル
井出 徹
IDE Toru
所属
ヘルスシステム統合科学学域 教授
職名
教授
外部リンク
 

論文

  • A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings. 国際誌

    Minako Hirano, Daiki Yamamoto, Mami Asakura, Tohru Hayakawa, Shintaro Mise, Akinobu Matsumoto, Toru Ide

    Micromachines   11 ( 12 )   2020年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.

    DOI: 10.3390/mi11121070

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  • Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity. 国際誌

    Tohru Hayakawa, Midoka Miyazaki, Syoya Harada, Mami Asakura, Toru Ide

    Applied microbiology and biotechnology   104 ( 20 )   8789 - 8799   2020年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane β-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane β-hairpin region. KEY POINTS: • Cry46Ab mutants were constructed by targeting the putative transmembrane β-hairpin region. • Charged residues within the β-hairpin control the flux of ions through channel pores. • Channel-pore cation selectivity is correlated with insecticidal activity.

    DOI: 10.1007/s00253-020-10893-5

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  • Single-molecule imaging of PI(4,5)P2 and PTEN in vitro reveals a positive feedback mechanism for PTEN membrane binding. 国際誌

    Daisuke Yoshioka, Seiya Fukushima, Hiroyasu Koteishi, Daichi Okuno, Toru Ide, Satomi Matsuoka, Masahiro Ueda

    Communications biology   3 ( 1 )   92 - 92   2020年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    PTEN, a 3-phosphatase of phosphoinositide, regulates asymmetric PI(3,4,5)P3 signaling for the anterior-posterior polarization and migration of motile cells. PTEN acts through posterior localization on the plasma membrane, but the mechanism for this accumulation is poorly understood. Here we developed an in vitro single-molecule imaging assay with various lipid compositions and use it to demonstrate that the enzymatic product, PI(4,5)P2, stabilizes PTEN's membrane-binding. The dissociation kinetics and lateral mobility of PTEN depended on the PI(4,5)P2 density on artificial lipid bilayers. The basic residues of PTEN were responsible for electrostatic interactions with anionic PI(4,5)P2 and thus the PI(4,5)P2-dependent stabilization. Single-molecule imaging in living Dictyostelium cells revealed that these interactions were indispensable for the stabilization in vivo, which enabled efficient cell migration by accumulating PTEN posteriorly to restrict PI(3,4,5)P3 distribution to the anterior. These results suggest that PI(4,5)P2-mediated positive feedback and PTEN-induced PI(4,5)P2 clustering may be important for anterior-posterior polarization.

    DOI: 10.1038/s42003-020-0818-3

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  • The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata. 国際誌

    Minako Hirano, Masumi Takebe, Tomoya Ishido, Toru Ide, Shigeru Matsunaga

    Scientific reports   9 ( 1 )   20262 - 20262   2019年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.

    DOI: 10.1038/s41598-019-56721-3

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  • Electrostatic state of the cytoplasmic domain influences inactivation at the selectivity filter of the KcsA potassium channel. 国際誌

    Minako Hirano, Toru Ide

    Biochimica et biophysica acta. Biomembranes   1861 ( 1 )   220 - 227   2019年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    KcsA is a proton-activated K+ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K+, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K+ selectivity, and permitted Na+ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.

    DOI: 10.1016/j.bbamem.2018.07.011

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  • A gold nano-electrode for single ion channel recordings. 国際誌

    Daichi Okuno, Minako Hirano, Hiroaki Yokota, Junya Ichinose, Takamitsu Kira, Taiki Hijiya, Chihiro Uozumi, Masahiro Yamakami, Toru Ide

    Nanoscale   10 ( 8 )   4036 - 4040   2018年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The artificial bilayer single channel recording technique is commonly used to observe the detailed physiological properties of various ion channel proteins. It permits easy control of the solution and membrane lipid composition, and is also compatible with pharmacological screening devices. However, its use is limited due to low measurement efficiency. Here, we developed a novel artificial bilayer single channel recording technique in which solubilized ion channel proteins immobilized on a gold nano-electrode are directly incorporated into a lipid bilayer at the same time as the bilayer is formed at the tip of it on coming in contact with an aqueous-oil interface. Using this technique, we measured the single channel currents of several types of channels including KcsA, MthK, hBK and P2X4. This technique requires only one action to simultaneously form the bilayers and reconstitute the channels into the membranes. This simplicity greatly increases the measurement efficiency and allows the technique to potentially be combined with high-throughput screening devices.

    DOI: 10.1039/c7nr08098k

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  • Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture. 国際誌

    Tohru Hayakawa, Akira Sakakibara, Sho Ueda, Yoshinao Azuma, Toru Ide, So Takebe

    Insect biochemistry and molecular biology   87   100 - 106   2017年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Cry46Ab is a Cry toxin derived from Bacillus thuringiensis TK-E6. Cry46Ab is not significantly homologous to other mosquitocidal Cry or Cyt toxins and is classified as an aerolysin-type pore-forming toxin based on structural similarity. In this study, the potency of Cry46Ab was assessed for its potential application to mosquito control. A synthetic Cry46Ab gene, cry46Ab-S1, was designed to produce recombinant Cry46Ab as a glutathione-S-transferase fusion in Escherichia coli. Recombinant Cry46Ab showed apparent toxicity to Culex pipiens larvae, with a 50% lethal dose of 1.02 μg/ml. In an artificial lipid bilayer, Cry46Ab activated by trypsin caused typical current transitions between open and closed states, suggesting it functions as a pore-forming toxin similar to other Cry and Cyt toxins. The single-channel conductance was 103.3 ± 4.1 pS in 150 mM KCl. Co-administration of recombinant Cry46Ab with other mosquitocidal Cry toxins, especially the combination of Cry4Aa and Cry46Ab, resulted in significant synergistic toxicity against C. pipiens larvae. Co-administration of multiple toxins exhibiting different modes of action is believed to prevent the onset of resistance in insects. Our data, taken in consideration with the differences in its structure, suggest that Cry46Ab could be useful in not only reducing resistance levels but also improving the insecticidal activity of Bt-based bio-insecticides.

    DOI: 10.1016/j.ibmb.2017.06.015

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  • A Simple Method for Ion Channel Recordings Using Fine Gold Electrode.

    Daichi Okuno, Minako Hirano, Hiroaki Yokota, Yukiko Onishi, Junya Ichinose, Toru Ide

    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry   32 ( 12 )   1353 - 1357   2016年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The artificial bilayer single-channel recording technique is commonly used to observe detailed pharmacological properties of various ion channel proteins. It permits easy control of the solution and membrane lipid composition, and is also compatible with pharmacological screening devices. However, its use is limited due to low measurement efficiency. Here, we develop a novel artificial bilayer single-channel recording technique in which bilayers are made and channels are reconstituted into the membranes by contacting a gold electrode to the lipid-solution interface. Using this technique, we measured the single-channel currents of two channel-forming peptides, gramicidin and alamethicin, and a channel-forming protein, α-hemolysin. This technique requires only one action, allowing the technique to potentially be combined with high-throughput screening devices.

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  • A single amino acid gates the KcsA channel. 国際誌

    Minako Hirano, Daichi Okuno, Yukiko Onishi, Toru Ide

    Biochemical and biophysical research communications   450 ( 4 )   1537 - 40   2014年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The KcsA channel is a proton-activated potassium channel. We have previously shown that the cytoplasmic domain (CPD) acts as a pH-sensor, and the charged states of certain negatively charged amino acids in the CPD play an important role in regulating the pH-dependent gating. Here, we demonstrate the KcsA channel is constitutively open independent of pH upon mutating E146 to a neutrally charged amino acid. In addition, we found that rearrangement of the CPD following this mutation was not large. Our results indicate that minimal rearrangement of the CPD, particularly around E146, is sufficient for opening of the KcsA channel.

    DOI: 10.1016/j.bbrc.2014.07.032

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  • Uncovering the protein translocon at the chloroplast inner envelope membrane. 国際誌

    Shingo Kikuchi, Jocelyn Bédard, Minako Hirano, Yoshino Hirabayashi, Maya Oishi, Midori Imai, Mai Takase, Toru Ide, Masato Nakai

    Science (New York, N.Y.)   339 ( 6119 )   571 - 4   2013年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Chloroplasts require protein translocons at the outer and inner envelope membranes, termed TOC and TIC, respectively, to import thousands of cytoplasmically synthesized preproteins. However, the molecular identity of the TIC translocon remains controversial. Tic20 forms a 1-megadalton complex at the inner membrane and directly interacts with translocating preproteins. We purified the 1-megadalton complex from Arabidopsis, comprising Tic20 and three other essential components, one of which is encoded by the enigmatic open reading frame ycf1 in the chloroplast genome. All four components, together with well-known TOC components, were found stoichiometrically associated with different translocating preproteins. When reconstituted into planar lipid bilayers, the purified complex formed a preprotein-sensitive channel. Thus, this complex constitutes a general TIC translocon.

    DOI: 10.1126/science.1229262

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  • Automated parallel recordings of topologically identified single ion channels. 国際誌

    Ryuji Kawano, Yutaro Tsuji, Koji Sato, Toshihisa Osaki, Koki Kamiya, Minako Hirano, Toru Ide, Norihisa Miki, Shoji Takeuchi

    Scientific reports   3   1995 - 1995   2013年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.

    DOI: 10.1038/srep01995

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  • A glass fiber sheet-based electroosmotic lateral flow immunoassay for point-of-care testing. 国際誌

    Yuriko Oyama, Toshihisa Osaki, Koki Kamiya, Ryuji Kawano, Tsutomu Honjoh, Haruki Shibata, Toru Ide, Shoji Takeuchi

    Lab on a chip   12 ( 24 )   5155 - 9   2012年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We have developed a quantitative immunoassay chip targeting point-of-care testing. To implement a lateral flow immunoassay, a glass fiber sheet was chosen as the material for the microfluidic channel in which the negative charge on the fiber surfaces efficiently generates the electroosmotic flow (EOF). The EOF, in turn, allows controllable bound/free separation of antigen/antibody interactions on the chip and enables precise determination of the antigen concentration. In addition, the defined size of the porous matrix was suitable for the filtration of undesired large particles. We confirmed the linear relationship between the concentration of analyte and the resulting fluorescence intensity from the immunoassay of two model analytes, C-reactive protein (CRP) and insulin, demonstrating that analyte concentration was quantitatively determined within the developed chip in 20 min. The limits of detection were 8.5 ng mL(-1) and 17 ng mL(-1) for CRP and insulin, respectively.

    DOI: 10.1039/c2lc40994a

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  • Role of the KcsA channel cytoplasmic domain in pH-dependent gating. 国際誌

    Minako Hirano, Yukiko Onishi, Toshio Yanagida, Toru Ide

    Biophysical journal   101 ( 9 )   2157 - 62   2011年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The KcsA channel is a representative potassium channel that is activated by changes in pH. Previous studies suggested that the region that senses pH is entirely within its transmembrane segments. However, we recently revealed that the cytoplasmic domain also has an important role, because its conformation was observed to change dramatically in response to pH changes. Here, to investigate the effects of the cytoplasmic domain on pH-dependent gating, we made a chimera mutant channel consisting of the cytoplasmic domain of the KcsA channel and the transmembrane region of the MthK channel. The chimera showed a pH dependency similar to that of KcsA, indicating that the cytoplasmic domain can act as a pH sensor. To identify how this region detects pH, we substituted certain cytoplasmic domain amino acids that are normally negatively charged at pH 7 for neutral ones in the KcsA channels. These mutants opened independently of pH, suggesting that electrostatic charges have a major role in the cytoplasmic domain's ability to sense and respond to pH.

    DOI: 10.1016/j.bpj.2011.09.024

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  • Direct manipulation of a single potassium channel gate with an atomic force microscope probe. 国際誌

    Mitsunori Kitta, Toru Ide, Minako Hirano, Hiroyuki Tanaka, Toshio Yanagida, Tomoji Kawai

    Small (Weinheim an der Bergstrasse, Germany)   7 ( 16 )   2379 - 83   2011年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Ion channels are membrane proteins that regulate cell functions by controlling the ion permeability of cell membranes. An ion channel contains an ion-selective pore that permeates ions and a sensor that senses a specific stimulus such as ligand binding to regulate the permeability. The detailed molecular mechanisms of this regulation, or gating, are unknown. Gating is thought to occur from conformational changes in the sensor domain in response to the stimulus, which results in opening the gate to permit ion conduction. Using an atomic force microscope and artificial bilayer system, a mechanical stimulus is applied to a potassium channel, and its gating is monitored in real time. The channel-open probability increases greatly when pushing the cytoplasmic domain toward the membrane. This result shows that a mechanical stimulus at the cytoplasmic domain causes changes in the gating and is the first to show direct evidence of coupling between conformational changes in the cytoplasmic domain and channel gating. This novel technology has the potential to be a powerful tool for investigating the activation dynamics in channel proteins.

    DOI: 10.1002/smll.201002337

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  • Channels formed by amphotericin B covalent dimers exhibit rectification. 国際誌

    Minako Hirano, Yuko Takeuchi, Nobuaki Matsumori, Michio Murata, Toru Ide

    The Journal of membrane biology   240 ( 3 )   159 - 64   2011年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Amphotericin B (AmB) is a widely used antifungal antibiotic with high specificity for fungi. We previously synthesized several covalently conjugated AmB dimers to clarify the AmB channel structure. Among these dimers, that with an aminoalkyl linker was found to exhibit potent hemolytic activity. We continue this work by investigating the channel activity of the dimer, finding that all channels comprised of AmB dimers show rectification. The direction of the dimer channel in the membrane depended on the electric potential at which the dimer channel was formed. On the other hand, only about half the monomer channels showed rectification. In addition, these channels were easily switched from a rectified to a nonrectified state following voltage stimulation, indicating instability. We propose a model to describe the AmB channel structure that explains why AmB dimer channels necessarily show rectification.

    DOI: 10.1007/s00232-011-9354-x

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  • Simultaneous optical and electrical recording of single molecule bonding to single channel proteins. 国際誌

    Toru Ide

    Chemphyschem : a European journal of chemical physics and physical chemistry   11 ( 16 )   3408 - 11   2010年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/cphc.201000560

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  • Simultaneous optical and electrical single channel recordings on a PEG glass. 国際誌

    Toru Ide, Yuko Takeuchi, Hiroyuki Noji, Kazuhito V Tabata

    Langmuir : the ACS journal of surfaces and colloids   26 ( 11 )   8540 - 3   2010年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Single molecule imaging of working ion-channels is much more difficult than that of water-soluble proteins because of the fragile nature of membranes and lateral diffusion of particles in the membranes, which does not allow fluorescent contamination for optical single channel recording. In this report, we reconstituted maxi-potassium channels from porcine uterine smooth muscle into artificial planar bilayers formed on poly(ethylene glycol) (PEG) modified glass and performed simultaneous optical and electrical recording of the single channels. The channels were immobilized in the membranes by anchoring to PEG molecules on the glass. The technique developed in this study should pave the way for single molecule pharmacology of ion-channels.

    DOI: 10.1021/la9045594

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  • Rearrangements in the KcsA cytoplasmic domain underlie its gating. 国際誌

    Minako Hirano, Yuko Takeuchi, Takaaki Aoki, Toshio Yanagida, Toru Ide

    The Journal of biological chemistry   285 ( 6 )   3777 - 83   2010年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    A change of cytosolic pH 7 to 4 opens the bacterial potassium channel KcsA. However, the overall gating mechanism leading to channel opening, especially the contribution of the cytoplasmic domain, remains unsolved. Here we report that deletion of the cytoplasmic domain resulted in changes in channel conductance and gating behavior at pH 4 without channel opening at pH 7. To probe for rearrangements in the cytoplasmic domain during channel opening, amino acid residues were substituted with cysteines and labeled with a fluorophore (tetramethylrhodamine maleimide) that exhibits increased fluorescence intensity upon transfer from a hydrophilic to hydrophobic environment. In all cases channel open probability (P(o)) was approximately 1 at pH 4 and approximately 0 at pH 7. Major increases in fluorescence intensity were observed for tetramethylrhodamine maleimide-labeled residues in the cytoplasmic domain as pH changed from 7 to 4, which suggests the fluorophores shifted from a hydrophilic to hydrophobic environment. Dipicrylamide, a lipid soluble quencher, reduced the fluorescence intensities of labeled residues in the cytosolic domain at pH 4. These results reveal that a decrease in pH introduces major conformational rearrangements associated with channel opening in the KcsA cytoplasmic domain.

    DOI: 10.1074/jbc.M109.084368

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  • Single channel properties of lysenin measured in artificial lipid bilayers and their applications to biomolecule detection.

    Takaaki Aoki, Minako Hirano, Yuko Takeuchi, Toshihide Kobayashi, Toshio Yanagida, Toru Ide

    Proceedings of the Japan Academy. Series B, Physical and biological sciences   86 ( 9 )   920 - 5   2010年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Single channel currents of lysenin were measured using artificial lipid bilayers formed on a glass micropipette tip. The single channel conductance for KCl, NaCl, CaCl(2), and Trimethylammonium-Cl were 474 ± 87, 537 ± 66, 210 ± 14, and 274 ± 10 pS, respectively, while the permeability ratio P(Na)/P(Cl) was 5.8. By adding poly(deoxy adenine) or poly(L-lysine) to one side of the bilayer, channel currents were influenced when membrane voltages were applied to pass the charged molecules through the channel pores. Current inhibition process was concentration-dependent with applied DNA. As the current fluctuations of α-hemolysin channels is often cited as the detector in a molecular sensor, these results suggest that by monitoring channel current changes, the lysenin channel has possibilities to detect interactions between it and certain biomolecules by its current fluctuations.

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  • Current recordings of ion channel proteins immobilized on resin beads. 国際誌

    Minako Hirano, Yuko Takeuchi, Takaaki Aoki, Toshio Yanagida, Toru Ide

    Analytical chemistry   81 ( 8 )   3151 - 4   2009年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Current ion channel current measurement techniques are cumbersome, as they require many steps and much time. This is especially true when reconstituting channels into liposomes and incorporating them into lipid bilayers. Here, we report a novel method that measures ion channel current more efficiently than current methods. We applied our method to KcsA and MthK channels by binding them to cobalt affinity gel beads with histidine tags and then forming a lipid bilayer membrane on the bead. This allowed channels to incorporate into the bilayer and channel currents to be measured quickly and easily. The efficiency was such that currents could be recorded with extremely low amounts of protein. In addition, the channel direction could be determined by the histidine tag. This method has the potential to be applied to various channel proteins and channel research in general.

    DOI: 10.1021/ac900286z

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  • Lipid bilayers at the gel interface for single ion channel recordings. 国際誌

    Toru Ide, Toshihide Kobayashi, Minako Hirano

    Analytical chemistry   80 ( 20 )   7792 - 5   2008年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Single-channel recording using artificial lipid bilayers is along with the patch-clamp technique a very powerful tool to physiologically and pharmacologically study ion channels. It is particularly advantageous in studying channels that are technically difficult to access with a patch pipet. However, the fragility of the bilayers and the difficulty to incorporate ion channels into them significantly compromises measurement efficiency. We have developed a novel method for forming artificial lipid bilayers on a hydrogel surface that significantly improves the measurement efficiency. Bilayers formed almost instantly (<1 s) and were able to incorporate various types of ion channel proteins within a short time (<30 s) enabling multichannel measurements. These results indicate that this method can potentially be applied to developing high-throughput screening devices for drug design.

    DOI: 10.1021/ac801224a

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  • GPHR is a novel anion channel critical for acidification and functions of the Golgi apparatus. 国際誌

    Yusuke Maeda, Toru Ide, Masato Koike, Yasuo Uchiyama, Taroh Kinoshita

    Nature cell biology   10 ( 10 )   1135 - 45   2008年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The organelles within secretory and endocytotic pathways in mammalian cells have acidified lumens, and regulation of their acidic pH is critical for the trafficking, processing and glycosylation of cargo proteins and lipids, as well as the morphological integrity of the organelles. How organelle lumen acidification is regulated, and how luminal pH elevation disturbs these fundamental cellular processes, is largely unknown. Here, we describe a novel molecule involved in Golgi acidification. First, mutant cells defective in Golgi acidification were established that exhibited delayed protein transport, impaired glycosylation and Golgi disorganization. Using expression cloning, a novel Golgi-resident multi-transmembrane protein, named Golgi pH regulator (GPHR), was identified as being responsible for the mutant cells. After reconstitution in planar lipid bilayers, GPHR exhibited a voltage-dependent anion-channel activity that may function in counterion conductance. Thus, GPHR modulates Golgi functions through regulation of acidification.

    DOI: 10.1038/ncb1773

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  • Cy3-3-acylcholine: a fluorescent analogue of acetylcholine for single molecule detection. 国際誌

    Kenzo Fujimoto, Yoshinaga Yoshimura, Makoto Ihara, Kazuhiko Matsuda, Yuko Takeuchi, Takaaki Aoki, Toru Ide

    Bioorganic & medicinal chemistry letters   18 ( 3 )   1106 - 9   2008年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We synthesized a novel fluorescent analogue of acetylcholine, Cy3-3-acylcholine. The molecular weight of the products agreed with structural predictions. Discrete intensity changes of fluorescent spots due to a single ligand binding/unbinding to nAChR were visualized by TIRF microscopy. The agonist effect of the Cy3-3-acylcholine on nicotinic acetylcholine receptor (nAChR) was confirmed electrophysiologically. This newly synthesized fluorescent analogue will enable us to conduct more elaborate studies on single channel interaction processes between nAChR and ligands.

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  • Lysenin forms a voltage-dependent channel in artificial lipid bilayer membranes. 国際誌

    Toru Ide, Takaaki Aoki, Yuko Takeuchi, Toshio Yanagida

    Biochemical and biophysical research communications   346 ( 1 )   288 - 92   2006年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Lysenin, a hemolytic protein derived from the body fluid of earthworm, was incorporated into artificial bilayer membranes. Upon insertion, it formed a voltage-dependent large conductance channel in asolectin bilayers in a sphingomyelin-dependent manner. The channel had low ion-selectivity. Single-channel conductance was calculated as approximately 550 pS in 100 mM KCl. The channel in asolectin bilayers closed when the membrane was held at a positive potential. In contrast, the channel showed no voltage dependency in membranes made of pure phosphatidylcholine and sphingomyelin, suggesting some lipid contents included in the asolectin membranes affected channel gating.

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  • Immobilizing single lipid and channel molecules in artificial lipid bilayers with annexin A5. 国際誌

    Takehiko Ichikawa, Takaaki Aoki, Yuko Takeuchi, Toshio Yanagida, Toru Ide

    Langmuir : the ACS journal of surfaces and colloids   22 ( 14 )   6302 - 7   2006年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The effects of annexin A5 on the lateral diffusion of single-molecule lipids and single-molecule proteins were studied in an artificial lipid bilayer membrane. Annexin A5 is a member of the annexin superfamily, which binds preferentially to anionic phospholipids in a Ca2+-dependent manner. In this report, we were able to directly monitor single BODIPY 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and ryanodine receptor type 2 (RyR2) labeled with Cy5 molecules in lipid bilayers containing phosphatidylserine (PS) by using fluorescence microscopy. The diffusion coefficients were calculated at various annexin A5 concentrations. The diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 in the absence of annexin A5 were 4.81 x 10(-8) cm(2)/s and 2.13 x 10(-8) cm(2)/s, respectively. In the presence of 1 microM annexin A5, the diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 were 2.2 x 10(-10) cm(2)/s and 9.5 x 10(-11) cm(2)/s, respectively. Overall, 1 microM of annexin A5 was sufficient to induce a 200-fold decrease in the lateral diffusion coefficient. Additionally, we performed electrophysiological examinations and determined that annexin A5 has little effect on the function of RyR2. This means that annexin A5 can be used to immobilize RyR2 in a lipid bilayer when imaging and analyzing RyR2.

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  • A novel method for artificial lipid-bilayer formation. 国際誌

    Toru Ide, Takehiko Ichikawa

    Biosensors & bioelectronics   21 ( 4 )   672 - 7   2005年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Many proposals have been made regarding the development of biosensors using single-channel recording with an artificial planar bilayer. The fragile nature of bilayer membranes is the major difficulty for the application of the artificial bilayer technique to the development of biosensors. We have developed an apparatus that promptly forms artificial bilayers. This technique is more efficient than other techniques for forming artificial bilayers. Bilayer membranes could be formed within 10s requiring 1 microl of analyte solution to record single-channel currents using our apparatus. A bilayer was formed by pressing the membrane on an agarose layer with hydraulic pressure. With this novel apparatus, we have recorded single-channel currents of various types of channels such as the BK-channel, the nicotinic receptor channel and the ryanodine receptor channel. The properties of the channels determined with this novel technique agreed well with those determined with conventional techniques.

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  • An amphotericin B-ergosterol covalent conjugate with powerful membrane permeabilizing activity. 国際誌

    Nobuaki Matsumori, Noritsugu Eiraku, Shigeru Matsuoka, Tohru Oishi, Michio Murata, Takaaki Aoki, Toru Ide

    Chemistry & biology   11 ( 5 )   673 - 9   2004年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Amphotericin B-sterol conjugates were synthesized and examined for their membrane permeabilizing activity. Ergosterol and cholesterol, each connected with amphotericin B via an ethylenecarbamate or hexamethylenecarbamate linker, were examined by K(+) flux assays using liposomes and by single-channel recording across phospholipid membrane. Among four conjugates tested, AmB-ergosterol bearing an ethylenecarbamate linker exhibited the most powerful activity, which substantially exceeded that of the cholesterol homolog. Single-channel recording clearly exhibited that the ergosterol conjugate elicited channel current with the conductance of 28 pS, which was comparable with those by AmB, and revealed a higher channel open probability than the cholesterol conjugate. These results imply that direct interaction between amphotericin B and ergosterol is reproduced by their conjugate, which may serve as a model compound for understanding the drug's selective toxicity.

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  • Simultaneous optical and electrical recording of a single ion-channel.

    Toru Ide, Yuko Takeuchi, Takaaki Aoki, Toshio Yanagida

    The Japanese journal of physiology   52 ( 5 )   429 - 34   2002年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    In recent years, the single-molecule imaging technique has proven to be a valuable tool in solving many basic problems in biophysics. The technique used to measure single-molecule functions was initially developed to study electrophysiological properties of channel proteins. However, the technology to visualize single channels at work has not received as much attention. In this study, we have for the first time, simultaneously measured the optical and electrical properties of single-channel proteins. The large conductance calcium-activated potassium channel (BK-channel) labeled with fluorescent dye molecules was incorporated into a planar bilayer membrane and the fluorescent image captured with a total internal reflection fluorescence microscope simultaneously with single-channel current recording. This innovative technology will greatly advance the study of channel proteins as well as signal transduction processes that involve ion permeation processes.

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