2022/07/02 更新

写真a

ヒラノ ミナコ
平野 美奈子
HIRANO Minako
所属
ヘルスシステム統合科学学域 准教授
職名
准教授
外部リンク

学位

  • 博士(理学) ( 大阪大学 )

研究キーワード

  • 光遺伝学

  • 1分子計測

  • 電気生理

  • イオンチャネル

研究分野

  • ライフサイエンス / 生物物理学

学歴

  • 大阪大学   Graduate School of Frontier Biosciences  

    2007年4月 - 2010年3月

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  • 名古屋大学   Graduate School of Science  

    2002年4月 - 2004年3月

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  • 名古屋大学   School of Science   Department of Biological Science

    1998年4月 - 2002年4月

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経歴

  • 岡山大学   学術研究院ヘルスシステム統合科学学域   准教授

    2022年4月 - 現在

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  • 光産業創成大学院大学 光産業創成研究科   講師

    2012年5月 - 2022年3月

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  • 理化学研究所   特別研究員

    2010年4月 - 2012年4月

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所属学協会

委員歴

  • 日本生物物理学会   分野別専門委員  

    2020年   

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  • 文部科学省 科学技術・学術政策研究所   科学技術専門家ネットワーク 専門調査員  

    2019年 - 2020年   

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  • 日本生物物理学会   分野別専門委員  

    2019年   

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    団体区分:学協会

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  • 日本生物物理学会   分野別専門委員  

    2017年   

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    団体区分:学協会

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  • 公益財団法人新世代研究所   バイオ単分子研究会 委員  

    2015年 - 現在   

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    団体区分:学協会

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論文

  • Development of an automated system to measure ion channel currents using a surface-modified gold probe. 査読 国際誌

    Minako Hirano, Masahisa Tomita, Chikako Takahashi, Nobuyuki Kawashima, Toru Ide

    Scientific reports   11 ( 1 )   17934 - 17934   2021年9月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Artificial lipid bilayer single-channel recording technique has been employed to determine the biophysical and pharmacological properties of various ion channels. However, its measurement efficiency is very low, as it requires two time-consuming processes: preparation of lipid bilayer membranes and incorporation of ion channels into the membranes. In order to address these problems, we previously developed a technique based on hydrophilically modified gold probes on which are immobilized ion channels that can be promptly incorporated into the bilayer membrane at the same time as the membrane is formed on the probes' hydrophilic area. Here, we improved further this technique by optimizing the gold probe and developed an automated channel current measurement system. We found that use of probes with rounded tips enhanced the efficiency of channel current measurements, and introducing a hydrophobic area on the probe surface, beside the hydrophilic one, further increased measurement efficiency by boosting membrane stability. Moreover, we developed an automated measurement system using the optimized probes; it enabled us to automatically measure channel currents and analyze the effects of a blocker on channel activity. Our study will contribute to the development of high-throughput devices to identify drug candidates affecting ion channel activity.

    DOI: 10.1038/s41598-021-97237-z

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  • Low-Light Photodetectors for Fluorescence Microscopy 査読

    Hiroaki Yokota, Atsuhito Fukasawa, Minako Hirano, Toru Ide

    APPLIED SCIENCES-BASEL   11 ( 6 )   2021年3月

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    記述言語:英語   出版者・発行元:MDPI  

    Over the years, fluorescence microscopy has evolved and has become a necessary element of life science studies. Microscopy has elucidated biological processes in live cells and organisms, and also enabled tracking of biomolecules in real time. Development of highly sensitive photodetectors and light sources, in addition to the evolution of various illumination methods and fluorophores, has helped microscopy acquire single-molecule fluorescence sensitivity, enabling single-molecule fluorescence imaging and detection. Low-light photodetectors used in microscopy are classified into two categories: point photodetectors and wide-field photodetectors. Although point photodetectors, notably photomultiplier tubes (PMTs), have been commonly used in laser scanning microscopy (LSM) with a confocal illumination setup, wide-field photodetectors, such as electron-multiplying charge-coupled devices (EMCCDs) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras have been used in fluorescence imaging. This review focuses on the former low-light point photodetectors and presents their fluorescence microscopy applications and recent progress. These photodetectors include conventional PMTs, single photon avalanche diodes (SPADs), hybrid photodetectors (HPDs), in addition to newly emerging photodetectors, such as silicon photomultipliers (SiPMs) (also known as multi-pixel photon counters (MPPCs)) and superconducting nanowire single photon detectors (SSPDs). In particular, this review shows distinctive features of HPD and application of HPD to wide-field single-molecule fluorescence detection.

    DOI: 10.3390/app11062773

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  • A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings. 査読 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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    担当区分:筆頭著者   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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  • The C-terminal region affects the activity of photoactivated adenylyl cyclase from Oscillatoria acuminata. 査読 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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    担当区分:筆頭著者, 責任著者   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. 査読 国際誌

    Minako Hirano, Toru Ide

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

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    担当区分:筆頭著者, 責任著者   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. 査読 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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  • Liposome chaperon in cell-free membrane protein synthesis: One-step preparation of KcsA-integrated liposomes and electrophysiological analysis by the planar bilayer method 査読

    M. Ando, M. Akiyama, D. Okuno, M. Hirano, T. Ide, S. Sawada, Y. Sasaki, K. Akiyoshi

    Biomaterials Science   4 ( 2 )   258 - 264   2016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)   出版者・発行元:Royal Society of Chemistry  

    Chaperoning functions of liposomes were investigated using cell-free membrane protein synthesis. KcsA potassium channel-reconstituted liposomes were prepared directly using cell-free protein synthesis. In the absence of liposomes, all synthesized KcsA protein aggregated. In the presence of liposomes, however, synthesized KcsA spontaneously integrated into the liposome membrane. The KscA-reconstituted liposomes were transferred to the planar bilayer across a small hole in a thin plastic sheet and the channel function of KcsA was examined. The original electrophysiological activities, such as voltage- and pH-dependence, were observed. These results suggested that in cell-free membrane protein synthesis, liposomes act as chaperones, preventing aggregation and assisting in folding and tetrameric formation, thereby allowing full channel activity.

    DOI: 10.1039/c5bm00285k

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

    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. 査読 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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    担当区分:筆頭著者, 責任著者   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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  • 3P222 イオンチャネルの機能の改変(生体膜・人工膜 : 興奮・チャネル,ポスター,第52回日本生物物理学会年会(2014年度))

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

    生物物理   54 ( 1 )   S285   2014年

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    記述言語:英語   出版者・発行元:一般社団法人 日本生物物理学会  

    DOI: 10.2142/biophys.54.S285_6

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  • Uncovering the protein translocon at the chloroplast inner envelope membrane. 査読 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. 査読 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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  • Intra/extracellular investigation for ion channels with lipid bilayer array at the single molecule level 査読

    R. Kawano, Y. Tsuji, M. Hirano, T. Osaki, K. Kamiya, N. Miki, T. Ide, S. Takeuchi

    Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems (MEMS)   57 - 58   2013年

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)  

    This paper describes an investigation of drug effects for ion channels with recognizing the intra/extracellular directions. We have previously reported the ion channel recordings with bilayer lipid membranes (BLMs) which formed on a parylene micropore using droplets contact method. However, the low reconstitution probability of ion channels into BLMs has been a major issue. In this study, BLMs area and position are regulated with changing the numbers and positions of the parylene micropores. As a result, the simultaneous single channel recordings of K+ channel expressed in nerve system with BLM array were able to be achieved at the high probability. © 2013 IEEE.

    DOI: 10.1109/MEMSYS.2013.6474175

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  • 3PT163 人工細胞膜アレイを用いた全自動イオンチャネル計測システム(日本生物物理学会第50回年会(2012年度))

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

    生物物理   52   S169   2012年

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    記述言語:英語   出版者・発行元:一般社団法人 日本生物物理学会  

    DOI: 10.2142/biophys.52.S169_2

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  • Role of the KcsA channel cytoplasmic domain in pH-dependent gating. 査読 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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    担当区分:筆頭著者   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. 査読 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. 査読 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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    担当区分:筆頭著者   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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  • Immobilizing channel molecules in artificial lipid bilayers for simultaneous electrical and optical single channel recordings 査読

    Toru Ide, Minako Hirano, Takehiko Ichikawa

    Cell Signaling Reactions: Single-Molecular Kinetic Analysis   107 - 120   2011年

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    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:Springer Netherlands  

    There has been much interest in imaging single drug bindings to ion channel proteins while simultaneously recording single channel current. We developed an experimental apparatus for simultaneous optical and electrical measurement of single channel proteins by combining the single molecule imaging technique and the artificial bilayer technique. However, one major problem is that single molecule imaging of drug bindings is limited by the innate thermal diffusion of channel proteins in the artificial bilayer. Therefore, immobilizing channel proteins in the bilayers is imperative for stable measurements of channel-drug interactions. For future studies on channel-drug interactions, we describe here three different methods for simultaneous optical and electrical observation of single channels in which channel proteins are immobilized. (i) Membrane binding protein annexin V reduces the lateral diffusion of single channel proteins in a concentration-dependent manner. (ii) Channel proteins are immobilized by anchorage through a polyethylene glycol (PEG) molecule to the glass substrate. (iii) Channels immobilized on a gel bead can be directly incorporated into artificial bilayers. © Springer Science+Business Media B.V. 2011.

    DOI: 10.1007/978-90-481-9864-1_5

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  • Rearrangements in the KcsA cytoplasmic domain underlie its gating. 査読 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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    担当区分:筆頭著者   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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  • A polysaccharide-based container transportation system powered by molecular motors. 査読 国際誌

    Youichi Tsuchiya, Tomotaka Komori, Minako Hirano, Tomohiro Shiraki, Akira Kakugo, Toru Ide, Jian-Ping Gong, Sunao Yamada, Toshio Yanagida, Seiji Shinkai

    Angewandte Chemie (International ed. in English)   49 ( 4 )   724 - 7   2010年

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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/anie.200904909

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

    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.

    PubMed

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  • Current recordings of ion channel proteins immobilized on resin beads. 査読 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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    担当区分:筆頭著者   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

    PubMed

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  • Ion Channels 査読

    Toru Ide, Minako Hirano, Yuko Takeuchi

    Single Molecule Dynamics in Life Science   87 - 97   2009年2月

     詳細を見る

    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:Wiley-VCH Verlag GmbH & Co. KGaA  

    DOI: 10.1002/9783527626137.ch4

    Scopus

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

    Toru Ide, Toshihide Kobayashi, Minako Hirano

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

     詳細を見る

    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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  • Lipid bilayers at gel/gel interface for ion channel recordings 査読

    Minako Hirano, Toshihide Kobayashi, Toru Ide

    e-Journal of Surface Science and Nanotechnology   6   130 - 133   2008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)   出版者・発行元:Surface Science Society Japan  

    We have developed a practical method to produce durable artificial lipid bilayers using a hydrogel-hydrogel interface for ion channel measurements. Bilayers were formed by forcing a hydrogel-bead into contact with the hydrogel layer (hydrogel plate) in a lipid solution. The immediate formation of a bilayer was observed (&lt
    1 s). This allows channel recordings to be repeated more easily and quickly as compared to conventional methods. Currents of various types of channel such as gramicidin, hemolysin and BK-channel have been recorded. Our channel property results mirrored those of other techniques and were reproducible. Hydrogel solutions containing gramicidin were extremely stable and could be used months after preparation for bilayer experiments. © 2008 The Surface Science Society of Japan.

    DOI: 10.1380/ejssnt.2008.130

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  • 3P-207 1分子構造・機能同時計測によるカリウムチャネル(KcsAチャネル)の開閉機構の解明(生体膜/人工膜・興奮,チャネル,第46回日本生物物理学会年会)

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

    生物物理   48   S159   2008年

     詳細を見る

    記述言語:英語   出版者・発行元:一般社団法人 日本生物物理学会  

    DOI: 10.2142/biophys.48.S159_5

    CiNii Article

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  • 3P-203 ハイドロゲル界面人工膜を用いたチャネル電流計測(生体膜/人工膜・興奮,チャネル,第46回日本生物物理学会年会)

    Ide Toru, Hirano Minako

    生物物理   48   S159   2008年

     詳細を見る

    記述言語:英語   出版者・発行元:一般社団法人 日本生物物理学会  

    DOI: 10.2142/biophys.48.S159_1

    CiNii Article

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産業財産権

  • 光活性化アデニル酸シクラーゼ

    平野 美奈子, 松永 茂, 建部 益美

     詳細を見る

    出願人:浜松ホトニクス株式会社、学校法人光産業創成大学院大学

    出願番号:特願2019-129374  出願日:2019年7月11日

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  • 人工生体膜を製造するデバイス及び製造方法

    平野美奈子, 井出徹

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    出願人:学校法人光産業創成大学院大学

    出願番号:特願2015-154014  出願日:2015年8月4日

    公開番号:特開2017-029090  公開日:2017年2月9日

    特許番号/登録番号:特許6632826  登録日:2019年12月20日 

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受賞

  • Hot Article Award

    2016年12月   Analytical Sciences  

    Okuno, D, Hirano, M, Yokota, H, Onishi, Y, Ichinose, J, Ide, T

     詳細を見る

  • ATI研究奨励賞

    2014年7月   (公財)新世代研究所   イオンチャネルの1分子計測・操作による構造機能相関の解明

    平野 美奈子

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  • 2012 Research Paper Award

    2013年11月   Rebeiz財団  

    M. Nakai, M. Hirano, S. Kikuchi, J. Bédard, Y. Hirabayashi, M. Oishi, M. Imai, M. Takase, T. Ide

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共同研究・競争的資金等の研究

  • 光活性化cAMP産生酵素の高時間分解能活性計測を基盤とした光遺伝学ツールの創製

    研究課題/領域番号:22K06173  2022年04月 - 2025年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    平野 美奈子

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    配分額:4160000円 ( 直接経費:3200000円 、 間接経費:960000円 )

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  • 光活性化タンパク質の活性制御機構の解明と細胞の光制御への応用

    2022年04月 - 2023年03月

    日本私立学校振興・共済事業団  学術研究振興資金 

    横田 浩章, 平野美奈子, 井出 徹

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    担当区分:研究分担者 

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  • 光活性化タンパク質を利用した嗅覚に関与するイオンチャネルの活性化機構の解明

    2021年

    公益財団法人小柳財団  研究助成 

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  • 光活性化タンパク質の活性制御機構の解明と細胞の光制御への応用

    2020年

    日本私立学校振興・共済事業団  学術研究振興資金 

    平野 美奈子

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    担当区分:研究代表者 

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  • 細胞内イオン環境の光制御のための新規光感受性イオンチャネルの創製

    研究課題/領域番号:18K06168  2018年04月 - 2021年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    平野 美奈子

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    配分額:4550000円 ( 直接経費:3500000円 、 間接経費:1050000円 )

    本研究では、非侵襲な光での細胞内イオン環境制御による細胞機能の操作を目指して、光照射で活性が制御される新規光感受性イオンチャネルを創製することを目的としている。本年度は、新規光感受性イオンチャネルの構成要素である光感受性タンパク質(PAC)の構造機能相関の解明と、得られていた候補となる青色光感受性キメラチャネルの特性を調べた。下記に詳細を記す。
    1. 光刺激によってcAMPを生産するタンパク質であるPACの特定の領域を欠損した変異体は、野生型と比べて低い光強度で活性を示した。このことから、この領域が光刺激に依存した活性制御に重要な部位であることが明らかとなった。よって、光感受性チャネル作製時にはこの領域に変異を導入することで、光感受性を変化できる可能性があることがわかった。
    2.特定のアミノ酸を標的とする蛍光色素を用いた標識実験により、PACの特定の領域が光照射時と暗所静置時で標識の度合いが明らかに異なる、つまり構造状態が異なることがわかった。このことから、この領域の光依存的な構造変化を利用してイオンチャネルを光制御できる可能性があることが示唆された。
    3. これまでに得られていた候補となる青色光感受性キメラチャネルを精製し、電気生理学的手法により活性の光依存性を調べた。しかしながら、今回検討した光強度では光依存性は見られなかった。今後、光強度や照射時間を詳細に検討し、光依存性の有無を再度調べる予定である。

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  • 新規光感受性イオンチャネルの創製による細胞機能の光制御

    研究課題/領域番号:15K07035  2015年04月 - 2019年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    平野 美奈子

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    配分額:5200000円 ( 直接経費:4000000円 、 間接経費:1200000円 )

    本研究では、イオン環境が鍵となる生命現象の基盤の解明のために、非侵襲な光で活性が制御される光感受性イオンチャネルを創製し、細胞機能を操作する系を確立することを目的とした。様々な長さのK+チャネル(KcsA)に、光感受性ドメインまたは様々な長さの光感受性蛋白質を付加した変異体の遺伝子ライブラリーを約100種類作製した。これらをK+輸送欠損酵母を用いて光刺激の有無で増殖能が異なる変異体をスクリーニングし、最終的に青色光照射下で増殖能が高い、つまりK+輸送能が高い変異体が6種類得られた。また、細胞の活性の光制御のため、作製した変異体をHEK細胞に導入し、パッチクランプ法で活性を測定する系を確立した。

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  • チャネル創薬支援に向けた1分子センサーの開発

    2015年

    日本私立学校振興・共済事業団  学術研究振興資金 

    平野 美奈子

      詳細を見る

    担当区分:研究代表者 

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  • 非侵襲な光による細胞機能制御に向けた新規光感受性イオンチャネルの創製

    2015年

    公益信託成茂神経科学研究助成基金  研究助成 

    平野 美奈子

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    担当区分:研究代表者 

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  • イオンチャネル創薬支援に向けた新規チャネル活性測定法の開発

    2015年

    一般財団法人東海産業技術振興財団  研究助成 

    平野 美奈子

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    担当区分:研究代表者 

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  • チャネル創薬支援に向けた1分子センサーの開発

    2014年

    日本私立学校振興・共済事業団  学術研究振興資金 

    平野 美奈子

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    担当区分:研究代表者 

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  • 光駆動性イオンチャネルの創製

    研究課題/領域番号:25840055  2013年04月 - 2016年03月

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    平野 美奈子

      詳細を見る

    配分額:4550000円 ( 直接経費:3500000円 、 間接経費:1050000円 )

    本研究では、細胞機能を光刺激で操作する系を確立するため、K+チャネル(KcsA)を改変し、光駆動型のK+チャネルやCa2+チャネルを創製することを目的とした。1)KcsAチャネルの活性制御に重要な細胞内領域を光感受性LOVドメインに置換した変異体では、リンカーの長さに応じて活性が変化し、35アミノ酸残基のリンカーを持つ変異体は青色光照射下と比べて暗所で高い活性を示した。2)KcsAチャネルの細胞内領域による活性制御では、E146の荷電状態が最も活性に影響を与えていることがわかった。3) KcsAチャネルのフィルター部位への変異導入により、Ca2+透過性へ改変することができた。

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  • 細胞機能の光制御を目指した光感受性イオンチャネルの創製

    2013年

    公益財団法人住友財団  基礎科学研究助成 

    平野 美奈子

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    担当区分:研究代表者 

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  • 光刺激による細胞内イオン環境の制御

    2013年

    公益財団法人上原記念生命科学財団  研究奨励金 

    平野 美奈子

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    担当区分:研究代表者 

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  • 細胞機能制御に向けた光感受性イオンチャネルの創製

    2013年

    公益財団法人興和生命科学振興財団  研究助成 

    平野 美奈子

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    担当区分:研究代表者 

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  • イオンチャネルの1分子計測・操作による構造機能相関の解明

    2012年

    公益財団法人新世代研究所  ATI研究助成 

    平野美奈子

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    担当区分:研究代表者 

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  • 蛍光色素を利用したイオンチャネルの開閉機構の可視化

    研究課題/領域番号:23113519  2011年04月 - 2013年03月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    平野 美奈子

      詳細を見る

    配分額:11440000円 ( 直接経費:8800000円 、 間接経費:2640000円 )

    イオンチャネルの機能は、イオンの流れを電流として捉えることで、1分子レベルで詳細に測定できる。しかしながら、チャネルの構造に関する情報は構造解析などからの特定の状態でのスナップショットしかなく、機能しているチャネルの構造の遷移は未だ明らかではない。本研究では、チャネルの分子実体を理解するために、以前我々が開発した構造・機能同時計測装置を用いて、機能しているチャネルの構造状態を1分子レベルで計測し、その構造機能相関を明らかにすることを目的とし、以下の4つを行った。
    1.蛍光色素を用いたKcsAチャネルの構造変化の検出. 以前多分子系でKcsAチャネルの開・閉状態を蛍光色素、テトラメチルローダミン(TMR)を用いて捉えることに成功した。今回、不活性化条件でも活性を示す開状態模倣変異体、E146Q、の構造状態を同様に調べた結果、E146Qの構造状態は野生型の開状態と異なるものであったことから、KcsAには中間状態が存在することが示唆された。
    2.KcsAチャネルの1分子レベルでの構造変化の可視化. 以前多分子系でTMRを用いてKcsAチャネルの開閉を明らかにした標識部位で、今回1分子レベルでも固体支持膜に再構成したKcsAの開閉を蛍光のオン・オフとして明確に捉えることができた。
    3. 同時計測系の改良. 最近、構造・機能同時計測装置に、新たなチャネル再構成法を組み込み、改良した。本研究では、事前にチャネルの疎水部を保護剤で保護することで、さらにチャネルの膜への組み込み効率を上げることに成功した。
    4. 1分子、実時間でのKcsAチャネルの構造変化と機能変化の同時計測. 3.で改良した同時計測装置を用い、膜に再構成されたTMR標識KcsAチャネルの電気的・光学的計測を行った。その結果、チャネル活性が見られ、TMRの蛍光を輝点として捉えることができた。

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  • 1分子計測・操作によるイオンチャネルの開閉機構の解明

    研究課題/領域番号:23770190  2011年 - 2012年

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    平野 美奈子

      詳細を見る

    配分額:4680000円 ( 直接経費:3600000円 、 間接経費:1080000円 )

    環境依存的な蛍光色素を用いて、カリウムチャネルの開閉に伴う構造変化を、1 分子レベルで蛍光のオン・オフとして明確に捉えることができた。また、イオンチャネルの構造変化と機能変化を1 分子レベルで同時計測して構造機能相関を明らかにするため、以前開発した同時計測装置に新たなチャネル電流測定法を組み込んだ。その結果、チャネルの膜中での拡散の問題が解消され、初めて安定にイオンチャネル1 分子の蛍光像を得ることに成功した。

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