伊原 誠(イハラ マコト)

農学部 応用生命化学科准教授

Last Updated :2024/06/18

■教員コメント

コメント

イオンチャネルを標的とする農薬の作用機構を研究しています。特に、ネオニコチノイド剤の昆虫選択的毒性発現の分子機構を、構造生理学的観点から重点的に解析しています。

■研究者基本情報

学位

  • 博士(農学)(近畿大学)

J-Global ID

研究キーワード

  • 生物有機化学   農薬科学   構造生理学   Structural Biology   

現在の研究分野(キーワード)

イオンチャネルを標的とする農薬の作用機構を研究しています。特に、ネオニコチノイド剤の昆虫選択的毒性発現の分子機構を、構造生理学的観点から重点的に解析しています。

研究分野

  • ライフサイエンス / 生物有機化学
  • ライフサイエンス / 機能生物化学
  • ライフサイエンス / 構造生物化学
  • ナノテク・材料 / 生物分子化学

■経歴

経歴

  • 2018年04月 - 現在  近畿大学農学部准教授
  • 2013年04月 - 2018年03月  近畿大学農学部 応用生命化学科講師
  • 2012年12月 - 2018年03月  独立行政法人理化学研究所放射光科学総合研究センター客員研究員
  • 2012年12月 - 2013年03月  岡山大学大学院医歯薬学総合研究科(薬学系)助教
  • 2006年10月 - 2012年11月  独立行政法人 理化学研究所 播磨研究所放射光科学総合研究センター 研究員
  • 2005年04月 - 2006年09月  近畿大学 研究員

■研究活動情報

受賞

  • 2018年03月 日本農芸化学会 農芸化学奨励賞
     
    受賞者: 伊原 誠
  • 2008年 日本農学進歩賞
     JPN
  • 2007年 望月喜多司記念賞 奨励賞
     JPN
  • 2007年 日本農薬学会 奨励賞
     JPN

論文

  • Keisuke Nishikawa; Yosuke Ono; Sumito Mori; Koichi Takayama; Makoto Ihara; Kazuhiko Matsuda; Yoshiki Morimoto
    The Journal of organic chemistry 2024年02月 
    Histrionicotoxin (HTX) alkaloids, which are isolated from Colombian poison dart frogs, are analgesic neurotoxins that modulate nicotinic acetylcholine receptors (nAChRs) as antagonists. Perhydrohistrionicotoxin (pHTX) is the potent synthetic analogue of HTX and possesses a 1-azaspiro[5.5]undecane skeleton common to the HTX family. Here, we show for the first time the divergent nine-step synthesis of pHTX and its three stereoisomers from the known aldehyde through a one-step construction of the 1-azaspiro[5.5]undecane framework from a linear amino ynone substrate. Surprisingly, some pHTX diastereomers exhibited antagonistic activities on the chicken α4β2-neuronal nAChRs that were more potent than pHTX.
  • Noritada Matsuo; Yukimi Sugisaka; Shiori Aoyama; Makoto Ihara; Harue Shinoyama; Munetaka Hosokawa; Yoshinobu Kamakura; Daisuke Tanaka; Yoo Tanabe; Kazuhiko Matsuda
    Journal of Medicinal Chemistry 66 12 7959 - 7968 2023年06月
  • Wataru Koizumi; Shuya Otsubo; Shogo Furutani; Kunihiro Niki; Koichi Takayama; Shota Fujimura; Takanobu Maekawa; Ryosuke Koyari; Makoto Ihara; Kenji Kai; Hideo Hayashi; Mohammad Shaokat Ali; Eriko Kage-Nakadai; David B Sattelle; Kazuhiko Matsuda
    Molecular pharmacology 2023年03月 
    The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs) but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive (L-type (UNC-38/UNC-29/UNC-63/LEV8/LEV-1)) nAChR than the nicotine-sensitive (N-type (ACR-16)) nAChR, a result consistent with in vivo studies on wild type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. Significance Statement Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.
  • Yuma Komori; Koichi Takayama; Naoki Okamoto; Masaki Kamiya; Wataru Koizumi; Makoto Ihara; Daitaro Misawa; Kotaro Kamiya; Yuto Yoshinari; Kazuki Seike; Shu Kondo; Hiromu Tanimoto; Ryusuke Niwa; David B Sattelle; Kazuhiko Matsuda
    PLoS genetics 19 2 e1010522  2023年02月 
    Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dβ1, and Dβ2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dβ3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dβ3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.
  • Koichi Takayama; Ryo Ito; Haruki Yamamoto; Shuya Otsubo; Rei Matsumoto; Hisanori Ojima; Yuma Komori; Kazuhiko Matsuda; Makoto Ihara
    Pesticide Biochemistry and Physiology 187 105177 - 105177 2022年10月
  • Yukimi Sugisaka; Shiori Aoyama; Konoka Kumagai; Makoto Ihara; Kazuhiko Matsuda
    Journal of agricultural and food chemistry 2022年07月 
    Natural pesticides pyrethrins biosynthesized by Tanacetum cinrerariifolium are biodegradable and safer insecticides for pest insect control. TcGLIP, a GDSL lipase underpinning the ester bond formation in pyrethrins, exhibits high stereo-specificity for acyl-CoA and alcohol substrates. However, it is unknown how the enzyme recognizes the other structural features of the substrates and whether such specificity affects the product amount and composition in T. cinrerariifolium. We report here that the cysteamine moiety in (1R,3R)-chrysanthemoyl CoA and the conjugated diene moiety in (S)-pyrethrolone play key roles in the interactions with TcGLIP. CoA released from chrysanthemoyl CoA in the pyrethrin-forming reaction reduces the substrate affinity for TcGLIP by feedback inhibition. (S)-Pyrethrolone shows the highest catalytic efficiency for TcGLIP, followed by (S)-cinerolone and (S)-jasmololone, contributing, at least in part, to determine the pyrethrin compositions in T. cinerariifolium.
  • Makoto Ihara; Keiji Tanaka; Kenji Kai; Hideo Hayashi; Kazuhiko Matsuda
    Pesticide Biochemistry and Physiology 183 105074 - 105074 2022年05月
  • Qiang Wang; Peng Xu; Felipe Andreazza; Yahui Liu; Yoshiko Nomura; Phil Duran; Lan Jiang; Mengli Chen; Genki Takamatsu; Makoto Ihara; Kazuhiko Matsuda; Rufus Isaacs; Eugenio E Oliveira; Yuzhe Du; Ke Dong
    PLoS genetics 17 7 e1009677  2021年07月 
    Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-β-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.
  • Feng Liu; Qiang Wang; Peng Xu; Felipe Andreazza; Wilson R Valbon; Elizabeth Bandason; Mengli Chen; Ru Yan; Bo Feng; Leticia B Smith; Jeffrey G Scott; Genki Takamatsu; Makoto Ihara; Kazuhiko Matsuda; James Klimavicz; Joel Coats; Eugenio E Oliveira; Yuzhe Du; Ke Dong
    Nature communications 12 1 2553 - 2553 2021年05月 
    Pyrethrum extracts from flower heads of Chrysanthemum spp. have been used worldwide in insecticides and repellents. While the molecular mechanisms of its insecticidal action are known, the molecular basis of pyrethrum repellency remains a mystery. In this study, we find that the principal components of pyrethrum, pyrethrins, and a minor component, (E)-β-farnesene (EBF), each activate a specific type of olfactory receptor neurons in Aedes aegypti mosquitoes. We identify Ae. aegypti odorant receptor 31 (AaOr31) as a cognate Or for EBF and find that Or31-mediated repellency is significantly synergized by pyrethrin-induced activation of voltage-gated sodium channels. Thus, pyrethrum exerts spatial repellency through a novel, dual-target mechanism. Elucidation of this two-target mechanism may have potential implications in the design and development of a new generation of synthetic repellents against major mosquito vectors of infectious diseases.
  • Makoto Ihara; Shogo Furutani; Sho Shigetou; Shota Shimada; Kunihiro Niki; Yuma Komori; Masaki Kamiya; Wataru Koizumi; Leo Magara; Mai Hikida; Akira Noguchi; Daiki Okuhara; Yuto Yoshinari; Shu Kondo; Hiromu Tanimoto; Ryusuke Niwa; David B Sattelle; Kazuhiko Matsuda
    Proceedings of the National Academy of Sciences of the United States of America 117 28 16283 - 16291 2020年07月 [査読有り]
     
    The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.
  • Sho Shigetou; Shota Shimada; Ihara Makoto; Kazuhiko Matsuda
    Pesticide biochemistry and physiology 166 104545 - 104545 2020年06月 [査読有り]
     
    Neonicotinoids targeting insect nicotinic acetylcholine (ACh) receptors (insect nAChRs) are used for crop protection, but there is a concern about adverse effects on pollinators such as honeybees (Apis mellifera). Thus, we investigated the agonist actions of neonicotinoids (imidacloprid, thiacloprid and clothianidin) on A. mellifera α1 (Amα1)/chicken β2 hybrid nAChRs in Xenopus laevis oocytes according to the subunit stoichiometry of (Amα1)3(β2)2 and (Amα1)2(β2)3 using voltage-clamp electrophysiology. ACh activated (Amα1)3(β2)2 and (Amα1)2(β2)3 nAChRs with similar current amplitude. We investigated the agonist activity of imidacloprid, thiacloprid and clothianidin for the two hybrid nAChRs and found that: 1) imidacloprid showed higher affinity than clothianidin, whereas clothianidin showed higher efficacy than imidacloprid for the nAChRs; 2) Thiacloprid showed the highest agonist affinity and the lowest efficacy for the nAChRs. The Amα1/β2 subunit ratio influenced the efficacy of imidacloprid and thiacloprid, but hardly affected that of clothianidin. Hydrogen bond formation by the NH group in clothianidin with the main chain carbonyl of the loop B may account, at least in part, for the unique agonist actions of clothianidin on the hybrid nAChRs tested.
  • Shota Shimada; Masaki Kamiya; Sho Shigetou; Kakeru Tomiyama; Yuma Komori; Leo Magara; Makoto Ihara; Kazuhiko Matsuda
    Scientific reports 10 1 7529 - 7529 2020年05月 
    Neonicotinoids selectively modulate insect nicotinic acetylcholine receptors (insect nAChRs). Studies have shown that serine with ability to form a hydrogen bond in loop C of some insect nAChR α subunits and glutamate with a negative charge at the corresponding position in vertebrate nAChRs may contribute to enhancing and reducing the neonicotinoid actions, respectively. However, there is no clear evidence what loop C properties underpin the target site actions of neonicotinoids. Thus, we have investigated the effects of S221A and S221Q mutations in loop C of the Drosophila melanogaster Dα1 subunit on the agonist activity of imidacloprid and thiacloprid for Dα1/chicken β2 nAChRs expressed in Xenopus laevis oocytes. The S221A mutation hardly affected either the affinity or efficacy for ACh and imidacloprid, whereas it only slightly reduced the efficacy for thiacloprid on the nAChRs with a higher composition ratio of β2 to Dα1 subunits. The S221Q mutation markedly reduced the efficacy of the neonicotinoids for the nAChRs with a higher composition of the β2 subunit lacking basic residues critical for binding neonicotinoids. Hence, we predict the possibility of enhanced neonicotinoid resistance in pest insect species by a mutation of the serine when it occurs in the R81T resistant populations lacking the basic residue in loop D of the β1 subunit.
  • Teruyuki Tahara; Ami Watanabe; Maho Yutani; Yuko Yamano; Mami Sagara; Shizu Nagai; Keita Saito; Mitsuaki Yamashita; Makoto Ihara; Akira Iida
    Bioorganic and Medicinal Chemistry 28 6 2020年03月 [査読有り]
     
    © 2020 Elsevier Ltd The extract of Tabebuia avellanedae has been used as a folk medicine, and the various biological activities of T. avellanedae have been extensively studied. However, few studies have reported which natural products play a role in their biological effects. In this study, we evaluated representative naphthoquinones isolated from T. avellanedae and found that furanonaphthoquinones were the key structures required to exhibit STAT3 phosphorylation inhibitory activities. Our SAR analysis indicated that removal of a hydroxyl group enhanced the STAT3 phosphorylation inhibitory activity. In addition, the combined results of a mobility shift assay, SH2 domain binding assay, and docking simulation by Autodock 4.2.6 suggested that (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione (1) could directly bind to the hinge region of STAT3.
  • Nozomu Sakurai; Hossein Mardani-Korrani; Masaru Nakayasu; Kazuhiko Matsuda; Kumiko Ochiai; Masaru Kobayashi; Yusuke Tahara; Takeshi Onodera; Yuichi Aoki; Takashi Motobayashi; Masakazu Komatsuzaki; Makoto Ihara; Daisuke Shibata; Yoshiharu Fujii; Akifumi Sugiyama
    Frontiers in Genetics 11 2020年02月 
    Inter-organismal communications below ground, such as plant–microbe interactions in the rhizosphere, affect plant growth. Metabolites are shown to play important roles in biological communication, but there still remain a large number of metabolites in soil to be uncovered. Metabolomics, a technique for the comprehensive analysis of metabolites in samples, may uncover the molecules that intermediate these interactions. We conducted a multivariate analysis using liquid chromatography (LC)—mass spectrometry (MS)-based untargeted metabolomics in several soil samples and also targeted metabolome analysis for the identification of the candidate compounds in soil. We identified okaramine A, B, and C in the rhizosphere soil of hairy vetch. Okaramines are indole alkaloids first identified in soybean pulp (okara) inoculated with Penicillium simplicissimum AK-40 and are insecticidal. Okaramine B was detected in the rhizosphere from an open field growing hairy vetch. Okaramine B was also detected in both bulk and rhizosphere soils of soybean grown following hairy vetch, but not detected in soils of soybean without hairy vetch growth. These results suggested that okaramines might be involved in indirect defense of plants against insects. To our knowledge, this is the first report of okaramines in the natural environment. Untargeted and targeted metabolomics would be useful to uncover the chemistry of the rhizosphere.
  • Kazuhiko Matsuda; Makoto Ihara; David B. Sattelle
    Annual Review of Pharmacology and Toxicology 60 241 - 255 2020年01月 
    Copyright © 2020 by Annual Reviews. All rights reserved. Neonicotinoids have been used to protect crops and animals from insect pests since the 1990s, but there are concerns regarding their adverse effects on nontarget organisms, notably on bees. Enhanced resistance to neonicotinoids in pests is becoming well documented. We address the current understanding of neonicotinoid target site interactions, selectivity, and metabolism not only in pests but also in beneficial insects such as bees. The findings are relevant to the management of both neonicotinoids and the new generation of pesticides targeting insect nicotinic acetylcholine receptors.
  • Makoto Ihara; Kazuhiko Matsuda
    Current Opinion in Insect Science 30 86 - 92 Elsevier {BV} 2018年12月 
    © 2018 Elsevier Inc. Neonicotinoids are insecticides that target insect nicotinic acetylcholine receptors (nAChRs), exhibiting high selective toxicity to insects over vertebrates and good systemic activity in crop plants. For these reasons, neonicotinoids currently make up ∼30% of insecticide sales worldwide. However, due to their adverse impact on pollinators such as honey bees and bumble bees, neonicotinoids are being banned from the EU, and other countries may follow. It is therefore crucial to understand the mechanism underlying neonicotinoid actions on pollinators as well as on the nAChRs of pests, with a view to understanding their selectivity. Here we review the molecular mechanisms of neonicotinoid actions at an atomic level, through structural and resistance mechanism studies and propose relevant research topics for further studies on the future of pest management.
  • Chen M; Du Y; Zhu G; Takamatsu G; Ihara M; Matsuda K; Zhorov BS; Dong K
    Pesticide biochemistry and physiology 151 82 - 89 2018年10月 [査読有り]
     
    © 2018 Elsevier Inc. Pyrethrin I, pyrethrin II, cinerin I, cinerin II, jasmolin I and jasmolin II are six closely related insecticidal active esters, known as pyrethrins, found in the pyrethrum extract from the dry flowers of Tanacetum cinerariifolium. The chemical structures of the six compounds differ only in the terminal moieties at the acid and alcohol ends, but the compounds’ in vivo toxicities are substantially different. Pyrethrins are lead compounds for pyrethroids, a large family of synthetic insecticides that alter nerve functions by prolonging the opening of voltage-gated sodium channels. However, data on the mechanism of action of natural pyrethrins are very limited. In this study, we examined the actions of all six pyrethrins on cockroach sodium channels expressed in Xenopus oocytes. Although the six compounds showed comparable potencies in inhibiting the inactivation of sodium channels, they had greatly variable potencies in inhibiting channel deactivation. Furthermore, unlike pyrethroids, the action of pyrethrins neither depend on nor were enhanced by repeated channel activation. We created a NavMs-based model of the cockroach sodium channel, in which pyrethrin II was docked at the pyrethroid receptor site 1 (PyR1), and proposed a rationale for the observed structure-activity relationship of the six pyrethrins. Our study sheds light on the molecular mechanism of pyrethrum action on sodium channels and reveled differences in the modes of action of the six bioactive constitutes of pyrethrum.
  • Mai Hikida; Shota Shimada; Ryo Kurata; Sho Shigetou; Makoto Ihara; David B. Sattelle; Kazuhiko Matsuda
    Pesticide Biochemistry and Physiology 151 47 - 52 Elsevier {BV} 2018年10月 [査読有り]
     
    © 2018 Elsevier Inc. Neonicotinoid insecticides interact with the orthosteric sites of nicotinic acetylcholine receptors (nAChRs) formed at the interfaces of (a) two adjacent α subunits and (b) α and non-α subunits. However, little is known of the detailed contributions of these two orthosteric sites to neonicotinoid actions. We therefore applied voltage-clamp electrophysiology to the Dα1/chicken β2 hybrid nAChR expressed in Xenopus laevis oocytes to explore the agonist actions of imidacloprid and thiacloprid on wild type receptors and following binding site mutations. First, we studied the S221E mutation in loop C of the ACh binding site of the Dα1 subunit. Secondly, we explored the impact of combining this mutation in loop C with others in the loop D-E-G triangle (R57S; E78K; K140T; S221E). The S221E loop C mutation alone reduced the affinity of the neonicotinoids tested, while hardly affecting the concentration-response curve for acetylcholine. Addition of the three R57S; E78K; K140T mutations in the loop D-E-G triangle led to a further reduction in neonicotinoid sensitivity, suggesting that all four binding site loops (C, D, E, G) in the Dα1 subunit, which are located upstream of loop B in the N-terminal, extracellular domain, contribute to the selective actions of neonicotinoid insecticides.
  • Aiichiro Muraoka; Yoshinori Matsuura; Hisashi Naitow; Makoto Ihara; Naoki Kunishima
    Analytical Biochemistry 557 46 - 58 Elsevier {BV} 2018年09月 [査読有り]
     
    © 2018 Elsevier Inc. It is known that the crystallizability of protein molecules may be improved by replacing their surface lysine residues with other residue types. Here an experimental method to identify surface lysine residues by NHS-biotin chemical modification combined with MALDI-TOF MS was proposed and was evaluated using PH1033 protein from Pyrococcus horikoshii. Interestingly, the biotinylation experiment with a protein-reagent molar ratio of 1:1 revealed that only seven of twenty-two lysine residues in the protein comprising 144 residues were labeled. To investigate the result, we analyzed structures from a molecular-dynamics simulation mimicking the experiment. A logistic regression analysis revealed that the biotinylation was significantly correlated with four factors relevant to the local environment of lysine residues: the solvent accessibility, the electrostatic energy, the number of hydrogen bonds, and the estimated pKa value. This result is overall in agreement with that from the same analysis on the crystal structure. However, reflecting the flexibility of the protein molecule in solution state, the factors except for the electrostatic energy were highly variable in the MD structures depending upon the protonation state of Tyr87. The present procedure of biotin-labeling can avoid lysine residues with extensive intramolecular interactions that are incompatible with the rational design of protein crystals.
  • Shogo Furutani; Makoto Ihara; Kristin Lees; Steven D. Buckingham; Frederick A. Partridge; Jonathan; A. David; Rohit Patel; Scott Warchal; Ian R. Mellor; Kazuhiko Matsuda; David B. Sattelle
    International Journal for Parasitology: Drugs and Drug Resistance 8 2 350 - 360 Elsevier {BV} 2018年08月 [査読有り]
     
    © 2018 A novel L-glutamate-gated anion channel (IscaGluCl1) has been cloned from the black-legged tick, Ixodes scapularis, which transmits multiple pathogens including the agents of Lyme disease and human granulocytic anaplasmosis. When mRNA encoding IscaGluCl1 was expressed in Xenopus laevis oocytes, we detected robust 50–400 nA currents in response to 100 μM L-glutamate. Responses to L-glutamate were concentration-dependent (pEC50 3.64 ± 0.11). Ibotenate was a partial agonist on IscaGluCl1. We detected no response to 100 μM aspartate, quisqualate, kainate, AMPA or NMDA. Ivermectin at 1 μM activated IscaGluCl1, whereas picrotoxinin (pIC50 6.20 ± 0.04) and the phenylpyrazole fipronil (pIC50 6.90 ± 0.04) showed concentration-dependent block of the L-glutamate response. The indole alkaloid okaramine B, isolated from fermentation products of Penicillium simplicissimum (strain AK40) grown on okara pulp, activated IscaGluCl1 in a concentration-dependent manner (pEC50 5.43 ± 0.43) and may serve as a candidate lead compound for the development of new acaricides.
  • Makoto Ihara; Mai Hikida; Hiroyuki Matsushita; Kyosuke Yamanaka; Yuya Kishimoto; Kazuki Kubo; Shun Watanabe; Mifumi Sakamoto; Koutaro Matsui; Akihiro Yamaguchi; Daiki Okuhara; Shogo Furutani; David B Sattelle; Kazuhiko Matsuda
    British Journal of Pharmacology 175 11 1999 - 2012 Wiley-Blackwell 2018年06月 [査読有り]
     
    © 2017 The British Pharmacological Society Background and Purpose: Neonicotinoid insecticides interact with the orthosteric site formed at subunit interfaces of insect nicotinic ACh (nACh) receptors. However, their interactions with the orthosteric sites at α–non α and α–α subunit interfaces remain poorly understood. The aim of this study was to elucidate the mechanism of neonicotinoid actions using the Drosophila Dα1-chicken β2 hybrid nACh receptor. Experimental Approach: Computer models of the (Dα1) 3 (β2) 2 nACh receptor in complex with imidacloprid and thiacloprid were generated. Amino acids in the Dα1 subunit were mutated to corresponding amino acids in the human α4 subunit to examine their effects on the agonist actions of neonicotinoids on (Dα1) 3 (β2) 2 and (Dα1) 2 (β2) 3 nACh receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology. Key Results: The (Dα1) 3 (β2) 2 nACh receptor models indicated that amino acids in loops D, E and G probably determine the effects of neonicotinoids. The amino acid mutations tested had minimal effects on the EC 50 for ACh. However, the R57S mutation in loop G, although having minimal effect on imidacloprid's actions, reduced the affinity of thiacloprid for the (Dα1) 3 (β2) 2 nACh receptor, while scarcely affecting thiacloprid's action on the (Dα1) 2 (β2) 3 nACh receptor. Both the K140T and the combined R57S;K140T mutations reduced neonicotinoid efficacy but only for the (Dα1) 3 (β2) 2 nACh receptor. Combining the E78K mutation with the R57S;K140T mutations resulted in a selective reduction of thiacloprid's affinity for the (Dα1) 3 (β2) 2 nACh receptor. Conclusions and Implications: These findings suggest that a triangle of residues from loops D, E and G contribute to the selective actions of neonicotinoids on insect-vertebrate hybrid nACh receptors. Linked Articles: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.
  • Kato N; Furutani S; Otaka J; Noguchi A; Kinugasa K; Kai K; Hayashi H; Ihara M; Takahashi S; Matsuda K; Osada H
    ACS chemical biology 13 3 561 - 566 2018年03月 [査読有り]
     
    © 2018 American Chemical Society. Prenylated indole alkaloid okaramines selectively target insect glutamate-gated chloride channels (GluCls). Because of their highly complex structures, including azocine and azetidine rings, total synthesis of okaramine A or B has not been achieved, preventing evaluation of the biological activities of okaramines. Biosynthetic approaches provide alternatives to accessing structurally diverse derivatives and enabling the elucidation of structure-activity relationships. To explore the biosynthetic potential of okaramines, gene knockout experiments of an okaramine-producer fungus were performed. The deletion mutants of the oxygenase genes okaB, okaD, okaE, and okaG provided analogues that were unlikely to be accumulated in the normal biosynthetic process of the wild-type strain. Analysis of the structure-activity relationships of okaramines collected from the fungal cultures revealed that 1,4-dihydroazocine and N-aliphatic group attached to the indole were crucial for GluCl-activating activity. This provided insights into further derivatization of the complex structure of okaramines in order to facilitate the development of new insecticides.
  • Okuhara D; Furutani S; Ito K; Ihara M; Matsuda K
    Molecular pharmacology 92 4 491 - 499 2017年10月 [査読有り]
     
    Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics. The pH-sensitive chloride channels (pHCls) are broadly expressed in insects, but little is known about their physiologic role, diversity, and sensitivity to insecticides acting on relevant chloride channels. Here we have sequenced 50 transcripts of the pHCl-1 gene from the brain, third thoracic ganglion (T3G), and midgut of larvae of silkworm Bombyx mori. It was found that .50 variants were expressed with distinct splicing in the T3G compared with the brain and midgut. Of the variants detected, variant 9, which was expressed most abundantly in the larvae, was reconstituted in Xenopus laevis oocytes to characterize its pH and ivermectin sensitivity. Variant 9 formed a functional pHCl with half-maximal activation at a pH of 7.87, and was activated by ivermectin irrespective of the extracellular pH. This was in contrast to variant 1, which was activated more profoundly at acidic rather than basic pH. To identify a key determinant for such differential ivermectin sensitivity, different amino acids in variants 1 and 9 were swapped, and the effects of the mutations on ivermectin sensitivity were investigated. The V275S mutation of variant 1 enhanced ivermectin sensitivity, whereas the S275V mutation of variant 9 caused a reduction in sensitivity. In homology models of the Bombyx pHCls, Val275 of variant 1 interacted more strongly with Ala273 than Ser275 of variant 9 at the channel gate. This interaction is likely to prevent ivermectin-induced opening of the channel, accounting, at least partially, for the differential macrolide action on the two variants.
  • Onozaki Y; Horikoshi R; Ohno I; Kitsuda S; Durkin KA; Suzuki T; Asahara C; Hiroki N; Komabashiri R; Shimizu R; Furutani S; Ihara M; Matsuda K; Mitomi M; Kagabu S; Uomoto K; Tomizawa M
    Journal of agricultural and food chemistry 65 36 7865 - 7873 2017年09月 [査読有り]
     
    © 2017 American Chemical Society. A novel chemotype insecticide flupyrimin (FLP) [N-[ (E)-1-(6-chloro-3-pyridinylmethyl)pyridin-2(1H)-ylidene]-2,2,2-trifluoroacetamide], discovered by Meiji Seika Pharma, has unique biological properties, including outstanding potency to imidacloprid (IMI)-resistant rice pests together with superior safety toward pollinators. Intriguingly, FLP acts as a nicotinic antagonist in American cockroach neurons, and [3H]FLP binds to the multiple high-affinity binding components in house fly nicotinic acetylcholine (ACh) receptor (nAChR) preparation. One of the [3H]FLP receptors is identical to the IMI receptor, and the alternative is IMI-insensitive subtype. Furthermore, FLP is favorably safe to rats as predicted by the very low affinity to the rat α4β2 nAChR. Structure-activity relationships of FLP analogues in terms of receptor potency, featuring the pyridinylidene and trifluoroacetyl pharmacophores, were examined, thereby establishing the FLP molecular recognition at the Aplysia californica ACh-binding protein, a suitable structural surrogate of the insect nAChR. These FLP pharmacophores account for the excellent receptor affinity, accordingly revealing differences in its binding mechanism from IMI.
  • Steven D. Buckingham; Makoto Ihara; David B Sattelle; Kazuhiko Matsuda
    Current Medicinal Chemistry 24 27 2935 - 2945 Bentham Science Publishers Ltd. 2017年08月 
    © 2017 Bentham Science Publishers. Background: ?-Aminobutyric acid (GABA) receptors play a central role in fast inhibitory neurotransmission in insects. Several classes of insecticides targeting insect GABA-gated chloride channels have been developed. The important resistant to dieldrin GABA receptor subunit (RDL) has been used to investigate insecticide sites of action using radioligands, electrophysiology and site-directed mutagenesis. Although this important subunit readily forms robust functional homomeric receptors when expressed, alternative splicing and RNA A-to-I editing can generate diverse forms of the receptor. Methods: We have reviewed studies on native and recombinant insect GABA-gated chloride channels, their interactions with ligands acting at orthosteric and allosteric sites and their interactions with insecticides. Since some GABA receptor modulators act on L-glutamate-gated chloride channels, some comparisons are included. Results: The actions on GABA-gated chloride channels of polychlorocycloalkanes, cyclodienes, macrocyclic lactones, phenylpyrazoles, isoxazolines, and metadiamides are described and the mechanisms of action of members of these insecticide classes are addressed. Mutations that lead to resistance are discussed as they can be important in developing field diagnostic tests. Toxicity issues relating to insecticides targeting GABA-gated chloride channels are also addressed. Conclusion: An overview of all major insecticide classes targeting insect GABA-gated chloride channels has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native GABA receptors remains unknown and studies to clarify this are needed. Also, the precise sites of action of the recently introduced isoxazolines and meta-diamides will be of interest to pursue.
  • Ihara M; Buckingham SD; Matsuda K; Sattelle DB
    Current medicinal chemistry 24 27 2925 - 2934 2017年08月 
    © 2017 Bentham Science Publishers. Background: Nicotinic acetylcholine receptors (nAChRs) of insects play a key role in fast excitatory neurotransmission. Several classes of insecticides target insect nAChRs, which are composed of subunit members of a family of multiple subunit encoding genes. Alternative splicing and RNA A-to-I editing can add further to receptor diversity. Native and recombinant receptors have been explored as sites of insecticide action using radioligands, electrophysiology and site-directed mutagenesis. Methods: We have reviewed the properties of native and recombinant insect nAChRs, the challenges of functional recombinant insect nAChR expression, nAChR interactions with ligands acting at orthosteric and allosteric sites and in particular their interactions with insecticides. Results: Actions on insect nAChRs of cartap, neonicotinoids, spinosyns, sulfoxamines, butenolides and mesoionic insecticides are reviewed and current knowledge of their modes of action are addressed. Mutations that add to our understanding of insecticide action and those leading to resistance are discussed. Co-crystallisation of neonicotinoids with the acetylcholine binding protein (AChBP), a surrogate for the nAChR ligand binding domain, has proved instructive. Toxicity issues relating to insecticides targeting nAChRs are also considered. Conclusion: An overview of insecticide classes targeting insect nAChRs has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native nAChRs remains poorly understood and functional expression still presents difficulties. These topics together with improved understanding of the precise sites of insecticide actions on insect nAChRs will be the subject of future research.
  • Furutani S; Ihara M; Kai K; Tanaka K; Sattelle D.B; Hayashi H; Matsuda K
    NeuroToxicology 60 240 - 244 2017年05月 [査読有り]
     
    © 2016 Elsevier B.V. The okaramine indole alkaloids were recently shown to be more selective than ivermectin in activating the glutamate-gated chloride channels of the silkworm larvae of Bombyx mori (BmGluCls). Those studies were carried out using the exon 3b variant as a representative of BmGluCls. However, it remains unknown whether okaramines are similarly effective on other silkworm GluCl variants and whether they share the same binding site as ivermectin on GluCls. To begin to address these questions, we examined the potency of four okaramines on the exon 3c variant of BmGluCls by two-electrode voltage clamp voltage recordings of glutamate-induced chloride currents. The potency of okaramines in activating the exon 3c BmGluCl agreed well with findings on the exon 3b BmGluCl and insecticidal potency. Okaramine B (10 μM) reduced the maximum binding (Bmax) but not the dissociation constant (KD) of [3H]ivermectin in studies on plasma membrane fractions of HEK293 cells expressing the exon 3c variant. These findings indicate that activation of GluCls is important in the insecticidal actions of okaramines.
  • Shogo Furutani; Makoto Ihara; Kenji Kai; Keiji Tanaka; David B. Sattelle; Hideo Hayashi; Kazuhiko Matsuda
    NEUROTOXICOLOGY 60 240 - 244 2017年05月 [査読有り]
     
    The okaramine indole alkaloids were recently shown to be more selective than ivermectin in activating the glutamate-gated chloride channels of the silkworm larvae of Bombyx mori (BmGluCls). Those studies were carried out using the exon 3b variant as a representative of BmGluCls. However, it remains unknown whether okaramines are similarly effective on other silkworm GluCl variants and whether they share the same binding site as ivermectin on GluCls. To begin to address these questions, we examined the potency of four okaramines on the exon 3c variant of BmGluCls by two-electrode voltage clamp voltage recordings of glutamate-induced chloride currents. The potency of okaramines in activating the exon 3c BmGluCI agreed well with findings on the exon 3b BmGluCl and insecticidal potency. Okaramine B (10 mu M) reduced the maximum binding (B-max) but not the dissociation constant (K-D) of [H-3]ivermectin in studies on plasma membrane fractions of HEK293 cells expressing the exon 3c variant. These findings indicate that activation of GluCls is important in the insecticidal actions of okaramines. (C) 2016 Elsevier B.V. All rights reserved.
  • Kobashi K; Harada T; Adachi Y; Mori M; Ihara M; Hayasaka D
    Ecotoxicology and Environmental Safety 138 122 - 129 2017年04月 [査読有り]
     
    © 2016 Elsevier Inc. There are growing concerns about the impacts of neonicotinoid insecticides on ecosystems worldwide, and yet ecotoxicity of many of these chemicals at community or ecosystem levels have not been evaluated under realistic conditions. In this study, effects of two neonicotinoid insecticides, imidacloprid and dinotefuran, on aquatic insect assemblages were evaluated in experimental rice mesocosms. During the 5-month period of the rice-growing season, residual concentrations of imidacloprid were 5–10 times higher than those of dinotefuran in both soil and water. Imidacloprid treatment (10 kg/ha) reduced significantly the populations of, Crocothemis servilia mariannae and Lyriothemis pachygastra nymphs, whereas those of Orthetrum albistylum speciosum increased slightly throughout the experimental period. However, Notonecta triguttata, which numbers were high from the start, later declined, indicating possible delayed chronic toxicity, while Guignotus japonicus disappeared. In contrast, dinotefuran (10 kg/ha) did not decrease the populations of any species, but rather increased the abundance of some insects, particularly Chironominae spp. larvae and C. servilia mariannae nymphs, with the latter being 1.7x higher than those of controls. This was an indirect effect resulting from increased prey (e.g., chironomid larvae) and lack of competition with other dragonfly species. The susceptibilities of dragonfly nymphs to neonicotinoids, particularly imidacloprid, were consistent with those reported elsewhere. In general, imidacloprid had higher impacts on aquatic insects compared to dinotefuran.
  • S. Furutani; M. Ihara; K. Kai; H. Hayashi; K. Matsuda
    ACS Symposium Series 1264 125 - 131 2017年 
    Although some fungi produce mycotoxins that render food on which they grow inedible, other fungi produce metabolites that, in addition, to human and animal healthcare, are useful in pest control. Penicillium simplicissimum AK-40 produces okaramines as an insecticide when grown on okara, a by-product of soybean curd production. Okaramines selectively activate glutamate-gated chloride channels expressed only in the nervous system of invertebrates. Asperparalines from Aspergillus japonicus JV-23 and chrodrimanins from Talaromyces sp. YO-2, both of which were isolated similarly to okaramines, selectively block insect nicotinic acetylcholine and γ-aminobutyric acid receptors, respectively. These results suggest that fungi induced by certain plant factors have potential for producing next-generation pesticide leads.
  • Shogo Furutani; Daiki Okuhara; Anju Hashimoto; Makoto Ihara; Kenji Kai; Hideo Hayashi; David B. Sattelle; Kazuhiko Matsuda
    Bioscience, Biotechnology, and Biochemistry 81 10 1861 - 1867 Informa {UK} Limited 2017年 [査読有り]
     
    © 2017 Japan Society for Bioscience, Biotechnology, and Agrochemistry. Okaramines produced by Penicillium simplicissimum AK-40 activate L-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to L-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.
  • Nakatani Y; Furutani S; Ihara M; Matsuda K
    Pesticide biochemistry and physiology 126 1 - 5 Elsevier {BV} 2016年01月 [査読有り]
     
    © 2015 Elsevier Inc.. The pH-sensitive chloride channels (pHCls) are expressed widely in the insect nervous system, but their physiological roles and pesticide sensitivity in Lepidoptera are poorly understood. Here, cDNAs of pHCl variants "A" and "B" were isolated from the head of silkworm (Bombyx mori) larvae and expressed in Xenopus laevis oocytes to characterize their functions and examine their pesticide sensitivity. Variant "A" possesses four entire transmembrane domains (TMs), while variant "B" lacks a part of the TMand the TM3-TM4 linker. Only the A variant formed a chloride channel in oocytes which was activated in response to an increase of pH in the extracellular solution.Neither fipronil nor ?-benzenehexachloride had a significant blocking effect on the A variantwhen tested at 10 μM. By contrast,macrolide ivermectin activated it at acidic pH but blocked it at pH 7.6 at concentrations higher than 300 nM, indicating a likely contribution to in vivo toxicity.
  • Sakamori K; Ono N; Ihara M; Suzuki H; Matsuura H; Tanaka K; Ohta D; Kanaya S; Matsuda K
    Plant signaling & behavior 11 4 e1149675  2016年 [査読有り]
     
    © 2016 Taylor & Francis Group, LLC. Natural pyrethrins are used to control household and agricultural pests, and it is of value to understand biosynthesis in Tanacetum cinerariifolium for enhanced production. We previously found that a blend of four green leaf volatiles (GLVs) and (E)-b-farnesene emitted by T. cinerariifolium seedlings enhanced gene expressions of certain biosynthetic enzymes in unwounded seedlings; however, the extent to which such a regulation facilitates pyrethrin biosynthesis remains unknown. Here we have investigated the effects of the blend of the volatile organic compounds (VOCs) on gene expressions of seven biosynthetic enzymes. VOC treatment resulted in enhanced chrysanthemyl diphosphate synthase (CDS), chrysanthemic acid synthase (CAS), Tanacetum cinerariifolium GDSL lipase (TcGLIP) and acyl-Coenzyme A oxidase 1 (ACX1) gene expressions that reached a peak at a 12 h VOC treatment, whereas the treatment minimally influenced the expressions of other biosynthetic genes. In undifferentiated Tanacetum tissues, such VOCinduced amplification of CDS, CAS, TcGLIP and ACX1 gene expressions were markedly reduced, suggesting that a high-resolution, VOC-mediated communication is an event selective to differentiated plants.
  • Makoto Ihara; David B. Sattelle; Kazuhiko Matsud
    Pesticide Biochemistry and Physiology 121 47 - 52 Elsevier {BV} 2015年06月 
    © 2015 Elsevier Inc. Neonicotinoid insecticides interact with the orthosteric site on the extracellular ligand binding domain (LBD) of nicotinic acetylcholine receptors (nAChRs), typically activating the cation permeable ion channels. In nAChRs consisting of two α and three non-α subunits, LBDs contain six loops (loops A, B and C on the α subunit and loops D, E and F on the non-α subunit) which make up the orthosteric binding site at the α/non-α subunit interfaces. Recently, an additional site (loop G) on the β1 strand has been identified. Also, when the α/non-α subunit ratio is 3/2, another binding site is generated at the interface of two adjacent α subunits. Roles for loop G and the α-α interface in the interactions with neonicotinoids are discussed with reference to recent structural and physiological data.
  • Yan Xu; Shogo Furutani; Makoto Ihara; Yun Ling; Xinling Yang; Kenji Kai; Hideo Hayashi; Kazuhiko Matsud
    PLoS} {ONE 10 4 e0122629  Public Library of Science ({PLoS}) 2015年04月 [査読有り]
     
    © 2015 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs.
  • Shogo Furutani; Makoto Ihara; Yuri Nishino; Miki Akamatsu; Andrew K. Jones; David B. Sattelle; Kazuhiko Matsuda
    MOLECULAR PHARMACOLOGY 86 6 686 - 695 2014年12月 [査読有り]
     
    Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. Insect GluCls show alternative splicing, and, to determine its impact on channel function and pharmacology, we isolated GluCl cDNAs from larvae of the silkworm (Bombyx mori). We show that six B. mori glutamate-gated chloride channel variants are generated by splicing in exons 3 and 9 and that exons 3b and 3c are common in the brain and third thoracic ganglion. When expressed in Xenopus laevis oocytes, the three functional exon 3 variants (3a, b, c) all had similar EC50 values for L-glutamate and ivermectin (IVM); however, I-max (the maximum L-glutamate- and IVM-induced response of the channels at saturating concentrations) differed strikingly between variants, with the 3c variant showing the largest L-glutamate- and IVM-induced responses. By contrast, a partial deletion detected in exon 9 had a much smaller impact on L-glutamate and IVM actions. Binding assays using [H-3] IVM indicate that diversity in IVM responses among the GluCl variants is mainly due to the impact on channel assembly, altering receptor cell surface numbers. GluCl variants expressed in HEK293 cells show that structural differences influenced B-max but not K-d values of [H-3] IVM. Domain swapping and site-directed mutagenesis identified four amino acids in exon 3c as hot spots determining the highest amplitude of the L-glutamate and IVM responses. Modeling the GluCl 3a and 3c variants suggested that three of the four amino acids contribute to intersubunit contacts, whereas the other interacts with the TM2-TM3 linker, influencing the receptor response.
  • M. Ihara; T. Okajima; A. Yamashita; T. Oda; T. Asano; M. Matsui; D. B. Sattelle; K. Matsud
    Molecular Pharmacology 86 6 736 - 746 American Society for Pharmacology {\&} Experimental Therapeutics ({ASPET}) 2014年12月 [査読有り]
     
    Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs). Their widespread use and possible risks to pollinators make it extremely urgent to understand the mechanisms underlying their actions on insect nAChRs. We therefore elucidated X-ray crystal structures of the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) and its Gln55Arg mutant, more closely resembling insect nAChRs, in complex with a nitromethylene imidacloprid analog (CH-IMI) and desnitro-imidacloprid metabolite (DN-IMI) as well as commercial neonicotinoids, imidacloprid, clothianidin, and thiacloprid. Unlike imidacloprid, clothianidin, and CH-IMI, thiacloprid did not stack with Tyr185 in the wild-type Ls-AChBP, but did in the Gln55Arg mutant, interacting electrostatically with Arg55. In contrast, DN-IMI lacking the NO2 group was directed away from Lys34 and Arg55 to form hydrogen bonds with Tyr89 in loop A and the main chain carbonyl of Trp143 in loop B. Unexpectedly, we found that several neonicotinoids interacted with Lys34 in loop G on the beta 1 strand in the crystal structure of the Gln55Arg mutant. Basic residues introduced into the alpha 7 nAChR at positions equivalent to AChBP Lys34 and Arg55 enhanced agonist actions of neonicotinoids, while reducing the actions of acetylcholine, (-)-nicotine, and DN-IMI. Thus, not only the basic residues in loop D, but also those in loop G determine the actions of neonicotinoids. These novel findings provide new insights into the modes of action of neonicotinoids and emerging derivatives.
  • Shogo Furutani; Yuri Nakatani; Yuka Miura; Makoto Ihara; Kenji Kai; Hideo Hayashi; Kazuhiko Matsuda
    SCIENTIFIC REPORTS 4 6190  2014年08月 [査読有り]
     
    In 1989, indole alkaloid okaramines isolated from the fermentation products of Penicillium simplicissimum were shown to be insecticidal, yet the mechanism of their toxicity to insects remains unknown. We therefore examined the action of okaramine B on silkworm larval neurons using patch-clamp electrophysiology. Okaramine B induced inward currents which reversed close to the chloride equilibrium potential and were blocked by fipronil. Thus it was tested on the silkworm RDL (resistant-to-dieldrin) gamma-aminobutyric-acid-gated chloride channel (GABACl) and a silkworm L-glutamate-gated chloride channel (GluCl) expressed in Xenopus laevis oocytes. Okaramine B activated GluCl, but not RDL. GluCl activation by okaramines correlated with their insecticidal activity, offering a solution to a long-standing enigma concerning their insecticidal actions. Also, unlike ivermectin, okaramine B was inactive at 10 mu M on human alpha 1 beta 2 gamma 2 GABACl and alpha 1 beta glycine-gated chloride channels and provides a new lead for the development of safe insect control chemicals.
  • Makoto Ihara; Yoshitaka Takano; Atsuko Yamashita
    PROTEIN SCIENCE 23 7 923 - 931 2014年07月 [査読有り]
     
    Transient receptor potential (TRP) channels are members of the voltage gated ion channel superfamily and display the unique characteristic of activation by diverse stimuli. We performed an expression analysis of fungal TRP channels, which possess relatively simple structures yet share the common functional characteristics with the other members, using a green fluorescent protein-based screening methodology. The analysis revealed that all the tested fungal TRP channels were severely digested in their cytosolic regions during expression, implying the common flexibility of this region, as observed in the recent structural analyses of the fungal member, TRPGz. These characteristics are likely to be important for their diverse functions.
  • Makoto Ihara; Naoya Shimazu; Mai Utsunomiya; Miki Akamatsu; David B. Sattelle; Kazuhiko Matsud
    Bioscience, Biotechnology and Biochemistry 78 4 543 - 549 Informa UK Limited 2014年04月 [査読有り]
     
    Polymorphisms are sometimes observed in native insect nicotinic acetylcholine receptor (nAChR) subunits, which are important insecticide targets, yet little is known of their impact on insecticide actions. Here we investigated the effects of a polymorphism involving the substitution of histidine108 by leucine in the Drosophila melanogaster D alpha 1 subunit on the agonist actions of the neurotransmitter acetylcholine (ACh) and two commercial neonicotinoid insecticides (imidacloprid and clothianidin). There was no significant impact of the H108L substitution on either the ACh EC50, the concentration leading to a half maximal ACh response, or the maximum current amplitude in response at 10 mu M ACh, of the D alpha 1-chicken beta 2 nAChR expressed in Xenopus laevis oocytes. However, the response amplitudes to imidacloprid and clothianidin were significantly enhanced, indicating a role of His108 in the selective interactions of D alpha 1 with these neonicotinoids.
  • Makoto Ihara; Shin Hamamoto; Yohei Miyanoiri; Mitsuhiro Takeda; Masatsune Kainosho; Isamu Yabe; Nobuyuki Uozumi; Atsuko Yamashita
    Journal of Biological Chemistry 288 21 15303 - 15317 2013年05月 [査読有り]
     
    Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca2+ elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
  • Yuji Ashikawa; Makoto Ihara; Noriko Matsuura; Yuko Fukunaga; Yuko Kusakabe; Atsuko Yamashita
    PROTEIN SCIENCE 20 10 1720 - 1734 2011年10月 [査読有り]
     
    Applications of the GFP-fusion technique have greatly facilitated evaluations of the amounts and qualities of sample proteins used for structural analyses. In this study, we applied the GFP-based sample evaluation to secreted protein expression by insect cells. We verified that a GFP variant, GFPuv, retains proper folding and monodispersity within all expression spaces in Sf9 cells, such as the cytosol, organelles, and even the extracellular space after secretion, and thus can serve as a proper folding reporter for recombinant proteins. We then applied the GFPuv-based system to the extracellular domains of class C G-protein coupled receptors (GPCRs) and examined their localization, folding, and oligomerization upon insect cell expression. The extracellular domain of metabotropic glutamate receptor 1 (mGluR1) exhibited good secreted expression by Sf9 cells, and the secreted proteins formed dimer with a monodisperse hydrodynamic state favorable for crystallization, consistent with the results from previous successful structural analyses. In contrast, the extracellular domains of sweet/umami taste receptors (T1R) almost completely remained in the cell. Notably, the T1R and mGluR1 subfractions that remained in the cellular space showed polydisperse hydrodynamic states with large aggregated fractions, without forming dimers. These results indicated that the proper folding and oligomerization of the extracellular domains of the class C GPCR are achieved through the secretory pathway.
  • Makoto Ihara; Noriko Matsuura; Atsuko Yamashita
    ANALYTICAL BIOCHEMISTRY 412 2 217 - 223 2011年05月 [査読有り]
     
    An improved native polyacrylamide gel electrophoresis (PAGE) method capable of evaluating the hydrodynamic states of membrane proteins and allowing in-gel fluorescence detection was established. In this method, bis(alkyl) sulfosuccinate is used to provide negative charges for detergent-solubilized membrane proteins to facilitate proper electrophoretic migration without disturbing their native hydrodynamic states. The method achieved high-resolution electrophoretic separation, in good agreement with the elution profiles obtained by size exclusion chromatography. The applicability of in-gel fluorescence detection for tagged green fluorescent protein (GFP) facilitates the analysis of samples without any purification. This method might serve as a general analytical technique for assessing the folding, oligomerization, and protein complex formation of membrane proteins. (C) 2011 Elsevier Inc. All rights reserved.
  • Ashikawa Yuji; Ihara Makoto; Matsuura Noriko; Fukunaga Yuko; Kusakabe Yuko; Yamashita Atsuko
    生物物理 51 S138  一般社団法人 日本生物物理学会 2011年
  • Makoto Ihara; Toshihide Okajima; Atsuko Yamashita; Takuma Oda; Koichi Hirata; Hisashi Nishiwaki; Takako Morimoto; Miki Akamatsu; Yuji Ashikawa; Shun'Ichi Kuroda; Ryosuke Mega; Seiki Kuramitsu; David B. Sattelle; Kazuhiko Matsuda
    Invertebrate Neuroscience 8 2 71 - 81 2008年06月 [査読有り]
     
    Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR-neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH-π interactions in the Ls-AChBP-CTD complex than in the Ls-AChBP-IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs. © 2008 The Author(s).
  • Kenzo Fujimoto; Yoshinaga Yoshimura; Makoto Ihara; Kazuhiko Matsuda; Yuko Takeuchi; Takaaki Aoki; Toru Ide
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 18 3 1106 - 1109 2008年02月 [査読有り]
     
    We synthesized a novel fluorescent analogue of acetylcholine, Cy3-3-acyleholine. 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. (C) 2007 Elsevier Ltd. All rights reserved.
  • Ihara Makoto; Okajima Toshihide; Yamashita Atsuko; Oda Takuma; Ashikawa Yuji; Sattelle David B.; Matsuda Kazuhiko
    生物物理 48 S58  一般社団法人 日本生物物理学会 2008年
  • TOSHIMA Kayoko; IHARA Makoto; KANAOKA Satoshi; TARUMOTO Kiyoshi; YAMADA Atsushi; SATTELLE David B; MATSUDA Kazuhiko
    Journal of pesticide science = 日本農薬学会誌 33 2 146 - 151 2008年 
    Effects of imidacloprid, clothianidin, thiacloprid and related compounds on the acetylcholine (ACh)-induced response of the recombinant, expressed chicken alpha 4 beta 2 nicotinic acetylcholine receptor (nAChR) were investigated using voltage-clamp electrophysiology. Imidacloprid and clothianidin enhanced the amplitude of the response to ACh of alpha 4 beta 2 nAChR. In complete contrast, thiacloprid attenuated the amplitude of the response to ACh of alpha 4 beta 2 nAChR. Replacing the nitro group of imidacloprid by a cyano group abolished the potentiating action, whereas exchanging the cyano group of thiacloprid for a nitro group conferred the ability to potentiate the ACh response. All three neonicotinoids shifted the ACh concentration-response curve without influencing the peak current amplitude of the ACh response. (C) Pesticide Science Society of Japan.
  • Makoto Ihara; Koichi Hirata; Chiharu Ishida; Shinzo Kagabu; Kazuhiko Matsuda
    NEUROSCIENCE LETTERS 425 3 137 - 140 2007年10月 [査読有り]
     
    Neonicotinoid insecticides target nicotinic acetylcholine receptors (nAChRs), which, in both vertebrates and invertebrates, mediate fast-acting synaptic neurotransmission in the nervous system. Recently, Kagabu et al. synthesized bis-neonicotinoids. The neural activities of bis-neonicotinoids have been evaluated on the central nerve cord of American cockroaches. However, the action of bis-neonicotinoids on nAChRs expressed by dissociated insect neurons has not yet been studied. Thus, the actions of several alkylene-tethered bis-neonicotinoids on the terminal abdominal ganglion neurons of the American cockroach, Periplaneta americana, were investigated using whole-cell patch-clamp electrophysiology. All of the ligands tested did not induce membrane currents, but reduced the responses to ACh when bath applied prior to co-application with ACh. Of the compounds tested, HK- 13, which possesses two imidacloprid units linked with a hexamethylene bridge, had the highest antagonist potency. The antagonist action was reduced, not only by elongating, but also by shortening the linker. (C) 2007 Elsevier Ireland Ltd. All rights reserved.
  • Ikuya Ohno; Koichi Hirata; Chiharu Ishida; Makoto Ihara; Kazuhiko Matsuda; Shinzo Kagabu
    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 17 16 4500 - 4503 2007年08月 [査読有り]
     
    Prod rugs of imidacloprid and the thiazolylmethyl analog masked with oxodioxolylniethyl group on the N3 site were prepared. The prodrugs decomposed in a buffer solution of pH 8.3 and in a physiological salt solution with half-lives of 10-15 h, releasing the parent insecticides. Being consistent with this, an inward current was evoked in dissociated cockroach neurons treated with the masked compound solutions, which were maintained for 24 h after preparation, as measured using patch-clamp electrophysiology, whereas no response was observed in neurons when the solutions were challenged immediately after preparation. The insecticidal test on the American cockroach showed that the minimum lethal dose for each compound at 24 h after injection was 6.4 x 10(-8) mol, which was similar to that for imidacloprid and the thiazolyl derivative. This result strongly suggested a regeneration of the active ingredients in vivo. (c) 2007 Elsevier Ltd. All rights reserved.
  • Makoto Ihara; Masaru Shimomura; Chiharu Ishida; Hisashi Nishiwaki; Miki Akamatsu; David B. Sattelle; Kazuhiko Matsuda
    Invertebrate Neuroscience 7 1 47 - 51 2007年03月 [査読有り]
     
    The low mammalian toxicity of neonicotinoid insecticides has been shown to be attributable, at least in part, to their selective actions on insect nicotinic acetylcholine receptors (nAChRs). There are multiple nAChRs in insects and a wealth of neonicotinoid chemicals. Studies to date have discribed a wide range of effects on nAChRs, notably partial agonist, super agonist and antagonist actions. Both the diversity of the neonicotinoid actions and their selectivity for insect over vertebrate nAChRs are the result of physicochemical and steric interactions at their molecular targets (nAChRs). In such interactions, the formation and breakage of hydrogen bond (HB) networks plays a key role. Therefore the loss or gain of even a single HB resulting from either structural changes in neonicotinoids, or the amino acid sequence of a particular nAChR subunit, could result in a drastic modification of neonicotinoid actions. In addition to the amino acid residues, the backbone carbonyl of nAChRs may also be involved in the formation of HB networks with neonicotinoids. © 2007 Springer-Verlag.
  • Makoto Ihara
    JOURNAL OF PESTICIDE SCIENCE 32 3 278 - 280 2007年 [査読有り]
     
    Ligand-gated ion channels (LGICs) mediate fast synaptic neurotransmission and are important targets for insecticides. Thus, the actions of several insecticides have been explored in electrophysiological studies on recombinant and native insect neuronal LGICs. I have shown that non-competitive antagonists of gamma-aminobutyric acid gated Cl- channels also act on glutamate-gated Cl- channels, albeit at higher concentrations. Neonicotinoids are more potent agonists on recombinant hybrid nicotinic acetylcholine receptors (nAChRs) consisting of Drosophila D alpha 2 and vertebrate beta 2 subunits than those consisting of only vertebrate nAChR subunits (alpha 4 beta 2). Using this hybrid nAChR, clothianidin and related compounds containing a acyclic guanidine moiety were found to be super-agonists. Similar super-agonist actions of neonicotinoids were also observed on cultured Drosophila cholinergic neurons. Single channel nAChR recordings show that a clothianidin analogue induces a high conductance state in channel opening more frequently than acetylcholine, thereby offering a possible explanation for its super-agonist action. Unlike the case for clothianidin, imidacloprid attenuates the acetylcholine-induced response of native neuronal nAChRs when co-applied with ACh. These new discoveries add to our understanding of both the selectivity and the diverse actions of insecticides targeting LGICs. (c) Pesticide Science Society of Japan.
  • Masaru Shimomura; Maiko Yokota; Makoto Ihara; Miki Akamatsu; David B. Sattelle; Kazuhiko Matsuda
    MOLECULAR PHARMACOLOGY 70 4 1255 - 1263 2006年10月 [査読有り]
     
    The insecticide imidacloprid and structurally related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (nAChRs). To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of mutations to basic amino acid residues in loop D of the nAChR acetylcholine (ACh) binding site on the interactions with imidacloprid. The receptors investigated are the recombinant chicken alpha 4 beta 2 nAChR and Drosophila melanogaster D alpha 2/chicken beta 2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of Thr77 in loop D of the beta 2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the alpha 4 beta 2 nAChR, T77R; E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Likewise, T77K; E79R and T77N; E79R double mutations in the D alpha 2 beta 2 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of Thr77 to basic residues. By contrast, these double mutations scarcely influenced the ACh concentration-response curve, suggesting selective interactions with imidacloprid of the newly introduced basic residues. Computational, homology models of the agonist binding domain of the wild-type and mutant alpha 4 beta 2 and D alpha 2 beta 2 nAChRs with imidacloprid bound were generated based on the crystal structures of acetylcholine binding proteins of Lymnaea stagnalis and Aplysia californica. The models indicate that the nitro group of imidacloprid interacts directly with the introduced basic residues at position 77, whereas those at position 79 either prevent or permit such interactions depending on their electrostatic properties, thereby explaining the observed functional changes resulting from site-directed mutagenesis.
  • Laurence A. Brown; Makoto Ihara; Steven D. Buckingham; Kazuhiko Matsuda; David B. Sattelle
    JOURNAL OF NEUROCHEMISTRY 99 2 608 - 615 2006年10月 [査読有り]
     
    Nicotinic acetylcholine receptors (nAChRs) are present in high density in insect nervous tissue and are targeted by neonicotinoid insecticides. Improved understanding of the actions of these insecticides will assist in the development of new compounds. Here, we have used whole-cell patch-clamp recording of cholinergic neurons cultured from the central nervous system of 3rd instar Drosophila larvae to examine the actions of acetylcholine (ACh) and nicotine, as well as the neonicotinoids imidacloprid, clothianidin and P-CH-clothianidin on native nAChRs of these neurons. Dose-response data yield an EC50 value for ACh of 19 mu M. Both nicotine and imidacloprid act as low efficacy agonists at native nAChRs, evoking maximal current amplitudes 10-14% of those observed for ACh. Conversely, clothianidin and P-CH-clothianidin evoke maximal current amplitudes up to 56% greater than those evoked by 100 mu M ACh in the same neurons. This is the first demonstration of 'super' agonist actions of an insecticide on native insect nAChRs. Cell-attached recordings indicate that super agonism results from more frequent openings at the largest (63.5 pS) conductance state observed.
  • Takeuchi Yuko; Aoki Takaaki; Ihara Makoto; Yoshimura Yoshinaga; Fujimoto Kenzo; Matsuda Kazuhiko; Ide Toru
    生物物理 46 2 S239  一般社団法人 日本生物物理学会 2006年
  • Kumiko Minami; Toru Nakasugi; Han-Dong Sun; Ai-Jun Hou; Makoto Ihara; Masanori Morimoto; Koichiro Komai
    JOURNAL OF NATURAL MEDICINES 60 2 138 - 140 2006年 [査読有り]
     
    Seven histamine-release inhibitors were isolated from Pistacia weinmannifolia J. Pisson ex. Franch. They were identified as gallic acid, 3-O-galloylquinic acid, methyl gallate, ethyl gallate, penta-O-galloyl-beta-D-glucopyrano side, myricetin 3-O-alpha-L-rhamnopyranoside, and myricetin-3-O-(3"-O-gallOyl)-alpha-L-rhamnopyranoside. These compounds suppressed the compound 48/80-induced histamine release from rat peritoneal mast cells.
  • M Ihara; LA Brown; C Ishida; H Okuda; DB Sattelle; K Matsuda
    JOURNAL OF PESTICIDE SCIENCE 31 1 35 - 40 2006年 [査読有り]
     
    The actions of neonicotinoid insecticides on nicotinic acetylcholine receptors (nAChRs) in the terminal abdominal ganglion neurons of the American cockroach were investigated using whole-cell patch-clamp electrophysiology. Neonicotinoids possessing a nitromethylene group showed higher agonist affinity than the corresponding nitroimine analogues, whereas compounds with an acyclic guanidine moiety showed greater agonist efficacy than the corresponding cyclic compounds. Imidacloprid showed the lowest agonist efficacy of all neonicotinoids and low concentrations of imidacloprid attenuated acetylcholine-induced currents. However, such blocking actions were minimal with other neonicotinoids. The diverse actions of neonicotinoids on nAChRs, combined with target accessibility based on hydrophobicity, appears to account for their insecticidal potency on cockroaches measured in the presence of metabolic inhibitors. (c) Pesticide Science Society of Japan.
  • Ihara Makoto; Ishida Chiharu; Okuda Hiroshi; Ozoe Yoshihisa; Matsuda Kazuhik
    Invertebrate Neuroscience 5 3-4 157 - 164 Springer-Verlag 2005年11月 [査読有り]
     
    4′-Ethynyl-4-n-propylbicycloorthobenzoate (EBOB) has been employed extensively as a radioligand in binding assays to evaluate the pharmacology of γ-aminobutyric acid (GABA)-gated Cl- channels (GABARs) of insects and mammals, and γ-hexachlorocyclohexane (γ-HCH) was used as an insecticide targeting insect GABARs. Since recent studies have shown that not only GABARs but also glutamate-gated chloride channels (GluCls) are blocked by picrotoxinin, dieldrin and fipronil, the actions of EBOB and γ-HCH on native GABARs and GluCls of terminal abdominal ganglion neurons in American cockroach (Periplaneta americana) were tested using patch-clamp electrophysiology. A marked run-down of the GABA- and glutamate-induced responses of the cockroach neurons occurred, when a standard pipette solution was employed, but addition of pyruvate to the solution permitted stable recordings of these responses. With this solution, EBOB and γ-HCH were found to block not only the GABA- but also glutamate-gated responses, with the actions augmented by repeated co-application with the agonists. It was also found that prolonged pre-application of EBOB and γ-HCH prior to co-application with GABA and glutamate resulted in enhanced blocking actions, indicating resting-state actions of the blockers. The blocking actions of EBOB and γ-HCH on the GABA- and glutamate-induced responses were compared by determining IC50 values under steady state condition. The IC50 values for the actions of EBOB on GABAR and GluCls differed less than those of γ-HCH. © Springer-Verlag 2005.
  • M Shimomura; H Satoh; M Yokota; M Ihara; K Matsuda; DB Sattelle
    NEUROSCIENCE LETTERS 385 2 168 - 172 2005年09月 [査読有り]
     
    A chimera based on the chicken alpha 4 nicotinic acetylcholine receptor (nAChR) subunit containing an insert from loop B to the N-terminus of the Drosophila melanogaster D alpha 2 (=SAD) subunit was constructed and co-expressed with the chicken beta 2 nAChR subunit in Xenopus laevis oocytes. The actions of the neonicotinoid insecticide imidacloprid were examined. Replacement of the region loop B to the N-terminus of the alpha 4 subunit by the corTesponding region of the D alpha 2 subunit had little effect on the concentration-response curve for imidacloprid. However, replacement of Glu219 by proline in the YXCC motif in loop C of the chimeric alpha 4 subunit resulted in a marked displacement to the left of the concentration-response curve for imidacloprid not seen when an equivalent mutation was made in the alpha 4 beta 2 nAChR. The results suggest that the region loop B to the N-terminus in the D alpha 2 subunit contributes to the high imidacloprid sensitivity of the hybrid D alpha 2 beta 2 nAChR. (c) 2005 Elsevier Ireland Ltd. All rights reserved.
  • K Matsuda; M Shimomura; M Ihara; M Akamatsu; DB Sattelle
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 8 1442 - 1452 2005年08月 [査読有り]
     
    Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.
  • IHARA Makoto; MATSUDA Kazuhiko; SHIMOMURA Masaru; SATTELLE David B; KOMAI Koichiro
    Bioscience, biotechnology, and biochemistry 68 3 761 - 763 社団法人 日本農芸化学会 2004年03月 [査読有り]
     
    To compare the actions of clothianidin, a neonicotinoid acting on insect nicotinic acetylcholine receptors, and related compounds with that of imidacloprid, the compounds were tested on the Drosophila SAD-chicken β2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. The maximum response of the SADβ2 nicotinic receptor to clothianidin was larger than that observed for acetylcholine. Ring breakage of the imidazolidine ring of imidacloprid resulting in the generation of a guanidine group was critical for this super agonist ...
  • Actions of clothianidin and related compounds on recombinant nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.
    伊原 誠; 松田 一彦; David B. Sattelle
    Neurotox 2003: Neurotoxicological Targets from Functional Genomics and Proteomics 19 - 24 Society for Chemical Industry 2004年
  • Valerie Raymond Delpech; Makoto Ihara; Claudio Coddou; Kazuhiko Matsuda; David B. Sattelle
    INVERTEBRATE NEUROSCIENCE 5 1 29 - 35 2003年11月 [査読有り]
     
    Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken alpha 7, chicken alpha 4 beta 2 and the Drosophila melanogaster/chicken hybrid receptors SAD/beta 2 and ALS/beta 2. No agonist action of NTX (0.1-100 mu M) was observed on chicken alpha 7, chicken alpha 4 beta 2 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (alpha 7) or heteromeric (alpha 4 beta 2) nicotinic AChRs. Block by NTX of the chicken a7, chicken alpha 4 beta 2 and the SAD/beta 2 and ALS/beta 2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.
  • M Ihara; K Matsuda; M Otake; M Kuwamura; M Shimomura; K Komai; M Akamatsu; Raymond, V; DB Sattelle
    NEUROPHARMACOLOGY 45 1 133 - 144 2003年07月 [査読有り]
     
    The 2-nitroimino-imidazolidine and related moieties are structural features of neonicotinoid insecticides acting on nicotinic acetylcholine receptors (nicotinic AChRs). To evaluate these moieties in neonicotinoid interactions with nicotinic AChR alpha subunits, the actions of imidacloprid and related compounds on the chicken alpha7, alpha4beta2 and Drosophila melanogaster-chicken hybrid (SADbeta2 and ALSbeta2) receptors expressed in Xenopus laevis oocytes were studied by voltage-clamp electrophysiology. Imidacloprid and nitenpyram were partial agonists and a nitromethylene analog of imidacloprid (CH-IMI) was a full agonist of the beta receptor, whereas their agonist actions on the alpha4beta2 receptor were very weak, contrasting with full agonist actions of DN-IMI, a desnitro derivative of imidacloprid. The neonicotinoids and DN-IMI were either full or partial agonists of the SADbeta2 receptors. Nitenpyram and DN-IMI were partial agonists of the ALSbeta2 receptor, whereas imidacloprid and CH-IMI scarcely activated the ALSbeta2 receptor. Imidacloprid and CH-IMI in fact suppressed ACh-induced responses of the ALSbeta2 receptor, whereas imidacloprid potentiated and CH-IMI suppressed ACh-induced responses of the alpha4beta2 receptor. These results suggest that interactions with alpha subunits of the 2-nitroimino-imidazolidine moiety of imidacloprid play a role in determining not only agonist and antagonist actions on all four receptors, but also the potentiation of ACh-induced responses of the alpha4beta2 receptor. (C) 2003 Elsevier Science Ltd. All rights reserved.
  • K Matsuda; M Ihara; K Nishimura; DB Sattelle; K Komai
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 7 1534 - 1541 2001年07月 [査読有り]
     
    Photoreactive derivatives of imidacloprid and its nitromethylene analogue were synthesized as candidate photoaffinity probes for identifying the amino acid residues of nicotinic acetylcholine receptors (nAChRs) that interact with the neonicotinoid insecticides. When the candidate probes were injected into American cockroaches, the nerve cord neural activity initially increased, then ceased and death of the insect followed. Both the nerve cord and toxicity were enhanced by changing the photoreactive substituent from the para position to the meta position on the spacer benzyl moiety. When tested on a Drosophila SAD/chicken beta2 hybrid, recombinant nAChR expressed in Xenopus oocytes, the nitromethylene candidate probes showed agonist activity similar to that previously observed for imidacloprid.
  • Matsuda Kazuhiko; Shimomura Masaru; Kondo Yumi; Ihara Makoto; Hashigami Kaori; Yoshida Naofumi; Raymond Valérie; Mongan Nigel P; Freeman John C; Komai Koichiro
    British Journal of Pharmacology 130 5 981 - 986 Wiley Blackwell (Blackwell Publishing) 2000年07月 [査読有り]
     
    1 The nitroguanidine insecticide imidacloprid along with a second generation of related compounds including nitenpyram, all nicotinic acetylcholine (ACh) receptor ligands, are used increasingly in many countries. Site-directed mutagenesis and heterologous expression in Xenopus laevis oocytes have been deployed to investigate mutants (G189D and G189E) of the chicken alpha 7 homomer-forming nicotinic receptor subunit which are predicted to enhance the negative charge at the negative subsite (loop D) of the ACh binding site. 2 Xenopus oocytes expressing wild-type a7 nicotinic receptors respond to imidacloprid with rapid inward currents. Imidacloprid and nitenpyram are partial agonists, whereas ACh, (-)-nicotine and (+)-epibatidine are full agonists. 3 Compared to wild-type alpha 7, the mutant G189D and G189E receptors are much less sensitive to the insecticides, whereas their sensitivity to (-)-nicotine, ACh and (+)-epibatidine is only slightly reduced. In contrast, G189N and G189Q mutants are sensitive not only to ACh, (-)-nicotine and (+)-epibatidine, but also to the two insecticides. Thus reduction of the insecticide-sensitivity by the mutations G189D and G189E are attributed to an increase in negativity of loop D. Desnitro-imidacloprid (DN-IMI), an imidacloprid derivative lacking the nitro group is a potent agonist on the G189D and G189E mutants suggesting an important role of loop D in nicotinic receptor interactions with the nitro group of nitroguanidine insecticides.

MISC

講演・口頭発表等

  • Actions of clothianidin and related neonicotinoids on recombinant and native nicotinic acetylcholine receptors.  [通常講演]
    Makoto Ihara
    XXII International Congress of Entomology, Brisbane, Australia 2004年 口頭発表(一般)
  • Structure activity relationship of clothianinidin and its related compounds  [通常講演]
    Makoto Ihara
    The third Pan-Pacific Conference on Pesticide Science, Hawaii, USA. 2003年 ポスター発表
  • Variations in agonist and antagonist profiles for neonicotinoids on recombinant nicotinic acetylcholine receptors expressed in Xenopus oocytes  [通常講演]
    Makoto Ihara
    10th IUPAC International Congress on the Chemistry of Crop Protection, Basel, Switzerland. 2002年 ポスター発表
  • Actions of clothianidin and related compounds on recombinant nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes  [通常講演]
    Makoto Ihara
    Neurotox 2003, Nottingham, UK. 口頭発表(一般)

担当経験のある科目_授業

  • 生命情報学実習近畿大学
  • 生命情報学近畿大学
  • 応用生命化学実験III(有機化学実験)近畿大学
  • 専門英語近畿大学
  • 有機反応化学近畿大学

所属学協会

  • 日本農芸化学会   日本農薬学会   

共同研究・競争的資金等の研究課題

  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2022年04月 -2026年03月 
    代表者 : 伊原 誠
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2016年04月 -2018年03月 
    代表者 : 伊原 誠; 疋田 麻衣; 福田 芳恵
     
    ネオニコチノイド系殺虫剤は,世界中で広く使用されている農業・園芸用殺虫剤であり,近年その毒性に注目が集まっている.本研究では,ネオニコチノイドの毒性発現を制御するニコチン性アセチルコリン受容体(nAChR)の構造因子を探索するために,nAChRのモデルタンパク質,アセチルコリン結合タンパク質を用いたX線結晶構造解析およびホモロジーモデリング法を用いて,ネオニコチノイドとnAChRの相互作用に重要な構造因子を探索した.その結果,昆虫のnAChRαサブユニット上に存在する,Loop領域のうち D,G,E領域のアミノ酸残基がネオニコチノイドとの相互作用に有利な構造を形成することを明らかにした.
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 松田 一彦; 平竹 潤; 松井 健二; 山下 敦子; 伊原 誠
     
    エステル構造をもつ殺虫性物質ピレスリンは、除虫菊の中で酸部とアルコール部とがGDSL リパーゼTcGLIPのはたらきにより連結されることにより生合成される。本研究では、本酵素の制御機構を解明するため、まずその触媒機構に必須のアミノ酸を大腸菌で発現させた組換え酵素を用いて同定し、結晶化に適した塩類溶液を明らかにした。また、低濃度で不可逆的に本酵素を阻害するホスホン酸エステル類を創出し、本酵素の遺伝子発現が傷害誘導的に生じる揮発性物質により促進されることを明らかにした。さらに、シロイヌナズナで本酵素遺伝子のホモログ遺伝子を破壊すると、防御遺伝子のプロモータ促進活性が低下することを見出した。

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