著者

丸田 聡 山岡 薫 大越 夏実 山下 まり

出版者

天然有機化合物討論会

雑誌

天然有機化合物討論会講演要旨集

巻号頁・発行日

no.49, pp.443-448, 2007-08-24

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Tetrodotoxin (TTX) and saxitoxin (STX) bind to a single site in the outer pore of the voltage-gated sodium channels (Na_vs), formed by the amino-acid residues in the outer-pore loops (p-loops) located between the S5 and S6 segments of each of the homologous domain (I-IV) of the a-subunit. Since puffer fish and newts accumulate TTX at high concentration in their tissues, they are thought to have special defense systems against their own TTX. We previously obtained a cDNA encoding Na_v from Fugu pardalis skeletal muscle (fMNa1=fNav1.4a). In fNav1.4a protein, the aromatic amino acid in p-loop region of Domain I in TTX-sensitive Nays was replaced by Asn. Also, Kaneko et al. reported that similar mutation was found in Na_v of retinal neuron of the newt, Cynops pyrrhogaster. In this study, we confirmed that these mutations are responsible to TTX-resistance of puffer fish and newts by evaluation of IC_<50>-TTX values of the corresponding mutants of rNav1.2a transiently expressed in HEK293 cells by electrophysiological study.

著者

秋元 隆史 篠原 涼子 岩本 理 山下 まり 山岡 薫 長澤 和夫

出版者

天然有機化合物討論会実行委員会

雑誌

天然有機化合物討論会講演要旨集

巻号頁・発行日

vol.53, pp.517-522, 2011

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Voltage-gated sodium channels (Na_vCh) are transmembrane proteins that provide inward current carried by sodium ions, and they contribute to the control of membrane excitability, as well as the propagation of action potentials along axons. To date, nine subtypes of sodium channels (NaChs) have been identified, which are closely related to life activity such as a sense of pain, a heartbeat, the muscle expansion and contraction. Since each of these subtypes has unique properties, subtype selective ligand is required for controlling and elucidation of these functions. Saxitoxin (STX) is a naturally occurring NavCh inhibitor, which is believed to bind to the P-loop region of the ion-selective filter in NavCh, and blocks ion influx of Na_vChs in a similar manner to tetrdotoxin (TTX). Recently, a binding model of STX with P-loop domain was proposed based on molecular docking studies by Zhorov. From this model, domain I and STX in C13 and N7 is crucial for the interaction. In this paper, we described the structure-activity relationship studies on STX derivatives with focusing on the C13 and N7 positions. New STX derivatives of 14, 16-18, 23 and 24 modified at C13 and N7 were synthesized from fully protected form of STX of 8 efficiently. Inhibitory activity of these new derivatives against Na_vChs, i.e., Na_v1.2, Na_v1.4 (these are TTX-sensitive) and Na_v1.5 (TTX-resistant), were evaluated by the who'e-cell patch clamp method. As shown in Table 1, these derivatives show moderate inhibitory activities against Na_v1.2, Na_v1.4, but no inhibitory activity was observed to Na_v1.5. Further SAR studies are in progress.

著者

丸田 聡 山岡 薫 大越 夏実 山下 まり

出版者

天然有機化合物討論会実行委員会

雑誌

天然有機化合物討論会講演要旨集 49 (ISSN:24331856)

巻号頁・発行日

pp.443-448, 2007-08-24 (Released:2017-08-18)

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Tetrodotoxin (TTX) and saxitoxin (STX) bind to a single site in the outer pore of the voltage-gated sodium channels (Na_vs), formed by the amino-acid residues in the outer-pore loops (p-loops) located between the S5 and S6 segments of each of the homologous domain (I-IV) of the a-subunit. Since puffer fish and newts accumulate TTX at high concentration in their tissues, they are thought to have special defense systems against their own TTX. We previously obtained a cDNA encoding Na_v from Fugu pardalis skeletal muscle (fMNa1=fNav1.4a). In fNav1.4a protein, the aromatic amino acid in p-loop region of Domain I in TTX-sensitive Nays was replaced by Asn. Also, Kaneko et al. reported that similar mutation was found in Na_v of retinal neuron of the newt, Cynops pyrrhogaster. In this study, we confirmed that these mutations are responsible to TTX-resistance of puffer fish and newts by evaluation of IC_<50>-TTX values of the corresponding mutants of rNav1.2a transiently expressed in HEK293 cells by electrophysiological study.

著者

藤本 吉範 山岡 薫

出版者

広島大学

雑誌

基盤研究(C)

巻号頁・発行日

1998

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破骨細胞は骨吸収において中心的な役割を演ずるが、その活性調節には細胞外Ca^<2+>濃度が関与している。骨吸収期において、破骨細胞は非常に高濃度な細胞外Ca^<2+>に曝露され、細胞内にCa^<2+>が流入することで細胞内のCa^<2+>storeからCa^<2+>が放出される。この結果、細胞内Ca^<2+>濃度は急速に上昇し、骨吸収抑制のシグナルとなる。細胞外からのCa^<2+>の流入経路としては細胞膜に発現したryanodine receptor(RyR)様Ca^<2+>チャネルの存在が細胞内Ca^<2+>濃度を測定する方法や分子生物学的手法での研究で示唆されているが、実際にそのCa^<2+>チャネルを通るイオンチャネル電流は記録されたことがない。我々は初めて破骨細胞の細胞膜に発現したRyRの性質を有したイオンチャネル電流を検出した。破骨細胞にruthenium red(RR)を投与した場合、高濃度(0.1mM)では細胞内Ca^<2+>濃度が上昇し、低濃度(0.5μM)では細胞内Ca^<2+>濃度の上昇が抑えられたことにより、破骨細胞の細胞膜にRyR様Ca^<2+>チャネルが存在することが示唆されている(Adebanjo OA et al.Am J Physiol 270:F469-F475,1996)。我々は破骨細胞の細胞膜に発現したRyR様Ca^<2+>チャネルの性質を検討するため、単一チャネル電流記録法のうちinside-out法を用いたが、細胞内液にRR0.1mMとMgCl_23mMを添加することによりRR感受性電流を惹起することができた。またこの電流は低濃度(10μM)のRRによりブロックされた。以上の結果は上記Adebanjoらの報告におけるRyR様Ca^<2+>チャネルの性質を有したチャネルの活動を初めて測定したものと考えられる。本実験で得られた電流は、保持電位-40mVで内向きに長い開口時間を有し、平衡電位は+5〜+10mVであった。イオン伝導度は20pS(電極内CaCl_2:10mM)と27pS(電極内CaCl_2:60mM)でCa^<2+>濃度依存性の性質を示し、この電流がCa^<2+>を運んでいることを示唆した。