1-B-SS05-1
クライオ電子顕微鏡を用いた薬物動態関連膜タンパク質P糖タンパク質の三次元立体構造解析
Three-dimensional structural analysis of pharmacokinetics-related membrane protein P-glycoprotein using cryo-electron microscopy
〇濱口 紀江1,2、安達 成彦3、守屋 俊夫3、川崎 政人3、安田 賢司2、千田 俊哉3、小笠原 諭2、村田 武士2
Hamaguchi Norie1,2, Naruhiko Adachi3, Toshio Moriya3, Masato Kawasaki3, Kenji Yasuda2, Toshiya Senda3, Satoshi Oagasawara2, Takeshi Murata2
1千葉大・院医薬・薬理学、2千葉大・理・構造生物学、3高エネ機構・物構研・構造生物
1Grad. Sch. of Med. and Pharm. Sci, Chiba Univ, 2Dept of chem, Grad Sch of sci, Chiba Univ, 3IMSS, KEK, SBRC
P-glycoprotein (P-gp) is mainly found in the cell membrane of the small intestine and blood-brain barrier in vivo, and is responsible for the extracellular transport of cytotoxic hydrophobic compounds. P-gp is known to transport many pharmaceutical compounds as substrates. If we can understand the substrate recognition mechanism of P-gp, it will be possible to design pharmaceutical compounds that are not recognized by P-gp. Recently, the complex structures of human P-gp with substrates and inhibitors have been reported by single-particle analysis using Cryo-EM, and the differences in the binding pockets of substrates and inhibitors have been clarified. However, a detailed understanding of how P-gp can identify compounds as substrates or inhibitors has not been achieved. In this study, we aim to elucidate the detailed substrate recognition mechanism by elucidating and comparing multiple complex structures of P-gp and compounds. First, we established a system for expression and purification of human P-gp. Further, we have established a simple system for reconstitution into Nanodisc. Recently, we succeeded to obtain the 3D structure at the highest resolution (2.93 Å) as human P-gp. In this presentation, we will introduce the expression, purification, and Nanodisc reconstruction systems of P-gp and the obtained 3D structures.
1-B-SS05-2
炎症性マクロファージにおいて、カベオリン1はP2X7受容体を介するATPシグナルを調節する。
Caveolin-1 regulates ATP signaling mediated by P2X7 receptor in pro-inflammatory macrophages.
〇澤井 優輝、鈴木 良明、近藤 るびい、今泉 祐治、山村 寿男
Yuuki Sawai, Yoshiaki Suzuki, Rubii Kondo, Yuji Imaizumi, Hisao Yamamura
名古屋市大・院薬・細胞分子薬効解析
Dept. Mol & Cell. Pharmacol., Grad. Sch. Pharmaceut. Sci., Nagoya City Univ.
[Background] Macrophage (Mφ) plays crucial roles in immunity and its dysfunction leads to the chronic inflammatory diseases such as arteriosclerosis. Several Mφ functions are modulated by the activation of ionotropic purinergic P2X7 receptor. Caveolin-1 (Cav-1) enables effective intracellular Ca2+ signaling by accumulating ion channels within caveolae domain. In this study, we analyzed the functional coupling between Cav-1 and P2X7 receptor using Cav-1 knockout (Cav-1 KO) mice.
[Methods] In murine bone marrow-derived Mφ (BMDM), the expression of Cav-1 was analyzed by real-time PCR and Western Blotting. Interaction of Cav-1 and P2X7 receptor was analyzed by proximal ligation assay. Ca2+ influx, K+ efflux and reactive oxygen species (ROS) production were measured with confocal microscopy. Cell death was analyzed by LDH assay.
[Results] The expression of Cav-1 was increased by LPS (lipopolysaccharide)-induced inflammatory stimulation in BMDM. Cav-1 was interacted with P2X7 receptor. Thereafter, ATP-evoked Ca2+ influx and K+ efflux were increased in Cav-1 KO BMDM. ROS production and cell death evoked by ATP were also enhanced in Cav-1 KO BMDM.
[Conclusion] Cav-1 suppresses the activation of P2X7 receptor and modulates immune responses in Mφ. This study may lead to the development of novel drugs for chronic inflammatory diseases.
1-B-SS05-3
電位センサーの移動ではなく、細孔の開口がhERG阻害剤による促進作用の電位依存性を生み出している
Pore opening, not voltage sensor movement, underpins the voltage-dependence of facilitation by a hERG blocker.
〇河野 諒太朗1、古谷 和春1,2、一藁 南1、足立 亮1、Clancy Colleen2、Sack Jon2、喜多 紗斗美1
Kawano Ryotaro1, Kazuharu Furutani1,2, Minami Ichiwara1, Ryo Adachi1, Colleen Clancy2, Jon Sack2, Satomi Kita1
1徳島文理大・薬・薬学科、2カリフォルニア大学デービス校・医・生理学
1Dept. Pharmacol, Sch. Pharmaceut. Sci., Tokushima Bunri Univ., 2Dept. Physiol & Mem Biol, Univ. Cal, Davis
A drug that blocks the cardiac myocyte voltage-gated K+ channels encoded by the hERG carries a potential risk of long QT syndrome and life-threatening cardiac arrhythmia. Interestingly, certain hERG blockers can also facilitate hERG activation to increase hERG currents, which may reduce proarrhythmic potential. However, the molecular mechanism remains unclear. The hallmark feature of the facilitation effect by hERG blockers is that a depolarizing preconditioning pulse shifts voltage-dependence of hERG activation to more negative voltages. Here we utilize a D540K hERG mutant to study the mechanism of the facilitation effect. D540K hERG is activated by not only depolarization but also hyperpolarization. With D540K hERG, we find that nifekalant, a hERG blocker and Class III antiarrhythmic agent, blocks and facilitates not only current activation by depolarization but also current activation by hyperpolarization, suggesting a shared gating process upon depolarization and hyperpolarization. Moreover, in response to hyperpolarizing conditionings, nifekalant facilitates D540K hERG currents but not wild-type currents. Our results indicate that induction of facilitation is coupled to pore opening, not voltage per se. We propose that gated access to the hERG central cavity underlies the voltage-dependence of induction of facilitation.
1-B-SS05-4
TRPC3/C6 チャネルを標的とした新規阻害剤の開発
Development of a novel drug targeting TRPC3/C6 channels
〇白戸 真美子
Shirato Mamiko
京都大・大学院工学研究科・合成生物・化学専攻
Dept. Synth. Chem. & Biol. Chem., Grad. Sch. Eng., Kyoto Univ
A group of TRPCs, TRPC3, TRPC6, and TRPC7, form Ca2+-permeable channels directly activated by diacylglycerol (DAG) and play important roles in regulating neuronal survival and dendritic growth, cardiovascular fibrosis in vitro and in vivo through regulation of Ca2+signaling. Various compounds targeting these TRPC channels have been developed for the treatment of serious diseases such as sudden pulmonary fibrosis and chronic nephropathy. However, none of these compounds have yet reached clinical application, and therefore development of new TRPC3/C6/C7 inhibitors has been much-needed. Here, we have developed a piperazine derivative targeting TRPC3/C6 channels. This compound suppressed receptor-activated Ca2+influx in a dose-dependent manner in human embryonic kidney cells 293 expressed with human TRPC3 or TRPC6 (TRPC3, IC50 = 0.086: TRPC6, IC50 = 0.034µM). This drug showed no significant inhibitory or stimulatory effect on other TRPs including TRPC7. Interestingly, during isolation of human TRPC7, we obtained a new splice variant of human TRPC7; we are in the process to characterize biophysical and pharmacological properties of the variant that has a deletion in one of the functionally critical domains.
1-B-SS05-5
カベオリン1の欠損はTMEM16Aチャネルを介した門脈ClCa電流を増大させる
TMEM16A-mediated Ca2+-activated Cl- currents is increased in portal vein smooth muscle cells from caveolin 1-deficient mice
〇川田 成紀、近藤 るびい、鈴木 良明、山村 寿男
Naoki Kawata, Rubii Kondo, Yoshiaki Suzuki, Hisao Yamamura
名古屋市大・院薬・細胞分子薬効解析
Dept. Mol. & Cell. Pharmacol., Grad. Sch. Pharmaceut. Sci., Nagoya City Univ.
In vascular smooth muscles, the activity of Ca2+-activated Cl- (ClCa) channels regulates the membrane excitability and myogenic tone. TMEM16A channels are predominantly form ClCa currents in vascular smooth muscles including portal vein smooth muscles (PVSMs). Caveola is a cholesterol-rich membrane invaginations and structurally contributes to effective and efficient signal transduction. Caveolin 1 (Cav1) is accumulated in the caveolin and plays a key role in forming the functional complex among enzymes, receptors, and ion channels. In this study, the functional roles of Cav1 on the expression and activity of TMEM16A ClCa channels were examined in portal vein smooth muscle cells (PVSMCs) from wild-type (WT) and Cav1-knockout (KO) mice. Spontaneous contractions of PVSMs were recorded using an isotonic transducer. TMEM16A-mediated ClCa currents were recorded by whole-cell patch-clamp configurations. The expression of TMEM16A channels was quantitatively analyzed by real-time PCR. The amplitude of spontaneous contractions of PVSMs was larger in Cav1-KO mice than WT mice. Whole-cell ClCa currents were also larger in Cav1-KO PVSMCs than WT PVSMCs. Importantly, Ani9 (a specific blocker for TMEM16A channels)-sensitive currents were increased in Cav1-KO PVSMCs compared to WT PVSMCs. The expression of TMEM16A channels was higher in Cav1-KO PVSMs than WT PVSMs. The present data strongly suggest that the caveola structure formed by Cav1 negatively regulates the expression and activity of TMEM16A-mediated ClCa channels in vascular smooth muscle cells.
1-B-SS05-6
マウス線条体間接路におけるL-DOPA受容体GPR143はD2Rとの連関を介して不安様行動を制御する
The L-DOPA receptor GPR143 in the indirect pathways regulates an anxiety-like behavior through GPR143-DRD2 coupling
〇田近 伶、増川 太輝、内村 放、五嶋 良郎
Rei Tajika, Daiki Masukawa, Hiraku Uchimura, Yoshio Goshima
横浜市立大・院医・薬理
Dept. Mol. Pharmacol. Neurobiol., Yokohama City Univ. Grad. Sch. Med.
We propose that L-DOPA by itself is a neurotransmitter. Recently, a G-protein coupled receptor GPR143, a gene product of ocular albinism1, was identified as a receptor for L-DOPA. In this study, to identify the physiological role of GPR143, we performed phenotypic analysis using Gpr143-gene deficient (GPR143-KO) mice. To assess anxiety- and exploration-related behaviors, we employed zero-maze test, and found that time spent in open arms was decreased in GPR143-KO mice when compared to wild-type (WT) mice. The time spent in open arms was also decreased in striatal indirect pathway specific GPR143-KO mice. To investigate the involvement of endogenous L-DOPA, we examined the effect of alpha-methyl-para-tyrosine, a synthetic inhibitor of L-DOPA on mouse behavior. We found that administration of α-MPT at the dose of 3mg/kg (i.p.) decreased the release of L-DOPA without affecting that of dopamine from the dorsal striatum. The administration of α-MPT decreased the time spent in open arms in WT mice, while this effect was not observed in GPR143-KO mice. Furthermore, intraventricular administration of a synthetic peptide, which inhibited the interaction between GPR143 and dopamine D2 receptor (DRD2), increased anxiety-like behavior. These results suggest that L-DOPA regulates anxiety-like behavior through GPR143 and DRD2 coupling in the striatal indirect pathway.