研究等業績 - その他 - 沼田　朋大

Direct mechano-stress sensitivity of TRPM7 channel

Tomohiro Numata, Takahiro Shimizu, Yasunobu Okada

CELLULAR PHYSIOLOGY AND BIOCHEMISTRY ( KARGER )  19 ( 1-4 ) 1 - 8   2007年

It has recently been shown that shear stress augments the heterologously expressed TRPM7 channel activity by exocytosis-mediated incorporation of TRPM7 into the plasma membrane. On the other hand, our recent study has shown that the TRPM7-like channel endogenously expressed in HeLa cells is activated by membrane expansion induced by membrane stretch or osmotic cell swelling. Thus, the present study was aimed at exploring the possibility that the heterogously expressed TRPM7 channel is activated directly by membrane expansion in a manner independent of exocytosis. Here, whole-cell currents of the TRPM7 channel heterologously expressed in HEK293T cells were found to be augmented not only by perfusion of bath solution but also by osmotic swelling even under the conditions where exocytotic events can hardly take place in the cytosol dialyzed with ATP-free, Ca(2+)- free and EGTA- containing pipette solution. In addition, shear stress- induced augmentation was not affected by a blocker of vesicular protein traffic, brefeldin A. Furthermore, in cell- free patches, membrane stretch directly augmented single- channel activity of TRPM7 by increasing Po value at <= 20mV. We thus conclude that the TRPM7 channel can be directly activated by mechano- stress in a manner independent of exocytosis- mediated incorporation of this channel protein into the plasma membrane. Copyright (c) 2007 S. Karger AG, Basel

DOI PubMed

Signalling events employed in the hypertonic activation of cation channels in HeLa cells

Frank Wehner, Tomohiro Numata, Muthangi Subramanyan, Nobuyuki Takahashi, Yasunobu Okada

CELLULAR PHYSIOLOGY AND BIOCHEMISTRY ( KARGER )  20 ( 1-4 ) 75 - 82   2007年

In the present study, the signalling network behind the hypertonic activation of cation channels in HeLa cells was analysed by use of various inhibitors. Channel activation was monitored in whole-cell patch-clamp recordings, whereas the role of the channel in cell volume regulation was determined by electronic cell sizing. It is found that channel activation and volume control probably employs tyrosine kinases, G-proteins, PLC, PKC and p38MAP kinase, and that the process appears to depend on an intact actin skeleton. In contrast, RhoA, PI 3-kinase, ERK 1/2, JNK 1/2 as well as the exocytotic insertion of channels into the plasma membrane are likely not part of the signalling machinery. Copyright (c) 2007 S. Karger AG, Basel.

DOI PubMed

高浸透圧刺激によって誘導されるカチオンチャネルの活性化に関わるシグナル経路

沼田 朋大, 岡田 泰伸, べーなー ふらんく

日本生理学会大会発表要旨集 ( 一般社団法人 日本生理学会 )  2007   126 - 126   2007年

It is known that human epithelial HeLa cells exhibit cell volume regulation after osmotic shrinkage, called the regulatory volume increase (RVI), by both mechanisms mediated by activation of hypertonicity-induced cation channels (HICC) and by parallel activation of Na<SUP>+</SUP>/H<SUP>+ </SUP>exchangers and anion exchangers. To analyse the signalling network behind the HICC activation, in the present study, we examined the effects of a variety of high-specificity blockers of signalling molecules on activation of HICC monitored by whole-cell patch-clamp recordings and on the RVI monitored by electronic cell sizing. These data suggest that HICC currents and HICC-mediated RVI involve tyrosine kinases, G-proteins, PLC, PKC and p38MAP kinase, and also they appear to depend on an intact actin cytoskeleton. In contrast, RhoA, PI 3-kinase, ERK 1/2, JNK 1/2 as well as exocytotic insertion of channels into the plasma membrane do not appear to participate in the signalling machinery. <b>[J Physiol Sci. 2007;57 Suppl:S126]</b>

DOI CiNii Research

TRPM7チャネルは、機械刺激で活性化する

沼田 朋大, 清水 貴浩, 岡田 泰伸

日本生理学会大会発表要旨集 ( 一般社団法人 日本生理学会 )  2006   153 - 153   2006年

Mechanical stress activates TRPM7 channels expressed in HEK293T cells Numata, Tomohiro; Shimizu, Takahiro; Okada, Yasunobu (Dept. Cell Physiol., Natl. Inst. Physiol. Sci., Okazaki, Japan)Stretch-activated cation channels play an essential role in sensing and transducing external mechanical stresses in living cells. In the previous meeting we reported that TRPM7 channels endogenously expressed in human epithelial HeLa cells are activated by membrane stretch or osmotic cell swelling. However, it has not been known whether TRPM7 shows mechanosensitivity when heterologously expressed. HEK293T cells overexpressed with TRPM7 exhibited whole-cell currents typical of TRPM7, such as outward rectification, conductivity to Ca<SUP>2+</SUP>, and sensitivity to Mg<SUP>2+</SUP> and ruthenium red. In addition, TRPM7 currents were augmented by following three kinds of mechanical stimuli: shear stress imposed by perfusion of extracellular solution, membrane stretch produced by patch membrane suction, cell swelling due to hypotonic stimulation. We thus conclude that the TRPM7 channel can be activated by mechanical stress in the heterologous expression system. <b>[J Physiol Sci. 2006;56 Suppl:S153]</b>

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Characterization of single L-type Ca2+ channels in myocytes isolated from the cricket lateral oviduct

T Numata, M Yoshino

JOURNAL OF COMPARATIVE PHYSIOLOGY B-BIOCHEMICAL SYSTEMIC AND ENVIRONMENTAL PHYSIOLOGY ( SPRINGER )  175 ( 4 ) 257 - 263   2005年05月

The single Ca2+ channel activity was obtained from cell-attached patch recordings with the use of pipettes filled with 100 mM Ba2+ as the charge carrier in myocytes isolated from the lateral oviduct of cricket Gryllus bimaculatus. The following results were obtained. (1) The channel had a unitary conductance of 18 pS. (2) The open time histogram of the channel could be fitted with a single exponential while the closed time histogram could be fitted with the sum of two exponentials, suggesting that there are at least one open state and two closed states for this channel. (3) The open probability of the channel increased with increasing membrane depolarization. (4) The mean current reconstructed by averaging individual current trace responses inactivated slowly and the current-voltage relationship for the peak mean current showed a bell-shaped relation. (5) The dihydropyridine (DHP) Ca2+ antagonist, nifedipine, reduced the mean current by increasing the proportion of "blank" sweeps. On the other hand, the DHP Ca2+ agonist, Bay K 8644, increased the mean current by increasing the mean open-times of the channel. These results confirm a presence of DHP-sensitive L-type Ca2+ channel in myocytes isolated from the lateral oviduct of cricket G. bimaculatus.

DOI PubMed

Characterization of stretch-activated calcium permeable cation channels in freshly isolated myocytes of the cricket (Gryllus bimaculatus) lateral oviduct

T Numata, M Yoshino

JOURNAL OF INSECT PHYSIOLOGY ( PERGAMON-ELSEVIER SCIENCE LTD )  51 ( 5 ) 481 - 488   2005年05月

Stretch-activated channels (SACs) were investigated in myocytes isolated from the lateral oviduct in cricket Gryllus bimaculatus using the cell-attached or excised inside-out patch clamp technique. Application of both negative and positive pressure (10-100 cm H2O) into the patch pipettes induced the unitary channel current openings. The open probability (NPo) of the channel increased, when negative pressure applied into the patch pipettes increased. The single channel conductance for this channel was approximately 20 pS with 140 mM Na+, K+, or Cs+ in the patch pipettes and was approximately 13 pS with 100 mM Ca2+ or Ba2+ in the patch pipettes. External application of Gd3+, La3+, Cd2+ and Zn2+ inhibited the channel with the IC50 values of 14, 15, 28, and 18 mu M respectively. Interestingly external application of TEA, a specific blocker of K+ channel, also inhibited this channel with IC50 value of 8.8 mM. These results show for the first time the presence of stretch activated Ca2+-permeable nonselective cation channel in myocytes isolated from the cricket lateral oviduct. The physiological significance of this channel in oviposition behavior is discussed. (C) 2005 Elsevier Ltd. All rights reserved.

DOI PubMed

若年性ミオクロニーてんかん(JME)責任遺伝子の同定

鈴木 俊光, Delgado-Escueta A.V, Aguan Kripamoy, Shi Jun, 原 雄二, 西田 基宏, 沼田 朋大, 竹内 環, Bai Dongsheng, 井上 有史, 大澤 真木子, 兼子 直, 小国 弘量, 森 泰生, 山川 和弘

てんかん研究 ( (一社)日本てんかん学会 )  23 ( 1 ) 43 - 44   2005年02月

Identification of the gene responsible for juvenile myoclonic epilepsy.

T Suzuki, AV Delgado-Escueta, K Aguan, J Shi, Y Hara, M Nishida, T Numata, T Takeuchi, DS Bai, Y Inoue, M Osawa, S Kaneko, H Oguni, Y Mori, K Yamakawa

EPILEPSIA ( BLACKWELL PUBLISHING )  46   15 - 16   2005年

DOI

TRPM7チャネルは、細胞膨張によって活性化されて容積調節に関与する

沼田 朋大, 清水 貴浩, 岡田 泰伸

日本生理学会大会発表要旨集 ( 一般社団法人 日本生理学会 )  2005   S76 - S76   2005年

In animal cells, it is known that under hypoosmotic conditions, an intracellular Ca<SUP>2+</SUP> increase initially occurs followed by a regulatory volume decrease (RVD) which is attained by effluxes of K<SUP>+</SUP>, Cl<sup>−</SUP> and organic osmolytes and the resulting extraction of osmotically obliged water. The present study was performed to identify the volume-regulatory Ca<SUP>2+</SUP> influx pathway under hypoosmotic conditions in HeLa cells. Whole-cell recordings showed that osmotic swelling activates TRPM7-like cation currents. The currents showed outward rectification and sharp sensitivity to Mg<SUP>2+</SUP>, Gd<SUP>3+</SUP>, SKF96365 and ruthenium red (RR). These TRPM7 channel blockers also inhibited the intracellular Ca<SUP>2+</SUP> increase in response to osmotic swelling and the following RVD. RT-PCR studies demonstrated expression of TRPM7 mRNA in HeLa cells. The siRNA silencing of TRPM7 significantly suppressed not only expression of TRPM7 mRNA but also whole-cell TRPM7-like channel currents, Ca<SUP>2+</SUP> influx and RVD in HeLa cells upon a hypotonic challenge. Thus, we conclude that the endogenous activity of swelling-activated cation channel exhibits the properties identical to the hallmark biophysical and pharmacological features of TRPM7, and that the TRPM7 channel plays an important role in the RVD process by serving as the volume-regulatory Ca<SUP>2+</SUP> influx pathway in the human epithelial cells. <b>[Jpn J Physiol 55 Suppl:S76 (2005)]</b>

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Store-operated Ca2+ channel in cricket lateral oviduct myocytes

Masami Yoshino, Tomohiro Numata, Yoshimi Nakazono, Yoichi Abiru, Takeshi Aihara, Minoru Tsukada

ZOOLOGICAL SCIENCE ( ZOOLOGICAL SOC JAPAN )  21 ( 12 ) 1327 - 1327   2004年12月

DOI

Mutations in EFHC1 cause juvenile myoclonic epilepsy

T Suzuki, AV Delgado-Escueta, K Aguan, ME Alonso, J Shi, Y Hara, M Nishida, T Numata, MT Medina, T Takeuchi, R Morita, DS Bai, S Ganesh, Y Sugimoto, J Inazawa, JN Bailey, A Ochoa, A Jara-Prado, A Rasmussen, J Ramos-Peek, S Cordova, F Rubio-Donnadieu, Y Inoue, M Osawa, S Kaneko, H Oguni, Y Mori, K Yamakawa

NATURE GENETICS ( NATURE PUBLISHING GROUP )  36 ( 8 ) 842 - 849   2004年08月

Juvenile myoclonic epilepsy (JME) is the most frequent cause of hereditary grand mal seizures(1,2). We previously mapped and narrowed a region associated with JME on chromosome 6p12-p11 (EJM1)(3-5). Here, we describe a new gene in this region, EFHC1, which encodes a protein with an EF-hand motif. Mutation analyses identified five missense mutations in EFHC1 that cosegregated with epilepsy or EEG polyspike wave in affected members of six unrelated families with JME and did not occur in 382 control individuals. Overexpression of EFHC1 in mouse hippocampal primary culture neurons induced apoptosis that was significantly lowered by the mutations. Apoptosis was specifically suppressed by SNX-482, an antagonist of R-type voltage-dependent Ca2+ channel (Ca(v)2.3). EFHC1 and Ca(v)2.3 immunomaterials overlapped in mouse brain, and EFHC1 coimmunoprecipitated with the Cav2.3 C terminus. In patch-clamp analysis, EFHC1 specifically increased R-type Ca2+ currents that were reversed by the mutations associated with JME.

DOI PubMed

若年性ミオクローヌスてんかん原因遺伝子の単離に向けて(Towards the identification of genes responsible for juvenile myoclonic epilepsy)

鈴木 俊光, Delgado-Escueta Av, Aguan K, 原 雄二, 西田 基宏, Numata T, 竹内 環, Bai D, 井上 有史, 大沢 真木子, 兼子 直, 小国 弘量, 森 泰生, 山川 和弘

神経化学 ( 日本神経化学会 )  43 ( 2-3 ) 512 - 512   2004年08月

A role of reactive oxygen species in apoptotic activation of volume-sensitive Cl- channel

T Shimizu, T Numata, Y Okada

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA ( NATL ACAD SCIENCES )  101 ( 17 ) 6770 - 6773   2004年04月

Apoptotic volume decrease is a pivotal event triggering a cell to undergo apoptosis and is induced by ionic effluxes resulting mainly from increased K+ and Cl- conductances. Here, we demonstrate that in human epithelia HeLa cells both mitochondrion- and death receptor-mediated apoptosis inducers [staurosporine and Fas ligand or tumor necrosis factor (TNF)-alpha] rapidly activate Cl- currents that show properties phenotypical of volume-sensitive outwardly rectifying Cl- channel currents, including outward rectification, voltage-dependent inactivation gating at large positive potentials, inhibition by osmotic shrinkage, sensitivity to classic Cl- channel blockers, and dependence on cytosolic ATP. Staurosporine, but not Fas ligand or TNF-alpha, rapidly (within 30 min) increased the intracellular level of reactive oxygen species (ROS). A ROS scavenger and an NAD(P)H oxidase inhibitor blocked the current activation by staurosporine but not by Fas ligand or TNF-alpha. A ROS scavenger also inhibited apoptotic volume decrease, caspase-3 activation, and apoptotic cell death induced by staurosporine. Thus, it is concluded that an apoptosis-triggering anion conductance is carried by the volume-sensitive outwardly rectifying Cl- channel and that the channel activation on apoptotic stimulation with staurosporine, but not with Fas ligand or TNF-alpha, is mediated by ROS.

DOI PubMed

ミトコンドリア仲介性アポトーシス誘導時のアニオンチャネル活性化における活性酸素の役割

清水 貴浩, 沼田 朋大, 岡田 泰伸

日本生理学会大会発表要旨集 ( 一般社団法人 日本生理学会 )  2004   S83 - S83   2004年

Apoptosis is essential for normal tissue development and homeostasis. The apoptotic volume decrease (AVD), which is induced by KCl efflux due to activation of K<SUP>+</SUP> and Cl<sup>−</SUP> channels, is an early-phase, prerequisite component of apoptosis. We previously demonstrated that volume-sensitive Cl<sup>−</SUP> currents are activated after application either of a mitochondrion-mediated inducer, staurosporine (STS), or of a death receptor-mediated inducer, TNF-α or Fas ligand, in HeLa cells without showing cell swelling. Here, we investigated a role of reactive oxygen species (ROS) in STS-induced activation of Cl<sup>−</SUP> channel. STS was found to increase the intracellular ROS level by using 2', 7'-dichlorofluorescein diacetate. The rise in ROS was inhibited by a ROS scavenger, N-acetyl-cysteine (NAC), or an NAD(P)H oxidase inhibitor, diphenylene-iodonium chloride (DPI). In the presence of NAC or DPI, STS failed to activate Cl<sup>−</SUP> currents. In addition, extracellular application of hydrogen peroxide directly increased Cl<sup>−</SUP> currents, which exhibited properties identical to those of volume-sensitive Cl<sup>−</SUP> currents. NAC and DPI could abolish the AVD induced by STS. STS-induced caspase-3 activation and reduction of cell viability were also suppressed by NAC and DPI. These results suggest that ROS is a key mediator for activation of volume-sensitive Cl<sup>−</SUP> channel during a mitochondrion-mediated apoptosis. <b>[Jpn J Physiol 54 Suppl:S83 (2004)]</b>

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