Research Achievements - Other -
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The deltaC splice-variant of TRPM2 is the hypertonicity-induced cation channel (HICC)in HeLa cells, and the ecto-enzyme CD38 mediates its activation
Numata Tomohiro, Sato Kaori, Christmann Jens, Marx Romy, Mori Yasuo, Okada Yasunobu, Wehner Frank
JOURNAL OF PHYSIOLOGICAL SCIENCES 63 S130 2013
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Using physiological temperature variations to perform a functional analysis of mutant HCN channels
Nakamura Yuki, Shi Xiuyu, Numata Tomohiro, Mori Yasuo, Inoue Ryuji, Hirose Shinichi
JOURNAL OF PHYSIOLOGICAL SCIENCES 63 S133 2013
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細胞生死スイッチに関与する細胞容積関連性イオンチャネル
岡田泰伸, 沼田(佐藤)かお理, 沼田朋大, 清水貴浩, 秋田天平, 岡田俊昭
日本Cell Death学会学術集会プログラム抄録集 22nd 28 2013
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The ?C splice-variant of TRPM2 is the hypertonicity-induced cation channel in HeLa cells, and the ecto-enzyme CD38 mediates its activation
Tomohiro Numata, Kaori Sato, Jens Christmann, Romy Marx, Yasuo Mori, Yasunobu Okada, Frank Wehner
JOURNAL OF PHYSIOLOGY-LONDON 590 ( 5 ) 1121 - 1138 2012.02
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TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade.
Koike Chieko, Obara Takehisa, Uriu Yoshitsugu, Numata Tomohiro, Sanuki Rikako, Miyata Kentarou, Koyasu Toshiyuki, Ueno Shinji, Funabiki Kazuo, Tani Akiko, Ueda Hiroshi, Kondo Mineo, Mori Yasuo, Tachibana Masao, Furukawa Takahisa
Proceedings of the National Academy of Sciences of the United States of America 107 ( 1 ) 332 - 337 2010.01
An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways byretinal bipolar cells(BCs). Glutamate released from photoreceptors modulates the photoresponse of ONBCsvia metabotropic glutamatereceptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and itsactivity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.
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TRPM1: a vertebrate TRP channel responsible for retinal ON bipolar function.
Koike Chieko, Numata Tomohiro, Ueda Hiroshi, Mori Yasuo, FurukawaTakahisa
Cell calcium 48 ( 2-3 ) 95 - 101 2010
The transient receptor potential (TRP) channels affect essential functions widely in sensory systems of various species, both invertebrates and vertebrates. The channel protein encoded by the trp gene, the first identified TRP superfamily molecule, is known to mediate the Drosophila light response. A vertebrate TRP channel playing a crucial role in the visual system has not yet been discovered, although numerous studies have revealed primal functions of TRP superfamily moleculesin various sensory systems other thanvision. In the retina, which is the entry tissue in the vertebrate visualpathway, the transduction cation channel in ONbipolar cells has been elusive, despite intensive investigation by many researchers over a long period of time. Recent studies finally revealed that TRPM1, the first member of the melanoma-related transient receptor potential (TRPM) subfamilyto be discovered, is a visualtransductionchannel in retinalON bipolar cells. This review coversthe significant discoveries on the physiological function and regulatory mechanism of the TRPM1 channel in retinal ON bipolar cells and the association of humanTRPM1 mutations with congenital stationary night blindness.
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AFTERNOON TEA
71 ( 6 ) 232 - 232 2009.06
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Structures and variable functions of TRP channels
Tomohiro Numata, Daisuke Kozai, Nobuaki Takahashj, Kenta Kato, Yoshitsugu Uriu, Shinichiro Yamamoto, Takeshi Kaneko, Tatsuo Shinmoto, Yasuo Mori
Seikagaku 81 ( 11 ) 962 - 983 2009
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Mechanosensor TRPM7 channel and its physiological role in cell volume regulation
Numata Tomohiro
Proceedings of Annual Meeting of the Physiological Society of Japan ( PHYSIOLOGICAL SOCIETY OF JAPAN ) 2008 009 - 009 2008
Stretch-activated cation (SAC) channels play an essential role in sensing and transducing external mechanical stresses in living cells. However, its molecular identity in mammalian cells is not as yet firmly established. In human epithelial HeLa cells, we found the activity of non-selective cation channel which is delicately sensitive to membrane stretch with a negative pressure for half-maximum activation of around 3 cm-H<SUB>2</SUB>O. Also, the whole-cell cation current was augmented by osmotic cell swelling. The Mg<SUP>2+</SUP>-sensitive, Ca<SUP>2+</SUP>-conducting, stretch-activated cation channel current exhibited the hallmark biophysical and pharmacological features of TRPM7 at both single-channel and whole-cell levels. The endogenous expression of TRPM7 in HeLa was confirmed by RT-PCR and western blotting. Treatment with siRNA targeted against TRPM7 led to abolition of stretch-activated single-channel cation currents and of swelling-activated whole-cell cation currents. When the TRPM7 cDNA was transfected into HEK-293T cells, the robust stretch-activated cation channel current exhibited similar biophysical and pharmacological features of endogenous TRPM7 at both single-channel and whole-cell levels. Suppression of the regulatory volume decrease (RVD) upon a hypotonic challenge was observed by application of siRNA for TRPM7, by elimination of extracellular Ca<SUP>2+</SUP> or by addition of a TRPM7 channel blocker. Thus, it is concluded that TRPM7 is the SAC channel endogenously expressed in human epithelial cells and is involved in volume regulation of the cells by serving as a swelling-induced Ca<SUP>2+</SUP> influx pathway. <b>[J Physiol Sci. 2008;58 Suppl:S9]</b>
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Rescue of apoptosis by activation of hypertonicity-induced cation channels in human epithelial cells
Okada Yasunobu, Wehner Frank, Numata Tomohiro
Proceedings of Annual Meeting of the Physiological Society of Japan ( PHYSIOLOGICAL SOCIETY OF JAPAN ) 2008 209 - 209 2008
Cell shrinkage, called apoptotic volume decrease (AVD), is one of the characteristic early events in the apoptosis processes. On the other hand, the hypertonicity-induced cation channel (HICC) plays a highly efficient role in the regulatory volume increase (RVI) of many cell types after osmotic shrinkage. By means of electronic cell sizing and whole-cell patch-clamp techniques, in the present study, effects of HICC activation on the staurosporine (STS)-induced AVD and apoptosis were examined in HeLa cells on the basis of a differential time protocol for activation of both processes and by use of blockers of the HICC. Hypertonic stress was found to reduce STS-induced AVD, cell death, caspases 3/7 activation and DNA fragmentation, and these effects were actually due to activation of the HICC. On the other hand, treatment with STS was found to significantly diminish osmotic HICC activation. Thus, it is concluded that the HICC plays not only a volume-restoring role in osmotic shrunken cells but also a death-rescuing role in apoptotic shrunken cells. <b>[J Physiol Sci. 2008;58 Suppl:S209]</b>
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Signalling events employed in activation of the hypertonicity-induced cation channels in HeLa cells
Numata Tomohiro, Okada Yasunobu, Wehner Frank
Proceedings of Annual Meeting of the Physiological Society of Japan ( PHYSIOLOGICAL SOCIETY OF JAPAN ) 2007 126 - 126 2007
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>
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Mechanical stress activates TRPM7 channels expressed in HEK293T cells
Numata Tomohiro, Shimizu Takahiro, Okada Yasunobu
Proceedings of Annual Meeting of the Physiological Society of Japan ( PHYSIOLOGICAL SOCIETY OF JAPAN ) 2006 153 - 153 2006
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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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 2005.02
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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
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TRPM7 channels are activated by cell swelling and involved in the cell volume regulation
Numata Tomohiro, Shimizu Takahiro, Okada Yasunobu
Proceedings of Annual Meeting of the Physiological Society of Japan ( PHYSIOLOGICAL SOCIETY OF JAPAN ) 2005 S76 - S76 2005
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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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 2004.08
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The role of reactive oxygen species in anion channel activation induced by a mitochondrion-mediated apoptosis inducer
Shimizu Takahiro, Numata Tomohiro, Okada Yasunobu
Proceedings of Annual Meeting of the Physiological Society of Japan ( PHYSIOLOGICAL SOCIETY OF JAPAN ) 2004 S83 - S83 2004
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>