研究等業績 - その他 - 沼田　朋大

The juvenile myoclonic epilepsy-related protein EFHC1 interacts with the redox-sensitive TRPM2 channel linked to cell death

Masahiro Katano, Tomohiro Numata, Kripamoy Aguan, Yuji Hara, Shigeki Kiyonaka, Shinichiro Yamamoto, Takafumi Miki, Seishiro Sawamura, Toshimitsu Suzuki, Kazuhiro Yamakawa, Yasuo Mori

CELL CALCIUM ( CHURCHILL LIVINGSTONE )  51 ( 2 ) 179 - 185   2012年02月

The transient receptor potential M2 channel (TRPM2) is the Ca2+-permeable cation channel controlled by cellular redox status via beta-NAD(+) and ADP-ribose (ADPR). TRPM2 activity has been reported to underlie susceptibility to cell death and biological processes such as inflammatory cell migration and insulin secretion. However, little is known about the intracellular mechanisms that regulate oxidative stress-induced cell death via TRPM2. We report here a molecular and functional interaction between the TRPM2 channel and EF-hand motif-containing protein EFHC1, whose mutation causes juvenile myoclonic epilepsy (JME) via mechanisms including neuronal apoptosis. In situ hybridization analysis demonstrates TRPM2 and EFHC1 are coexpressed in hippocampal neurons and ventricle cells, while immunoprecipitation analysis demonstrates physical interaction of the N- and C-terminal cytoplasmic regions of TRPM2 with the EFHC1 protein. Coexpression of EFHC1 significantly potentiates hydrogen peroxide (H2O2)- and ADPR-induced Ca2+ responses and cationic currents via recombinant TRPM2 in HEK293 cells. Furthermore, EFHC1 enhances TRPM2-conferred susceptibility of HEK293 cells to H2O2-induced cell death, which is reversed by JME mutations. These results reveal a positive regulatory action of EFHC1 on TRPM2 activity, suggesting that TRPM2 contributes to the expression of JME phenotypes by mediating disruptive effects of JME mutations of EFHC1 on biological processes including cell death. (C) 2011 Elsevier Ltd. All rights reserved.

DOI PubMed

The ΔC splice-variant of TRPM2 is the hypertonicity-induced cation channel in HeLa cells, and the ecto-enzyme CD38 mediates its activation.

Numata T, Sato K, Christmann J, Marx R, Mori Y, Okada Y, Wehner F

The Journal of physiology ( WILEY-BLACKWELL )  590 ( 5 ) 1121 - 1138   2012年02月

Hypertonicity-induced cation channels (HICCs) are key-players in proliferation and apoptosis but their molecular correlate remains obscure. Furthermore, the activation profile of HICCs is not well defined yet. We report here that, in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr (cADPr), as supposed activators of TRPM2, elicited cation currents that were virtually identical to the osmotic activation of HICCs. Silencing of the expression of TRPM2 and of the ecto-enzyme CD38 (as a likely source of ADPr and cADPr) inhibited HICC as well as nucleotide-induced currents and, in parallel, the hypertonic volume response of cells (the regulatory volume increase, RVI) was attenuated. Quantification of intracellular cADPr levels and the systematic application of extra-vs. intracellular nucleotides indicate that the outwardly directed gradient rather than the cellular activity of ADPr and cADPr triggers TRPM2activation, probably alongwith a simultaneous biotransformation ofnucleotides. Cloning of TRPM2 identified the C-splice variant as the molecular correlate of the HICC, which could be strongly supported by a direct comparison of the respective Ca2+ selectivity. Finally, immunoprecipitation and high-resolution FRET/ FLIM imaging revealed the interaction of TRPM2 and CD38 in the native as well as in a heterologous (HEK293T) expression system. We propose transport-related nucleotide export via CD38 as a novel mechanism of TRPM2/ HICC activation. With the biotransformation of nucleotides running in parallel, continuous zero trans-conditions are achieved which will render the system infinitely sensitive.

DOI PubMed

Activation of TRPA1 channels by transnitrosylating reagents

Daisuke Kozai, Maximilian Ebert, Fumika Karaki, Yuko Otani, Yoji Kabasawa, Nobuaki Takahashi, Tomohiro Numata, Shigeki Kiyonaka, Tomohiko Ohwada, Yasuo Mori

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC )  118   69P - 69P   2012年

Identification of oxygen sensitivity of TRPA1 channels using redox-sensitive chemical library

Shigeki Kiyonaka, Nobuaki Takahashi, Daisuke Kozai, Tomohiro Numata, Yusuke Mizuno, Jun-ichi Yoshida, Yasuo Mori

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC )  118   239P - 239P   2012年

TRPA1 senses oxygen availability

Nobuaki Takahashi, Shigeki Kiyonaka, Tomohiro Numata, Daisuke Kozai, Yasuo Mori

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC )  118   10P - 10P   2012年

"TRPA1 チャネルの酸素感受機構の解明”

沼田朋大, 高橋重成, 清中茂樹, 香西大輔, 森泰生

第89 回日本生理学会大会, 2012.3.29     2012年

“血中酸素飽和度を感知するTRPAl ”

高橋重成, 清中茂樹, 沼田朋大, 香西大輔, 森泰生

第85回日本薬理学会, 2012.3.14     2012年

TRPA1 underlies a sensing mechanism for O-2

Nobuaki Takahashi, Tomoyuki Kuwaki, Shigeki Kiyonaka, Tomohiro Numata, Daisuke Kozai, Yusuke Mizuno, Shinichiro Yamamoto, Shinji Naito, Ellen Knevels, Peter Carmeliet, Toru Oga, Shuji Kaneko, Seiji Suga, Toshiki Nokami, Jun-ichi Yoshida, Yasuo Mori

NATURE CHEMICAL BIOLOGY ( NATURE PUBLISHING GROUP )  7 ( 10 ) 701 - 711   2011年10月

Oxygen (O-2) is a prerequisite for cellular respiration in aerobic organisms but also elicits toxicity. To understand how animals cope with the ambivalent physiological nature of O-2, it is critical to elucidate the molecular mechanisms responsible for O-2 sensing. Here our systematic evaluation of transient receptor potential (TRP) cation channels using reactive disulfides with different redox potentials reveals the capability of TRPA1 to sense O-2. O-2 sensing is based upon disparate processes: whereas prolyl hydroxylases (PHDs) exert O-2-dependent inhibition on TRPA1 activity in normoxia, direct O-2 action overrides the inhibition via the prominent sensitivity of TRPA1 to cysteine-mediated oxidation in hyperoxia. Unexpectedly, TRPA1 is activated through relief from the same PHD-mediated inhibition in hypoxia. In mice, disruption of the Trpa1 gene abolishes hyperoxia- and hypoxia-induced cationic currents in vagal and sensory neurons and thereby impedes enhancement of in vivo vagal discharges induced by hyperoxia and hypoxia. The results suggest a new O-2-sensing mechanism mediated by TRPA1.

DOI

Development of a GPCR Based Electrophysiological Biosensor

Masato Suzuki, Shigeki Kiyonaka, Tomohiro Numata, Ken Shimono, Hiroaki Oka, Yasuo Mori

BIOPHYSICAL JOURNAL ( CELL PRESS )  100 ( 3 ) 621 - 621   2011年02月

Expression of N-type calcium channels in human adrenocortical cells and their contribution to corticosteroid synthesis

Shizuka Aritomi, Hirotaka Wagatsuma, Tomohiro Numata, Yoshitsugu Uriu, Yasuko Nogi, Akira Mitsui, Tomoyuki Konda, Yasuo Mori, Michihiro Yoshimura

HYPERTENSION RESEARCH ( NATURE PUBLISHING GROUP )  34 ( 2 ) 193 - 201   2011年02月

The inhibition of aldosterone activity is a useful approach for preventing the progression of cardiovascular and renal diseases in hypertensive patients. Although the results of our previous in vivo study suggested that N-type calcium channels may have a role in regulating plasma aldosterone levels, the direct relationship between N-type calcium channels and aldosterone production in adrenocortical cells has not been examined. In this study, the analysis of quantitative reverse transcription-PCR, western blotting, and immunocytological staining indicated the possible presence of N-type calcium channels in human adrenocortical cells (H295R cell line). Patch clamp analysis indicated that omega-conotoxin GVIA (CnTX), an N-type calcium channel inhibitor, suppressed voltage-dependent barium currents. During steroidogenesis, CnTX significantly reduced the transient calcium signaling induced by angiotensin II (Ang II) and partially prevented Ang II-induced aldosterone and cortisol formation with no significant influence on CYP11B2 and CYP11B1 mRNA expression. In addition, in alpha 1B calcium channel subunits, knockdown significantly decreased Ang II-induced aldosterone formation with increments in CYP11B2 mRNA expression. We also investigated the inhibitory activities of some types of dihydropyridine calcium channel blockers (CCBs; cilnidipine: L-/N-type CCB, efonidipine: L-/T-type CCB, and nifedipine: L-type CCB), and these agents showed a dose-dependent inhibition effect on Ang II-induced aldosterone and cortisol production. Furthermore, only cilnidipine failed to suppress CYP11B1 expression in H295R cells. These results suggest that N-type calcium channels have a significant role in transducing the Ang II signal for aldosterone (and cortisol) biosynthesis, which may explain the mechanism by which N-type calcium channels regulate plasma aldosterone levels. Hypertension Research (2011) 34, 193-201; doi: 10.1038/hr.2010.191; published online 28 October 2010

DOI PubMed

V₂ receptor-mediated autocrine role of somatodendritic release of AVP in rat vasopressin neurons under hypo-osmotic conditions.

Sato K, Numata T, Saito T, Ueta Y, Okada Y

Science signaling   4 ( 157 ) ra5   2011年01月

Arginine vasopressin (AVP) neurons in the hypothalamus are osmosensory neurons that respond to increased or decreased plasma osmolarity by releasing more or less AVP, respectively, from their axon terminals. Here, we found that, in contrast, hypo-osmotic stress enhanced somatodendritic AVP secretion from isolated rat AVP neurons, and this somatodendritic release depended on actin depolymerization. In AVP neurons identified by transgenic expression of green fluorescent protein, hypo-osmotic stimulation led to activation of anion currents and a slow regulatory volume decrease (RVD). Bath application of AVP increased the volume-sensitive anion current and accelerated RVD
these effects were abolished by inhibition of adenylate cyclase or by a specific antagonist of the V2-type vasopressin receptor. The V2 receptor antagonist slowed the RVD rate of AVP neurons even in the absence of exogenous AVP when the volume of bath solution was reduced. Reverse transcription polymerase chain reaction and immunostaining both indicated that the V 2 receptor was present in AVP neurons.We conclude that somatodendritic release of AVP under hypo-osmotic conditions acts through the V2 receptor as an autocrine signal to enhance volume-sensitive anion channel activity and thereby facilitate cell volume regulation.

DOI PubMed

Activation of TRPA1 channels by novel N-nitrosamines

Daisuke Kozai, Ebert Maximilian, Fumika Karaki, Yuko Otani, Yoji Kabasawa, Nobuaki Takahashi, Tomohiro Numata, Shigeki Kiyonaka, Tomohiko Ohwada, Yasuo Mori

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC )  115   148P - 148P   2011年

VOLUME-REGULATORY AUTOCRINE ACTION OF VASOPRESSIN RELEASED FROM SOMATA AND DENDRITES IN VASOPRESSIN NEURONS IS MEDIATED BY THE V-2 RECEPTOR AND CAMP

Kaori Sato, Tomohiro Numata, Takeshi Saito, Yoichi Ueta, Yasunobu Okada

CELLULAR PHYSIOLOGY AND BIOCHEMISTRY ( KARGER )  28 ( 6 ) 1325 - 1325   2011年

TRPA1チャネルが担う生体における新しいO2 セン サー機構

Nobuaki Takahashi, Tomoyuki Kuwaki, Shigeki Kiyonaka, Tomohiro Numata, Daisuke Kozai, Yusuke Mizuno, Shinichiro Yamamoto, Shinji Naito, Ellen Knevels, Peter Carmeliet, Toru Oga, Shuji Kaneko, Seiji Suga, Toshiki Nokami, Jun-ichi Yoshida, Yasuo Mori

ライフサイエンス新着論文レビュー 高橋重成 一森泰生 (京都大学大学院工学研究科合成・生物化学専攻分子生物化学分野) TRPA1 underlies a sensing mechanism for O2. first lifesc fenoedb. j)/archives/5598     2011年

Rim2 alpha Determines Docking and Priming States in Insulin Granule Exocytosis

Takao Yasuda, Tadao Shibasaki, Kohtaro Minami, Harumi Takahashi, Akira Mizoguchi, Yoshitsugu Uriu, Tomohiro Numata, Yasuo Mori, Jun-ichi Miyazaki, Takashi Miki, Susumu Seino

CELL METABOLISM ( CELL PRESS )  12 ( 2 ) 117 - 129   2010年08月

Insulin secretion is essential for maintenance of glucose homeostasis, but the mechanism of insulin granule exocytosis, the final step of insulin secretion, is largely unknown. Here, we investigated the role of Rim2 alpha in insulin granule exocytosis, including the docking, priming, and fusion steps. We found that interaction of Rim2 alpha and Rab3A is required for docking, which is considered a brake on fusion events, and that docking is necessary for K(+)-induced exocytosis, but not for glucose-induced exocytosis. Furthermore, we found that dissociation of the Rim2 alpha/Munc13-1 complex by glucose stimulation activates Syntaxin1 by Munc13-1, indicating that Rim2 alpha primes insulin granules for fusion. Thus, Rim2 alpha determines docking and priming states in insulin granule exocytosis depending on its interacting partner, Rab3A or Munc13-1, respectively. Because Rim2 alpha(-/-) mice exhibit impaired secretion of various hormones stored as dense-core granules, including glucose-dependent insulinotropic polypeptide, growth hormone, and epinephrine, Rim2 alpha plays a critical role in exocytosis of these dense-core granules.

DOI PubMed

TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade

Chieko Koike, Takehisa Obara, Yoshitsugu Uriu, Tomohiro Numata, Rikako Sanuki, Kentarou Miyata, Toshiyuki Koyasu, Shinji Ueno, Kazuo Funabiki, Akiko Tani, Hiroshi Ueda, Mineo Kondo, Yasuo Mori, Masao Tachibana, Takahisa Furukawa

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA ( NATL ACAD SCIENCES )  107 ( 1 ) 332 - 337   2010年01月

An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 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 its activity 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.

DOI PubMed

A pathogenic C-terminus-truncated polycystin-2 mutant enhances receptor-activated Ca2+ entry via association with TRPC3 and TRPC7

Tomohiro Numata, Shigeki Kiyonaka, Yoshikatsu Kanai, Yasuo Mori

JOURNAL OF PHYSIOLOGICAL SCIENCES ( SPRINGER TOKYO )  60   S7 - S7   2010年

Pathogenic C-terminus-truncated polycystin-2 mutant enhances receptor-activated Ca2+ entry via association with TRPC3 and TRPC7

Shigeki Kiyonaka, Kyoko Miyagi, Kazunori Yamada, Takafumi Miki, Emiko Mori, Kenta Kato, Tomohiro Numata, Yuichi Sawaguchi, Toru Kimura, Yoshikatsu Kanai, Yasuo Mori

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC )  112   202P - 202P   2010年

The modulation of TRPM7 currents by nafamostat mesilate depends directly upon extracellular concentrations of divalent cations

Xuanmao Chen, Tomohiro Numata, Minghua Li, Yasuo Mori, Beverley A. Orser, Michael F. Jackson, Zhi-Gang Xiong, John F. MacDonald

MOLECULAR BRAIN ( BIOMED CENTRAL LTD )  3   38   2010年

Concentrations of extracellular divalent cations (Ca2+ and Mg2+) fall substantially during intensive synaptic transmission as well as during some pathophysiological conditions such as epilepsy and brain ischemia. Here we report that a synthetic serine protease inhibitor, nafamostat mesylate (NM), and several of its analogues, block recombinant TRPM7 currents expressed in HEK293T cells in inverse relationship to the concentration of extracellular divalent cations. Lowering extracellular Ca2+ and Mg2+ also evokes a divalent-sensitive non-selective cation current that is mediated by TRPM7 expression in hippocampal neurons. In cultured hippocampal neurons, NM blocked these TRPM7-mediated currents with an apparent affinity of 27 mu M, as well as the paradoxical Ca2+ influx associated with lowering extracellular Ca2+. Unexpectedly, pre-exposure to NM strongly potentiated TRPM7 currents. In the presence of physiological concentrations of extracellular divalent cations, NM activates TRPM7. The stimulating effects of NM on TRPM7 currents are also inversely related to extracellular Ca2+ and Mg2+. DAPI and HSB but not netropsin, blocked and stimulated TRPM7. In contrast, mono-cationic, the metabolites of NM, p-GBA and AN, as well as protease inhibitor leupeptin and gabexate failed to substantially modulate TRPM7. NM thus provides a molecular template for the design of putative modulators of TRPM7.

DOI PubMed

The role of vasopressin receptor in the AVP neurons under hypotonic conditions

Kaori Sato, Tomohiro Numata, Yoichi Ueta, Yasunobu Okada

JOURNAL OF PHYSIOLOGICAL SCIENCES ( SPRINGER TOKYO )  60   S122 - S122   2010年