研究等業績 - その他 - 沼田　朋大

ヒト心音図と心電図の測定に基づく中学校理科2学年2分野における「動物の体のつくりと働き」の授業 : ICSTシステムの適用

吉野 正巳, 勝木 知昭, 朝日 俊介, 沼田 朋大, 松川 正樹, 原田 和雄, 長谷川 正

東京学芸大学紀要. 自然科学系 ( 東京学芸大学教育実践研究推進本部 )  75   63 - 75   2023年09月

論文(Article)

CiNii Research

イオンチャネルの細胞容積調節能における意義と医療応用を目指した新たな活性制御法の開発

沼田 朋大

秋田医学 ( 秋田医学会 )  49 ( 1 ) 1 - 9   2022年06月

細胞生理機能制御のための光誘導性分子による脱分極機構の解明

沼田 朋大, 佐藤 かお理[沼田]

日本生理学雑誌 ( (一社)日本生理学会 )  84 ( 2 ) 36 - 36   2022年05月

Evaluation of crystallinity in carbon fiber-reinforced poly(ether ether ketone) by using infrared low frequency Raman spectroscopy

Yamaguchi M.

Journal of Applied Polymer Science ( Journal of Applied Polymer Science )  139 ( 8 )   2022年02月

DOI

心筋Sigma-1受容体を介したミトコンドリアCa²⁺シグナル調節機構

田頭 秀章, 篠田 康晴, 沼田 朋大, 福永 浩司

日本薬理学会年会要旨集 ( 公益社団法人 日本薬理学会 )  96   1-B-P-020   2022年

Cardiovascular disease (CVD) is a leading cause of death worldwide. We previously reported that the Sigma-1 receptor (Sigmar1) is down-regulated in mice with cardiac dysfunction. Recent study suggested that Sigmar1 deficient mice display cardiac dysfunction via impairment of mitochondrial function. However, the mechanism of mitochondrial quality control mediated by Sigmar1 has not been investigated in detail. In this study, we investigated the role of Sigmar1 for ER-mitochondrial tethering and mitochondrial Ca²⁺ signaling using a Sigmar1-knockdown cardiomyocytes. We found that disruption of ER-mitochondrial tethering and reduction of ER-mitochondrial Ca²⁺ transport was induced by Sigmar1 knockdown in cardiomyocytes. We also demonstrated that Endothelin-1-induced cardiomyocyte hypertrophy is aggravated associated with induction of mitophagy in Sigmar1 knockdown cardiomyocytes. These data suggest that reduction of cardiac Sigmar1 is involved in myocyte hypertrophy by maintaining of intracellular Ca²⁺ signaling mediated by regulation of ER-mitochondrial tethering.

DOI CiNii Research

二つのタンパク質の協力で細胞の大きさを一定に保つメカニズムを解明

沼田 朋大, 岡田 泰伸

日本生理学雑誌 ( (一社)日本生理学会 )  83 ( 3 ) 49 - 50   2021年08月

二つのタンパク質の協力で細胞の大きさを一定に保つメカニズムを解明

沼田 朋大, 岡田 泰伸

日本生理学雑誌 ( (一社)日本生理学会 )  83 ( 3 ) 49 - 50   2021年08月

Impaired TRIM16-Mediated lysophagy in chronic obstructive pulmonary disease pathogenesis

Araya J.

Journal of Immunology ( Journal of Immunology )  207 ( 1 ) 65 - 76   2021年07月

DOI

TRPM7 is an essential regulator for volume-sensitive outwardly rectifying anion channel.

Tomohiro Numata, Kaori Sato-Numata, Meredith C Hermosura, Yasuo Mori, Yasunobu Okada

Communications biology ( NATURE RESEARCH )  4 ( 1 ) 599 - 599   2021年05月

Animal cells can regulate their volume after swelling by the regulatory volume decrease (RVD) mechanism. In epithelial cells, RVD is attained through KCl release mediated via volume-sensitive outwardly rectifying Cl- channels (VSOR) and Ca2+-activated K+ channels. Swelling-induced activation of TRPM7 cation channels leads to Ca2+ influx, thereby stimulating the K+ channels. Here, we examined whether TRPM7 plays any role in VSOR activation. When TRPM7 was knocked down in human HeLa cells or knocked out in chicken DT40 cells, not only TRPM7 activity and RVD efficacy but also VSOR activity were suppressed. Heterologous expression of TRPM7 in TRPM7-deficient DT40 cells rescued both VSOR activity and RVD, accompanied by an increase in the expression of LRRC8A, a core molecule of VSOR. TRPM7 exerts the facilitating action on VSOR activity first by enhancing molecular expression of LRRC8A mRNA through the mediation of steady-state Ca2+ influx and second by stabilizing the plasmalemmal expression of LRRC8A protein through the interaction between LRRC8A and the C-terminal domain of TRPM7. Therefore, TRPM7 functions as an essential regulator of VSOR activity and LRRC8A expression.

DOI PubMed

BK Channels Are Activated by Functional Coupling With L-Type Ca2+ Channels in Cricket Myocytes

Tomohiro Numata, Kaori Sato-Numata, Masami Yoshino

Frontiers in Insect Science ( Frontiers Media SA )  1   2021年04月

Large-conductance calcium (Ca²⁺)-activated potassium (K⁺) (BK) channel activation is important for feedback control of Ca²⁺influx and cell excitability during spontaneous muscle contraction. To characterize endogenously expressed BK channels and evaluate the functional relevance of Ca²⁺sources leading to BK activity, patch-clamp electrophysiology was performed on cricket oviduct myocytes to obtain single-channel recordings. The single-channel conductance of BK channels was 120 pS, with increased activity resulting from membrane depolarization or increased intracellular Ca²⁺concentration. Extracellular application of tetraethylammonium (TEA) and iberiotoxin (IbTX) suppressed single-channel current amplitude. These results indicate that BK channels are endogenously expressed in cricket oviduct myocytes. Ca²⁺release from internal Ca²⁺stores and Ca²⁺influx via the plasma membrane, which affect BK activity, were investigated. Extracellular Ca²⁺removal nullified BK activity. Administration of ryanodine and caffeine reduced BK activity. Administration of L-type Ca²⁺channel activity regulators (Bay K 8644 and nifedipine) increased and decreased BK activity, respectively. Finally, the proximity between the L-type Ca²⁺channel and BK was investigated. Administration of Bay K 8644 to the microscopic area within the pipette increased BK activity. However, this increase was not observed at a sustained depolarizing potential. These results show that BK channels are endogenously expressed in cricket oviduct myocytes and that BK activity is regulated by L-type Ca²⁺channel activity and Ca²⁺release from Ca²⁺stores. Together, these results show that functional coupling between L-type Ca²⁺and BK channels may underlie the molecular basis of spontaneous rhythmic contraction.

DOI

Vasopressin Neurons Respond to Hyperosmotic Stimulation with Regulatory Volume Increase and Secretory Volume Decrease by Activating Ion Transporters and Ca2+ Channels.

Kaori Sato-Numata, Tomohiro Numata, Yoichi Ueta, Yasunobu Okada

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology   55 ( S1 ) 119 - 134   2021年03月

BACKGROUND/AIMS: Arginine vasopressin (AVP) neurons play an important role for sensing a change in the plasma osmolarity and thereby responding with regulated AVP secretion in order to maintain the body fluid homeostasis. The osmo-sensing processes in magnocellular neurosecretory cells (MNCs) including AVP and oxytocin (OXT) neurons of the hypothalamus were reported to be coupled to sustained osmotic shrinkage or swelling without exhibiting discernible cell volume regulation. Since increasing evidence has shown some important differences in properties between AVP and OXT neurons, osmotic volume responses are to be reexamined with distinguishing these cell types from each other. We previously reported that AVP neurons identified by transgenic expression of enhanced green fluorescence protein (eGFP) possess the ability of regulatory volume decrease (RVD) after hypoosmotic cell swelling. Thus, in the present study, we examined the ability of regulatory volume increase (RVI) after hyperosmotic cell shrinkage in AVP neurons. METHODS: Here, we used eGFP-identified AVP neurons acutely dissociated from AVP-eGFP transgenic rats. We performed single-cell size measurements, cytosolic RT-PCR analysis, AVP secretion measurements, and patch-clamp studies. RESULTS: The AVP neurons were found to respond to a hyperosmotic challenge with physiological cell shrinkage caused by massive secretion of AVP, called a secretory volume decrease (SVD), superimposed onto physical osmotic cell shrinkage, and also to exhibit the ability of RVI coping with osmotic and secretory cell shrinkage. Furthermore, our pharmacological and molecular examinations indicated that AVP secretion and its associated SVD event are triggered by activation of T-type Ca2+ channels, and the RVI event is attained by parallel operation of Na+/H+ exchanger and Cl-/HCO3- anion exchanger. CONCLUSION: Thus, it is concluded that AVP neurons respond to hyperosmotic stimulation with the regulatory volume increase and the secretory volume increase by activating ion transporters and Ca2+ channels, respectively.

DOI PubMed

Cell Death Induction and Protection by Activation of Ubiquitously Expressed Anion/Cation Channels. Part 2: Functional and Molecular Properties of ASOR/PAC Channels and Their Roles in Cell Volume Dysregulation and Acidotoxic Cell Death.

Yasunobu Okada, Kaori Sato-Numata, Ravshan Z Sabirov, Tomohiro Numata

Frontiers in cell and developmental biology ( FRONTIERS MEDIA SA )  9   702317 - 702317   2021年

For survival and functions of animal cells, cell volume regulation (CVR) is essential. Major hallmarks of necrotic and apoptotic cell death are persistent cell swelling and shrinkage, and thus they are termed the necrotic volume increase (NVI) and the apoptotic volume decrease (AVD), respectively. A number of ubiquitously expressed anion and cation channels play essential roles not only in CVR but also in cell death induction. This series of review articles address the question how cell death is induced or protected with using ubiquitously expressed ion channels such as swelling-activated anion channels, acid-activated anion channels, and several types of TRP cation channels including TRPM2 and TRPM7. In the Part 1, we described the roles of swelling-activated VSOR/VRAC anion channels. Here, the Part 2 focuses on the roles of the acid-sensitive outwardly rectifying (ASOR) anion channel, also called the proton-activated chloride (PAC) anion channel, which is activated by extracellular protons in a manner sharply dependent on ambient temperature. First, we summarize phenotypical properties, the molecular identity, and the three-dimensional structure of ASOR/PAC. Second, we highlight the unique roles of ASOR/PAC in CVR dysfunction and in the induction of or protection from acidotoxic cell death under acidosis and ischemic conditions.

DOI PubMed

Expression and functions of N-type Cav2.2 and T-type Cav3.1 channels in rat vasopressin neurons under normotonic conditions.

Kaori Sato-Numata, Tomohiro Numata, Yoichi Ueta, Yasunobu Okada

The journal of physiological sciences : JPS ( BMC )  70 ( 1 ) 49 - 49   2020年10月

Arginine vasopressin (AVP) neurons play essential roles in sensing the change in systemic osmolarity and regulating AVP release from their neuronal terminals to maintain the plasma osmolarity. AVP exocytosis depends on the Ca2+ entry via voltage-gated Ca2+ channels (VGCCs) in AVP neurons. In this study, suppression by siRNA-mediated knockdown and pharmacological sensitivity of VGCC currents evidenced molecular and functional expression of N-type Cav2.2 and T-type Cav3.1 in AVP neurons under normotonic conditions. Also, both the Cav2.2 and Cav3.1 currents were found to be sensitive to flufenamic acid (FFA). TTX-insensitive spontaneous action potentials were suppressed by FFA and T-type VGCC blocker Ni2+. However, Cav2.2-selective ω-conotoxin GVIA failed to suppress the firing activity. Taken together, it is concluded that Cav2.2 and Cav3.1 are molecularly and functionally expressed and both are sensitive to FFA in unstimulated rat AVP neurons. Also, it is suggested that Cav3.1 is primarily involved in their action potential generation.

DOI PubMed

Elucidation of the Mechanisms for the Underlying Depolarization and Reversibility by Photoactive Molecule.

Tomohiro Numata, Ryosuke Fukuda, Mitsuru Hirano, Kazuma Yamaguchi, Kaori Sato-Numata, Hiroshi Imahori, Tatsuya Murakami

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology   54 ( 5 ) 899 - 916   2020年09月

BACKGROUND/AIMS: Light-induced control of the cell membrane potential has enabled important advances in the study of biological processes involving the nervous system and muscle activity. The use of these light-induced modifications is expected in various medical applications, including the control of physiological responses and the recovery of lost functions by regulating nerve activity. In particular, charge-separating linkage molecules (Charge-Separation (CS) molecules) can depolarize cells by photoexcitation without genetic processing. However, the molecular mechanisms underlying cell membrane depolarization are unknown and have hindered its application. Here, we show that CS molecules localized in the cell membrane of PC12 cells using a high-density lipoprotein (HDL)-based drug carrier can excite the cells through a novel membrane current regulation mechanism by light irradiation. METHODS: Membrane potential, channel activity, and membrane capacitance were measured by patch clamp method in rat adrenal gland pheochromocytoma (PC12) cells and KV-overexpressing PC12 cells. CS molecules localized in the cell membrane of PC12 cells using HDL-based drug carrier. The localization of CS molecule was measured by a confocal microscopy. The mRNA expression was tested by RT-PCR. RESULTS: Current clamp measurements revealed that the photo-activated CS molecule causes a sharp depolarization of about 15 mV. Furthermore, it was shown by voltage clamp measurement that this mechanism inactivates the voltage-dependent potassium current and simultaneously generates photo-activated CS molecule induced (PACS) current owing to the loss of the cell membrane capacitance. This activity continues the depolarization of the target cell, but is reversible via a regenerative mechanism such as endocytosis and exocytosis because the cell membrane is intact. CONCLUSION: Thus, the mechanism of photo-induced depolarization concludes that photo-activated TC1 causes depolarization by generating PACS current in parallel with the suppression of the K+ current. Moreover, the depolarization slowly restores by internalization of TC1 from the membrane and insertion of new lipids into the cell membrane, resulting in the restoration of KV to normal activity and eliminating PACS currents, without cell damage. These results suggest the possibility of medical application that can safely control membrane excitation.

DOI PubMed

Chaperone-mediated autophagy suppresses apoptosis via regulation of the unfolded protein response during chronic obstructive pulmonary disease pathogenesis

Hosaka Y.

Journal of Immunology ( Journal of Immunology )  205 ( 5 ) 1256 - 1267   2020年09月

DOI

O2-Dependent Protein Internalization Underlies Astrocytic Sensing of Acute Hypoxia by Restricting Multimodal TRPA1 Channel Responses

Makoto Uchiyama, Akito Nakao, Yuki Kurita, Isato Fukushi, Kotaro Takeda, Tomohiro Numata, Ha Nam Tran, Seishiro Sawamura, Maximilian Ebert, Tatsuki Kurokawa, Reiko Sakaguchi, Alexander J. Stokes, Nobuaki Takahashi, Yasumasa Okada, Yasuo Mori

Current Biology ( Elsevier BV )  30 ( 17 ) 3378 - 3396   2020年07月

Hypoxia sensors are essential for regulating local oxygen (O2) homeostasis within the body. This is especially pertinent within the CNS, which is particularly vulnerable to O2 deprivation due to high energetic demand. Here, we reveal hypoxia-monitoring function exerted by astrocytes through an O2-regulated protein trafficking mechanism within the CNS. Strikingly, cultured mouse astrocytes isolated from the parafacial respiratory group (pFRG) and retrotrapezoid nucleus (RTN) region are capable of rapidly responding to moderate hypoxia via the sensor cation channel transient receptor potential (TRP) A1 but, unlike multimodal sensory neurons, are inert to hyperoxia and other TRPA1 activators (carbon dioxide, electrophiles, and oxidants) in normoxia. Mechanistically, O2 suppresses TRPA1 channel activity by protein internalization via O2-dependent proline hydroxylation and subsequent ubiquitination by an E3 ubiquitin ligase, NEDD4-1 (neural precursor cell-expressed developmentally down-regulated protein 4). Hypoxia inhibits this process and instantly accumulates TRPA1 proteins at the plasma membrane, inducing TRPA1-mediated Ca2+ influx that triggers ATP release from pFRG/RTN astrocytes, potentiating respiratory center activity. Furthermore, astrocyte-specific Trpa1 disruption in a mouse brainstem-spinal cord preparation impedes the amplitude augmentation of the central autonomic respiratory output during hypoxia. Thus, reversible coupling of the TRPA1 channels with O2-dependent protein translocation allows astrocytes to act as acute hypoxia sensors in the medullary respiratory center.

DOI PubMed

Cryo-EM structure of the volume-regulated anion channel LRRC8D isoform identifies features important for substrate permeation.

Ryoki Nakamura, Tomohiro Numata, Go Kasuya, Takeshi Yokoyama, Tomohiro Nishizawa, Tsukasa Kusakizako, Takafumi Kato, Tatsuya Hagino, Naoshi Dohmae, Masato Inoue, Kengo Watanabe, Hidenori Ichijo, Masahide Kikkawa, Mikako Shirouzu, Thomas J Jentsch, Ryuichiro Ishitani, Yasunobu Okada, Osamu Nureki

Communications biology   3 ( 1 ) 240 - 240   2020年05月

Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC). LRRC8A and at least one of the other LRRC8 isoforms assemble into heteromers to generate VRAC transport activities. Despite the availability of the LRRC8A structures, the structural basis of how LRRC8 isoforms other than LRRC8A contribute to the functional diversity of VRAC has remained elusive. Here, we present the structure of the human LRRC8D isoform, which enables the permeation of organic substrates through VRAC. The LRRC8D homo-hexamer structure displays a two-fold symmetric arrangement, and together with a structure-based electrophysiological analysis, revealed two key features. The pore constriction on the extracellular side is wider than that in the LRRC8A structures, which may explain the increased permeability of organic substrates. Furthermore, an N-terminal helix protrudes into the pore from the intracellular side and may be critical for gating.

DOI PubMed

潤腸湯に含まれる生薬成分による分泌性細胞縮小作用の検討

沼田 朋大, 佐藤 かお理[沼田], 岡田 泰伸, 井上 隆司

日本生理学雑誌 ( (一社)日本生理学会 )  82 ( 2 ) 40 - 40   2020年05月

J-GLOBAL

組織特異的TRPM6/7機能抑制マウスの血管機能特性

喜多 知, 田頭 秀章, 沼田 朋大, 喜多 紗斗美, 岩本 隆宏

血管 ( 日本心脈管作動物質学会 )  43 ( 1 ) 39 - 39   2020年01月

J-GLOBAL

Cell Death Induction and Protection by Activation of Ubiquitously Expressed Anion/Cation Channels. Part 1: Roles of VSOR/VRAC in Cell Volume Regulation, Release of Double-Edged Signals and Apoptotic/Necrotic Cell Death.

Yasunobu Okada, Ravshan Z Sabirov, Kaori Sato-Numata, Tomohiro Numata

Frontiers in cell and developmental biology ( FRONTIERS MEDIA SA )  8   614040 - 614040   2020年

Cell volume regulation (CVR) is essential for survival and functions of animal cells. Actually, normotonic cell shrinkage and swelling are coupled to apoptotic and necrotic cell death and thus called the apoptotic volume decrease (AVD) and the necrotic volume increase (NVI), respectively. A number of ubiquitously expressed anion and cation channels are involved not only in CVD but also in cell death induction. This series of review articles address the question how cell death is induced or protected with using ubiquitously expressed ion channels such as swelling-activated anion channels, acid-activated anion channels and several types of TRP cation channels including TRPM2 and TRPM7. The Part 1 focuses on the roles of the volume-sensitive outwardly rectifying anion channels (VSOR), also called the volume-regulated anion channel (VRAC), which is activated by cell swelling or reactive oxygen species (ROS) in a manner dependent on intracellular ATP. First we describe phenotypical properties, the molecular identity, and physical pore dimensions of VSOR/VRAC. Second, we highlight the roles of VSOR/VRAC in the release of organic signaling molecules, such as glutamate, glutathione, ATP and cGAMP, that play roles as double-edged swords in cell survival. Third, we discuss how VSOR/VRAC is involved in CVR and cell volume dysregulation as well as in the induction of or protection from apoptosis, necrosis and regulated necrosis under pathophysiological conditions.

DOI PubMed

Short TRPM2 prevents the targeting of full-length TRPM2 to the surface transmembrane by hijacking to ER associated degradation.

Yamamoto S, Ishii T, Mikami R, Numata T, Shimizu S

Biochemical and biophysical research communications ( ACADEMIC PRESS INC ELSEVIER SCIENCE )  520 ( 3 ) 520 - 525   2019年12月

Membrane proteins are targeted to the surface transmembrane after folding and assembling in the endoplasmic reticulum (ER). Misfolded- and unassembled-proteins are degraded by proteasomes following ubiquitination, termed ER-associated degradation (ERAD). Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive channel. One of the TRPM2 splicing variants, short TRPM2 (TRPM2-S) having only the N-terminus and first two transmembrane domains, was reported to prevent full-length TRPM2 (TRPM2-L) activation. Although TRPM2-S interacts with TRPM2-L, the inhibitory mechanisms of TRPM2-S are unclear. We found that TRPM2-S prevents transmembrane expression of TRPM2-L by targeting ERAD. TRPM2-S expression was lower than that of TRPM2-L, and was increased by an ERAD inhibitor. TRPM2-S was not expressed at the transmembrane. This suggests that TRPM2-S is a substrate for ERAD. Upon the simultaneous expression of TRPM2-S, the transmembrane expression of TRPM2-L was attenuated and the poly-ubiquitination of TRPM2-L was facilitated. Our study may clarify why TRPM2-S inhibits oxidative stress-induced TRPM2-L activation.

DOI PubMed

SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation.

Komatsu S, Nomiyama T, Numata T, Kawanami T, Hamaguchi Y, Iwaya C, Horikawa T, Fujimura-Tanaka Y, Hamanoue N, Motonaga R, Tanabe M, Inoue R, Yanase T, Kawanami D

Endocrine journal   67 ( 1 ) 99 - 106   2019年11月

Cancer is currently one of the major causes of death in patients with type 2 diabetes mellitus. We previously reported the beneficial effects of the glucagon-like peptide-1 receptor agonist exendin-4 against prostate and breast cancer. In the present study, we examined the anti-cancer effect of the sodium-glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin using a breast cancer model. In human breast cancer MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. Ipragliflozin at 1-50 μM significantly and dose-dependently suppressed the growth of MCF-7 cells. BrdU assay also revealed that ipragliflozin attenuated the proliferation of MCF-7 cells in a dose-dependent manner. Because the effect of ipragliflozin against breast cancer cells was completely canceled by knocking down SGLT2, ipragliflozin could act via inhibiting SGLT2. We next measured membrane potential and whole-cell current using the patch clamp technique. When we treated MCF-7 cells with ipragliflozin or glucose-free medium, membrane hyperpolarization was observed. In addition, glucose-free medium and knockdown of SGLT2 by siRNA suppressed the glucose-induced whole-cell current of MCF-7 cells, suggesting that ipragliflozin inhibits sodium and glucose cotransport through SGLT2. Furthermore, JC-1 green fluorescence was significantly increased by ipragliflozin, suggesting the change of mitochondrial membrane potential. These findings suggest that the SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation via membrane hyperpolarization and mitochondrial membrane instability.

DOI PubMed

Herbal components of Japanese Kampo medicines exert laxative actions in colonic epithelium cells via activation of BK and CFTR channels.

Numata T, Sato-Numata K, Okada Y

Scientific reports ( NATURE PUBLISHING GROUP )  9 ( 1 ) 15554 - 15554   2019年10月

Japanese Kampo medicines Junchoto and Mashiningan are mixtures of numerous herbal plant extracts and empirically known to exert laxative actions by stimulating fluid secretion in the colonic epithelium. However, it is unknown which and how the herbal components of these crude Kampo drugs are effective to stimulate ion effluxes causing fluid secretion. Here, we selected four herbal components of Junchoto and Mashiningan, Mashinin (MSN), Kyonin (KYN), Tonin (TON), and Daio (DIO), which are putatively laxatives, and examined their effects on the ion channel activity of human colonic epithelial Caco-2 cells. Patch clamp analyses revealed that MSN activated whole-cell current characteristics of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, whereas KYN, TON, and DIO activated the large-conductance and voltage-activated K+ (BK) channel. Furthermore, electronic cell sizing showed that MSN induced secretory volume decrease (SVD) sensitivity to a CFTR blocker, whereas TON, KYN, and DIO induced SVD sensitivity to a K+ channel blocker. In conclusion, MSN and TON, KYN, and DIO promote fluid secretion from colonic epithelial cells by activating CFTR and BK channels. Thus, Japanese Kampo medicines, Junchoto and Mashiningan, exert anti-constipation actions by inducing KCl efflux through the combined actions of CFTR- and BK-stimulating herbal components.

DOI PubMed

Efficacy of mepolizumab for patients with severe asthma and eosinophilic chronic rhinosinusitis

Numata T.

BMC Pulmonary Medicine ( BMC Pulmonary Medicine )  19 ( 1 )   2019年10月

DOI

Construction of a Fluorescent Screening System of Allosteric Modulators for the GABA(A) Receptor Using a Turn-On Probe

Seiji Sakamoto, Kei Yamaura, Tomohiro Numata, Fumio Harada, Kazuma Amaike, Ryuji Inoue, Shigeki Kiyonaka, Itaru Hamachi

ACS CENTRAL SCIENCE ( AMER CHEMICAL SOC )  5 ( 9 ) 1541 - 1553   2019年09月

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The fast inhibitory actions of GABA are mainly mediated by GABA(A) receptors (GABA(A)Rs), which are widely recognized as clinically relevant drug targets. However, it remains difficult to create screening systems for drug candidates that act on GABA A Rs because of the existence of multiple ligand-binding sites and the delicate pentameric structures of GABA(A)Rs. We here developed the first turn-on fluorescent imaging probe for GABA(A)Rs, which can be used to quantitatively evaluate ligand-receptor interactions under live cell conditions. Using noncovalent labeling of GABA(A)Rs with this turn-on probe, a new imaging-based ligand assay system, which allows discovery of positive allosteric modulators (PAMs) for the GABA(A)R, was successfully constructed. Our system is applicable to high-throughput ligand screening, and we discovered new small molecules that function as PAMs for GABA(A)Rs. These results highlight the power of the use of a turn-on fluorescent probe to screen drugs for complicated membrane proteins that cannot be addressed by conventional methods.

DOI PubMed

TRPM4チャネルと心血管の生理・病態生理

井上隆司, 胡耀鵬, 沼田朋大

医学のあゆみ ( 医歯薬出版 )  270 ( 10 ) 954 - 960   2019年09月

TRPM4チャネルはTRP蛋白質スーパーファミリーのなかにあって、一価陽イオンを選択的に透過させるユニークな性質をもつ。全身に広く発現し、種々の生体機能の制御に関与するカルシウム(Ca2+)依存性陽イオンチャネル(CAN)の分子実体と考えられている。最近、TRPM4蛋白質の分子立体構造が解明され、そのイオン選択性や活性化機構についての理解が大きく進んだ。また、TRPM4ゲーティングの数理化により、細胞内Ca2+動態に連結した実用性のある細胞興奮モデルが構築されている。本稿では、これらの新しい知見を紹介しつつ、このチャネルが心血管系の生理機能や病態生理に果たす役割について述べる。(著者抄録)

J-GLOBAL

TRPM7 is involved in acid-induced necrotic cell death in a manner sensitive to progesterone in human cervical cancer cells.

Numata T, Sato-Numata K, Okada Y

Physiological reports ( Wiley )  7 ( 13 ) e14157   2019年07月

Because intravaginal pH is strongly acidic, it is important to investigate the effects of acidosis on cervical cancer cells. Recently, in response to strong acidosis, human cervical cancer HeLa cells were shown to exhibit necrosis after showing persistent cell swelling induced by Cl- influx. Since cation influx should be accompanied with Cl- influx to drive water inflow causing cell swelling, we here studied on the nature of acidotoxic cation conductance. The mRNA/protein expression was assessed by RT-PCR and Western blotting. Ionic currents were measured by patch-clamping techniques. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and caspase 3/7 activity, respectively. Cell volume and size were measured by electronic sizing and video-microscopic measurements, respectively. Acid exposure enhanced TRPM7 activity endogenously expressed in HeLa cells and exogenously overexpressed in HEK293T cells. Gene silencing of TRPM7 abolished acid-induced cell swelling and necrosis but rather induced activation of apoptotic caspase 3/7 in HeLa cells. Overexpression with the pore charge-neutralizing D1054A mutant suppressed acid-enhanced cation currents, acid-induced cell swelling, and acidotoxic necrosis in HEK293T cells. Progesterone treatment was surprisingly found to suppress molecular and functional expression of TRPM7 and cell proliferation in HeLa cells. Furthermore, in the progesterone-treated cells, acid exposure did not induce persistent cell swelling followed by necrosis but induced persistent cell shrinkage and apoptotic cell death. These results indicate that in the human cervical cancer cells, TRPM7 is essentially involved in acidotoxic necrotic cell death, and progesterone inhibits TRPM7 expression thereby inhibiting acidotoxic necrosis by switching to apoptosis.

DOI PubMed

TRPM7 channels mediate spontaneous Ca2+ fluctuations in growth plate chondrocytes that promote bone development.

Nianchao Qian, Atsuhiko Ichimura, Daisuke Takei, Reiko Sakaguchi, Akihiro Kitani, Ryohei Nagaoka, Masato Tomizawa, Yuu Miyazaki, Hitoshi Miyachi, Tomohiro Numata, Sho Kakizawa, Miyuki Nishi, Yasuo Mori, Hiroshi Takeshima

Science signaling ( American Association for the Advancement of Science ({AAAS}) )  12 ( 576 )   2019年04月

During endochondral ossification of long bones, the proliferation and differentiation of chondrocytes cause them to be arranged into layered structures constituting the epiphyseal growth plate, where they secrete the cartilage matrix that is subsequently converted into trabecular bone. Ca2+ signaling has been implicated in chondrogenesis in vitro. Through fluorometric imaging of bone slices from embryonic mice, we demonstrated that live growth plate chondrocytes generated small, cell-autonomous Ca2+ fluctuations that were associated with weak and intermittent Ca2+ influx. Several genes encoding Ca2+-permeable channels were expressed in growth plate chondrocytes, but only pharmacological inhibitors of transient receptor potential cation channel subfamily M member 7 (TRPM7) reduced the spontaneous Ca2+ fluctuations. The TRPM7-mediated Ca2+ influx was likely activated downstream of basal phospholipase C activity and was potentiated upon cell hyperpolarization induced by big-conductance Ca2+-dependent K+ channels. Bones from embryos in which Trpm7 was conditionally knocked out during ex vivo culture exhibited reduced outgrowth and displayed histological abnormalities accompanied by insufficient autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the growth plate. The link between TRPM7-mediated Ca2+ fluctuations and CaMKII-dependent chondrogenesis was further supported by experiments with chondrocyte-specific Trpm7 knockout mice. Thus, growth plate chondrocytes generate spontaneous, TRPM7-mediated Ca2+ fluctuations that promote self-maturation and bone development.

DOI PubMed

光誘発性電荷分離分子を利用した神経興奮制御

沼田 朋大

日本生理学雑誌 ( (一社)日本生理学会 )  81 ( 1 ) 1 - 2   2019年02月

J-GLOBAL

徐脈性不整脈遺伝子変異TRPM4-E7Kチャネルの数理モデルシミュレーション

胡 耀鵬, 平石 敬三, 倉原 琳, 沼田 朋大, 井上 隆司

日本生理学雑誌 ( (一社)日本生理学会 )  81 ( 1 ) 4 - 4   2019年02月

J-GLOBAL

Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties.

Okada Y, Okada T, Sato-Numata K, Islam MR, Ando-Akatsuka Y, Numata T, Kubo M, Shimizu T, Kurbannazarova RS, Marunaka Y, Sabirov RZ

Pharmacological reviews   71 ( 1 ) 49 - 88   2019年01月

There are a number of mammalian anion channel types associated with cell volume changes. These channel types are classified into two groups: volume-activated anion channels (VAACs) and volume-correlated anion channels (VCACs). VAACs can be directly activated by cell swelling and include the volume-sensitive outwardly rectifying anion channel (VSOR), which is also called the volume-regulated anion channel; the maxi-anion channel (MAC or Maxi-Cl); and the voltage-gated anion channel, chloride channel (ClC)-2. VCACs can be facultatively implicated in, although not directly activated by, cell volume changes and include the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, the Ca2+-activated Cl- channel (CaCC), and the acid-sensitive (or acid-stimulated) outwardly rectifying anion channel. This article describes the phenotypical properties and activation mechanisms of both groups of anion channels, including accumulating pieces of information on the basis of recent molecular understanding. To that end, this review also highlights the molecular identities of both anion channel groups; in addition to the molecular identities of ClC-2 and CFTR, those of CaCC, VSOR, and Maxi-Cl were recently identified by applying genome-wide approaches. In the last section of this review, the most up-to-date information on the pharmacological properties of both anion channel groups, especially their half-maximal inhibitory concentrations (IC50 values) and voltage-dependent blocking, is summarized particularly from the standpoint of pharmacological distinctions among them. Future physiologic and pharmacological studies are definitely warranted for therapeutic targeting of dysfunction of VAACs and VCACs.

DOI PubMed

Roles of volume-regulatory anion channels, VSOR and Maxi-Cl, in apoptosis, cisplatin resistance, necrosis, ischemic cell death, stroke and myocardial infarction.

Okada Y, Numata T, Sato-Numata K, Sabirov RZ, Liu H, Mori SI, Morishima S

Current topics in membranes ( ELSEVIER ACADEMIC PRESS INC )  83   205 - 283   2019年

Two types of anion channels are directly activated by osmotic swelling and are involved in the regulatory volume decrease (RVD) in most types of mammalian cells, and they include the volume-sensitive outwardly rectifying anion channel (VSOR), also called the volume-regulated anion channel (VRAC), and the large-conductance maxi-anion channel (Maxi-Cl). In cardiomyocytes, a splice variant of cystic fibrosis transmembrane conductance regulator anion channel (cardiac CFTR) participates in the RVD mechanism under β-adrenergic stimulation. VSOR and Maxi-Cl are also involved in facilitation of the RVD process by releasing extracellular autocrine/paracrine signals, glutamate and ATP. Apoptotic cell death starts with cell shrinkage, called the apoptotic volume decrease (AVD), which is also caused by activation of VSOR. Since VSOR is implicated not only in the AVD induction but also in the uptake of an anti-cancer drug, cisplatin, downregulation of VSOR activity is causatively involved in acquisition of cisplatin resistance in cancer cells. Necrotic cell death exhibits persistent cell swelling, called the necrotic volume increase (NVI), which is coupled to RVD dysfunction due to impaired VSOR function. Acidotoxic and lactacidosis-induced necrotic cell death is induced both by glutamate release mediated by astroglial VSOR and Maxi-Cl and by exaggerated Cl- influx mediated by neuronal VSOR under prolonged depolarization caused by activation of ionotropic glutamate receptor (iGluR) cation channels. Both VSOR and Maxi-Cl are elaborately involved, in a manner as double-edged swords, in ischemia- and ischemia-reperfusion-induced apoptotic or necrotic cell death in cerebral and myocardial cells by mediating not only Cl- transport but also release of glutamate and/or ATP. Cardiac CFTR exerts a protective action against ischemia(-reperfusion)-induced cardiac injury, called myocardial infarction (MI), which is largely necrotic. Cardiac Maxi-Cl activity may participate in protection against ischemia(-reperfusion) injury by mediating ATP release.

DOI PubMed

がん SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松 志保, 野見山 崇, 沼田 朋大, 堀川 剛, 川波 賢子, 濱口 百合子, 高橋 貴理子, 岩屋 智加予, 田中 智子, 井上 隆司, 柳瀬 敏彦

糖尿病合併症 ( (一社)日本糖尿病合併症学会 )  32 ( Suppl.1 ) 224 - 224   2018年10月

J-GLOBAL

【消化器病漢方治療-理論と実践】理論 漢方薬作用機序の科学的解明 潤腸湯/麻子仁丸

沼田 朋大, 井上 隆司

臨床消化器内科 ( (株)日本メディカルセンター )  33 ( 11 ) 1371 - 1376   2018年09月

＜文献概要＞便秘は日常生活に大きなストレスをもたらす.便秘の症状を緩和するためにいくつかの作用機序の異なる薬物が使用されているが,漢方薬をはじめそれらの分子作用基盤はよく理解されていないことが多い.最近,慢性便秘漢方薬である潤腸湯/麻子仁丸の効果機序が分子生理学的手法を用いた機能解析によって明らかにされた.興味深いことに潤腸湯の投与は,細胞内cAMP上昇によってCFTRチャネルの活性化を介したクロライドイオン流出を誘発し,腸管内への水分泌を促進することが明らかとなった.本稿では,この話題を中心に,類似の作用機序を有し,近年新しい範疇の緩下剤として注目されている小腸刺激性下剤,ルビプロストンについても議論したい.

J-GLOBAL

TRPM7-mediated spontaneous Ca²⁺ entry regulates the proliferation and differentiation of human leukemia cell line K562.

Takahashi K, Umebayashi C, Numata T, Honda A, Ichikawa J, Hu Y, Yamaura K, Inoue R

Physiological reports ( WILEY )  6 ( 14 ) e13796   2018年07月

Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real-time RT-PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+ ]i ) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+ ]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2-APB, Gd3+ , and FTY720 dose dependently decreased basal [Ca2+ ]i . A spontaneously active inward current (Ispont ) contributive to basal [Ca2+ ]i was identified by the nystatin-perforated whole-cell recording. Ispont permeated Ca2+ comparably to Na+ , and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA-mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin-induced γ-globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.

DOI PubMed

Cellular mechanism for herbal medicine Junchoto to facilitate intestinal Cl−/water secretion that involves cAMP-dependent activation of CFTR

Tomohiro Numata, Kaori Sato-Numata, Yasunobu Okada, Ryuji Inoue

Journal of Natural Medicines ( Springer Nature )  72 ( 3 ) 694 - 705   2018年06月

Constipation is a common symptom frequently compromising the quality of daily life. Several mechanistically different drugs have been used to mitigate constipation, including Japanese herbal (Kampo) medicines. However, the mechanisms of their actions are often not well understood. Here we aimed to investigate the molecular mechanisms underlying the effects of Junchoto (JCT), a Kampo medicine empirically prescribed for chronic constipation. Cl− channel activity was measured by the patch-clamp method in human cystic fibrosis transmembrane conductance regulator (CFTR)-expressing HEK293T cells and human intestinal Caco-2 cells. cAMP was measured by a luciferase-based assay. Cell volume change was measured by a particle-sizing and particle-counting analyzer and video-microscopic measurement. In both CFTR-expressing HEK293T and Caco-2 cells, JCT dose-dependently induced whole-cell currents showing typical biophysical and pharmacological features of CFTR. Robust expression of CFTR was confirmed by RT-PCR and Western blotting in Caco-2 cells. Luciferase-based measurement revealed that JCT increases intracellular cAMP levels. Administration of the adenylate cyclase inhibitor SQ22536 or CFTR inhibitor-172, or treatment with small interfering RNAs (siRNA) targeting CFTR, abolished JCT-induced whole-cell currents, suggesting that elevated intracellular cAMP by JCT causes activation of CFTR in Caco-2 cells. Finally, blockade of CFTR activity by CFTR inhibitor-172 or siRNA-knockdown of CFTR or application of SQ22536 markedly reduced the degree of cell volume decrease induced by JCT. JCT can induce a Cl− efflux through the CFTR channel to promote water secretion, and this effect is likely mediated by increased cAMP production.

DOI PubMed

プロポフォールは敗血症時の血管炎症反応を抑制する

高橋 貴理子, 沼田 朋大, 山浦 健, 井上 隆司

日本生理学雑誌 ( (一社)日本生理学会 )  80 ( 2 ) 31 - 32   2018年05月

J-GLOBAL

慢性便秘薬「潤腸湯」の作用メカニズムの解明

沼田 朋大, 佐藤 かお理[沼田], 井上 隆司

日本生理学雑誌 ( (一社)日本生理学会 )  80 ( 2 ) 32 - 33   2018年05月

J-GLOBAL

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松 志保, 野見山 崇, 沼田 朋大, 岩屋 智加予, 川波 賢子, 濱口 百合子, 田中 智子, 田邉 真紀人, 井上 隆司, 柳瀬 敏彦

糖尿病 ( (一社)日本糖尿病学会 )  61 ( 4 ) 291 - 291   2018年04月

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松 志保, 野見山 崇, 沼田 朋大, 堀川 剛, 川波 賢子, 濱口 百合子, 高橋 貴理子, 岩屋 智加予, 田中 智子, 井上 隆司, 柳瀬 敏彦

糖尿病 ( (一社)日本糖尿病学会 )  61 ( Suppl.1 ) S - 232   2018年04月

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松 志保, 野見山 崇, 沼田 朋大, 堀川 剛, 川波 賢子, 濱口 百合子, 高橋 貴理子, 岩屋 智加予, 田中 智子, 井上 隆司, 柳瀬 敏彦

日本内分泌学会雑誌 ( (一社)日本内分泌学会 )  94 ( 1 ) 421 - 421   2018年04月

Functional Charaterization of Zebrafish Transient Receptor Potential Melastatin 2

Tran Ha Nam, Hederih Jure, Numata Tomohiro, Mori Masayuki X, Maegawa Shingo, Hosokawa Hiroshi, Mori Yasuo

BIOPHYSICAL JOURNAL ( CELL PRESS )  114 ( 3 ) 641A - 642A   2018年02月

慢性便秘漢方薬「潤腸湯」の作用機序の解明

沼田朋大, 佐藤(沼田)かお理, 岡田泰伸, 井上隆司

生体機能と創薬シンポジウム要旨集   2018   2018年

J-GLOBAL

生理数理科学アプローチによるPKD2L1チャネルの新たな電位感受性機構の発見

沼田朋大

トランスポーター研究会年会抄録集   13th   2018年

J-GLOBAL

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松志保, 野見山崇, 沼田朋大, 岩屋智加予, 川波賢子, 濱口百合子, 田中智子, 田邉真紀人, 井上隆司, 柳瀬敏彦

糖尿病(Web)   61 ( 4 )   2018年

J-GLOBAL

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松 志保, 野見山 崇, 沼田 朋大, 堀川 剛, 川波 賢子, 濱口 百合子, 高橋 貴理子, 田中 智子, 井上 隆司, 柳瀬 敏彦

日本内分泌学会雑誌 ( (一社)日本内分泌学会 )  93 ( 4 ) 1292 - 1292   2017年12月

J-GLOBAL

Distinct Mechanism of Cysteine Oxidation-Dependent Activation and Cold Sensitization of Human Transient Receptor Potential Ankyrin 1 Channel by High and Low Oxaliplatin

Takahito Miyake, Saki Nakamura, Zhao Meng, Satoshi Hamano, Keisuke Inoue, Tomohiro Numata, Nobuaki Takahashi, Kazuki Nagayasu, Hisashi Shirakawa, Yasuo Mori, Takayuki Nakagawa, Shuji Kaneko

FRONTIERS IN PHYSIOLOGY ( FRONTIERS MEDIA SA )  8 ( NOV ) 878 - 878   2017年11月

Oxaliplatin, a third-generation platinum-based chemotherapeutic agent, displays unique acute peripheral neuropathy triggered or enhanced by cold, and accumulating evidence suggests that transient receptor potential ankyrin 1 (TRPA1) is responsible. TRPA1 is activated by oxaliplatin via a glutathione-sensitive mechanism. However, oxaliplatin interrupts hydroxylation of a proline residue located in the N-terminal region of TRPA1 via inhibition of prolyl hydroxylase (PHD), which causes sensitization of TRPA1 to reactive oxygen species (ROS). Furthermore, PHD inhibition endows cold-insensitive human TRPA1 (hTRPA1) with ROS-dependent cold sensitivity. Since cysteine oxidation and proline hydroxylation regulate its activity, their association with oxaliplatin-induced TRPA1 activation and acquirement of cold sensitivity were investigated in the present study. A high concentration of oxaliplatin (1mM) induced outward-rectifier whole-cell currents and increased the intracellular Ca2+ concentration in hTRPA1-expressing HEK293 cells, but did not increase the probability of hTRPA1 channel opening in the inside-out configuration. Oxaliplatin also induced the rapid generation of hydrogen peroxide, and the resultant Ca2+ influx was prevented in the presence of glutathione and in cysteine-mutated hTRPA1 (Cys641Ser)-expressing cells, whereas proline-mutated hTRPA1 (Pro394Ala)-expressing cells showed similar whole-cell currents and Ca2+ influx. By contrast, a lower concentration of oxaliplatin (100 mu M) did not increase the intracellular Ca2+ concentration but did confer cold sensitivity on hTRPA1-expressing cells, and this was inhibited by PHD2 co-overexpression. Cold sensitivity was abolished by the mitochondria-targeting ROS scavenger mitoTEMPO and was minimal in cysteine-mutated hTRPA1 (Cys641Ser or Cys665Ser)-expressing cells. Thus, high oxaliplatin evokes ROS-mediated cysteine oxidation-dependent hTRPA1 activation independent of PHD activity, while a lower concentration induces cold-induced cysteine oxidation-dependent opening of hTRPA1 via PHD inhibition.

DOI PubMed

糖尿病とがん SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松 志保, 野見山 崇, 沼田 朋大, 川波 賢子, 濱口 百合子, 高橋 貴理子, 田中 智子, 井上 隆司, 柳瀬 敏彦

糖尿病合併症 ( (一社)日本糖尿病合併症学会 )  31 ( Suppl.1 ) 190 - 190   2017年10月

J-GLOBAL

血管・動脈硬化 SGLT-2阻害薬によるマクロファージ活性抑制を介した糖尿病大血管症進展抑制効果の解析

松村 剛, 村上 彩子, 瀬ノ口 隆文, 石井 規夫, 山田 沙理恵, 守田 雄太郎, 西田 周平, 久木留 大介, 本島 寛之, 近藤 龍也, 沼田 朋大, 荒木 栄一

糖尿病合併症 ( (一社)日本糖尿病合併症学会 )  31 ( Suppl.1 ) 240 - 240   2017年10月

Integrative Approach with Electrophysiological and Theoretical Methods Reveals a New Role of S4 Positively Charged Residues in PKD2L1 Channel Voltage-Sensing

Tomohiro Numata, Kunichika Tsumoto, Kazunori Yamada, Tatsuki Kurokawa, Shinichi Hirose, Hideki Nomura, Mitsuhiro Kawano, Yoshihisa Kurachi, Ryuji Inoue, Yasuo Mori

SCIENTIFIC REPORTS ( NATURE PUBLISHING GROUP )  7 ( 1 ) 9760   2017年08月

Numerical model-based simulations provide important insights into ion channel gating when experimental limitations exist. Here, a novel strategy combining numerical simulations with patch clamp experiments was used to investigate the net positive charges in the putative transmembrane segment 4 (S4) of the atypical, positively-shifted voltage-dependence of polycystic kidney disease 2-like 1 (PKD2L1) channel. Charge-neutralising mutations (K452Q, K455Q and K461Q) in S4 reduced gating charges, positively shifted the Boltzmann-type activation curve [i.e., open probability (P-open)-V curve] and altered the time-courses of activation/deactivation of PKD2L1, indicating that this region constitutes part of a voltage sensor. Numerical reconstruction of wild-type (WT) and mutant PKD2L1-mediated currents necessitated, besides their voltage-dependent gating parameters, a scaling factor that describes the voltage-dependence of maximal conductance, G(max). Subsequent single-channel conductance (gamma) measurements revealed that voltage-dependence of G(max) in WT can be explained by the inward-rectifying property of gamma, which is greatly changed in PKD2L1 mutants. Homology modelling based on PKD2 and Na(V)Ab structures suggest that such voltage dependence of P-open and gamma in PKD2L1 could both reflect the charged state of the S4 domain. The present conjunctive experimental and theoretical approaches provide a framework to explore the undetermined mechanism(s) regulating TRP channels that possess non-classical voltage-dependent properties.

DOI PubMed

Strategy to Attain Remarkably High Photoinduced Charge-Separation Yield of Donor−Acceptor Linked Molecules in Biological Environment via Modulating Their Cationic Moieties.

Ning Cai, Yuta Takano, Tomohiro Numata, Ryuji Inoue, Yasuo Mori, Tatsuya Murakami, Hiroshi Imahori

Physical Chemistry ( AMER CHEMICAL SOC )  121 ( 32 ) 17457 - 17465   2017年08月

A series of ferrocene-porphyrin-fullerene linked triads (TC1, TC2, and TC4) possessing different numbers of cationic moieties were designed and prepared to achieve a high photoinduced charge-separation (CS) yield in a biological environment. In a solution, TC1, TC2, and TC4 demonstrated the formation of their nanoaggregates. Among the new triads, TC4 possessing the four cationic moieties exhibited the formation of a long-lived charge-separated state with the highest CS yield (86%) ever reported in cell membrane-like lipid bilayers, which is consistent with the largest change in the cell membrane potential of PC12 cells via the photoinduced CS under green light illumination. The highest CS yield in the biological environment can be rationalized by the well-tailored balance in hydrophobicity and hydrophilicity of TC4. This finding provides a strategy to improve greatly the photoinduced charge-separation yield of donor-acceptor linked molecules in the biological environment and also will be informative for extracting the full potential of the photoinduced charge-separated state toward biological applications.

DOI

Uncovering the arrhythmogenic potential of TRPM4 activation in atrial-derived HL-1 cells using novel recording and numerical approaches

Yaopeng Hu, Yubin Duan, Ayako Takeuchi, Lin Hai-Kurahara, Jun Ichikawa, Keizo Hiraishi, Tomohiro Numata, Hiroki Ohara, Gentaro Iribe, Michio Nakaya, Masayuki X. Mori, Satoshi Matsuoka, Genshan Ma, Ryuji Inoue

CARDIOVASCULAR RESEARCH ( OXFORD UNIV PRESS )  113 ( 10 ) 1243 - 1255   2017年08月

Aims Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4), a Ca2+-activated nonselective cation channel abundantly expressed in the heart, has been implicated in conduction block and other arrhythmic propensities associated with cardiac remodelling and injury. The present study aimed to quantitatively evaluate the arrhythmogenic potential of TRPM4.
Methods and results Patch clamp and biochemical analyses were performed using expression system and an immortalized atrial cardiomyocyte cell line (HL-1), and numerical model simulation was employed. After rapid desensitization, robust reactivation of TRPM4 channels required high micromolar concentrations of Ca2+. However, upon evaluation with a newly devised, ionomycin-permeabilized cell-attached (Iono-C/A) recording technique, submicromolar concentrations of Ca2+ (apparent K-d = similar to 500 nM) were enough to activate this channel. Similar submicromolar Ca2+ dependency was also observed with sharp electrode whole-cell recording and in experiments coexpressing TRPM4 and L-type voltage-dependent Ca2+ channels. Numerical simulations using a number of action potential (AP) models (HL-1, Nygren, Luo-Rudy) incorporating the Ca2+-and voltage-dependent gating parameters of TRPM4, as assessed by Iono-C/A recording, indicated that a few-fold increase in TRPM4 activity is sufficient to delay late AP repolarization and further increases (>= six-fold) evoke early afterdepolarization. These model predictions are consistent with electrophysiological data from angiotensin II-treated HL-1 cells in which TRPM4 expression and activity were enhanced.
Conclusions These results collectively indicate that the TRPM4 channel is activated by a physiological range of Ca2+ concentrations and its excessive activity can cause arrhythmic changes. Moreover, these results demonstrate potential utility of the first AP models incorporating TRPM4 gating for in silico assessment of arrhythmogenicity in remodelling cardiac tissue.

DOI PubMed

SGLT2 Inhibitor Ipragliflozin Inhibits Breast Cancer via Membrane Hyperpolarization

Shiho Komatsu, Takashi Nomiyama, Tomohiro Numata, Takako Kawanami, Yuriko Hamaguchi, Kiriko Takahashi, Tomoko Tanaka, Ryuji Inoue, Toshihiko Yanase

DIABETES ( AMER DIABETES ASSOC )  66   A532 - A532   2017年06月

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松 志保, 野見山 崇, 沼田 朋大, 川波 賢子, 濱口 百合子, 高橋 貴理子, 田中 智子, 井上 隆司, 柳瀬 敏彦

日本内分泌学会雑誌 ( (一社)日本内分泌学会 )  93 ( 1 ) 348 - 348   2017年04月

J-GLOBAL

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松 志保, 野見山 崇, 沼田 朋大, 川波 賢子, 濱口 百合子, 高橋 貴理子, 井上 隆司, 柳瀬 敏彦

糖尿病 ( (一社)日本糖尿病学会 )  60 ( Suppl.1 ) S - 394   2017年04月

FUNCTIONAL CHARATERIZATION OF ZEBRAFISH TRANSIENT RECEPTOR POTENTIAL MELASTATIN 2 IN HEK 293 CELL LINE

Ha Nam Tran, Numata Tomohiro, Mori Masayuki X, Maegawa Shingo, Hosokawa Hiroshi, Tanaka Shingo, Mori Yasuo

JOURNAL OF PHARMACOLOGICAL SCIENCES   133 ( 3 ) S126   2017年03月

The regulatory effect of oxaliplatin on human TRPA1 cold sensitivity

Miyake Takahito, Nakamura Saki, Zhao Meng, So Kanako, Inoue Keisuke, Numata Tomohiro, Takahashi Nobuaki, Shirakawa Hisashi, Mori Yasuo, Nakagawa Takayuki, Kaneko Shuji

JOURNAL OF PHARMACOLOGICAL SCIENCES   133 ( 3 ) S85   2017年03月

K562細胞の増殖・赤芽球分化におけるTRPM7を介したイオン流入の役割

高橋 貴理子, 沼田 朋大, 山浦 健, 井上 隆司

日本生理学雑誌 ( (一社)日本生理学会 )  79 ( 1 ) 34 - 34   2017年02月

J-GLOBAL

共培養系を用いた線維芽細胞による心房興奮伝播制御機構の検討

胡 耀鵬, 平石 敬三, 倉原 琳, 市川 純, 沼田 朋大, 井上 隆司

日本生理学雑誌 ( (一社)日本生理学会 )  79 ( 1 ) 28 - 28   2017年02月

J-GLOBAL

Distinct contributions of LRRC8A and its paralogs to the VSOR anion channel from those of the ASOR anion channel

Kaori Sato-Numata, Tomohiro Numata, Ryuji Inoue, Ravshan Z. Sabirov, Yasunobu Okada

CHANNELS ( TAYLOR & FRANCIS INC )  11 ( 2 ) 167 - 172   2017年

Volume- and acid-sensitive outwardly rectifying anion channels (VSOR and ASOR) activated by swelling and acidification exhibit voltage-dependent inactivation and activation time courses, respectively. Recently, LRRC8A and some paralogs were shown to be essentially involved in the activity and inactivation kinetics of VSOR currents in human colonic HCT116 cells. In human cervix HeLa cells, here, inactivation of VSOR currents was found to become accelerated by RNA silencing only of LRRC8A but never decelerated by that of any LRRC8 isoform. These data suggest that LRRC8A is associated with the deceleration mechanism of VSOR inactivation, while none of LRRC8 members is related to the acceleration mechanism. Activation kinetics of ASOR currents was unaffected by knockdown of any LRRC8 family member. Double, triple and quadruple gene-silencing studies indicated that combinatory expression of LRRC8A with LRRC8D and LRRC8C is essential for VSOR activity, whereas none of LRRC8 family members is involved in ASOR activity.

DOI PubMed

SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する

小松志保, 野見山崇, 沼田朋大, 川波賢子, 濱口百合子, 高橋貴理子, 井上隆司, 柳瀬敏彦

糖尿病(Web)   60 ( Suppl )   2017年

J-GLOBAL

Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

Shigeki Kiyonaka, Ryou Kubota, Yukiko Michibata, Masayoshi Sakakura, Hideo Takahashi, Tomohiro Numata, Ryuji Inoue, Michisuke Yuzaki, Itaru Hamachi

NATURE CHEMISTRY ( NATURE PUBLISHING GROUP )  8 ( 10 ) 958 - 967   2016年10月

The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2'-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

DOI PubMed

Discovery of allosteric modulators for GABA(A) receptors by ligand-directed chemistry

Kei Yamaura, Shigeki Kiyonaka, Tomohiro Numata, Ryuji Inoue, Itaru Hamachi

NATURE CHEMICAL BIOLOGY ( NATURE PUBLISHING GROUP )  12 ( 10 ) 822 - +   2016年10月

The fast inhibitory actions of gamma-aminobutyric acid (GABA) are mainly mediated by GABA(A) receptors (GABA(A)Rs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABA(A)Rs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABA(A)R ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABA(A)Rs. Electrophysiological measurements revealed that one hit, 4,4',4 ''-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABA(A)R-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.

DOI PubMed

Cold sensitivity of TRPA1 is unveiled by the prolyl hydroxylation blockade-induced sensitization to ROS

Takahito Miyake, Saki Nakamura, Meng Zhao, Kanako So, Keisuke Inoue, Tomohiro Numata, Nobuaki Takahashi, Hisashi Shirakawa, Yasuo Mori, Takayuki Nakagawa, Shuji Kaneko

NATURE COMMUNICATIONS ( NATURE PUBLISHING GROUP )  7   12840   2016年09月

Mammalian transient receptor potential ankyrin 1 (TRPA1) is a polymodal nociceptor that plays an important role in pain generation, but its role as a cold nociceptor is still controversial. Here, we propose that TRPA1 can sense noxious cold via transduction of reactive oxygen species (ROS) signalling. We show that inhibiting hydroxylation of a proline residue within the N-terminal ankyrin repeat of human TRPA1 by mutation or using a prolyl hydroxylase (PHD) inhibitor potentiates the cold sensitivity of TRPA1 in the presence of hydrogen peroxide. Inhibiting PHD in mice triggers mouse TRPA1 sensitization sufficiently to sense cold-evoked ROS, which causes cold hypersensitivity. Furthermore, this phenomenon underlies the acute cold hypersensitivity induced by the chemotherapeutic agent oxaliplatin or its metabolite oxalate. Thus, our findings provide evidence that blocking prolyl hydroxylation reveals TRPA1 sensitization to ROS, which enables TRPA1 to convert ROS signalling into cold sensitivity.

DOI PubMed

Identification of MMP1 as a novel risk factor for intracranial aneurysms in ADPKD using iPSC models

Tomonaga Ameku, Daisuke Taura, Masakatsu Sone, Tomohiro Numata, Masahiro Nakamura, Fumihiko Shiota, Taro Toyoda, Satoshi Matsui, Toshikazu Araoka, Tetsuhiko Yasuno, Shin-Ichi Mae, Hatasu Kobayashi, Naoya Kondo, Fumiyo Kitaoka, Naoki Amano, Sayaka Arai, Tomoko Ichisaka, Norio Matsuura, Sumiko Inoue, Takuya Yamamoto, Kazutoshi Takahashi, Isao Asaka, Yasuhiro Yamada, Yoshifumi Ubara, Eri Muso, Atsushi Fukatsu, Akira Watanabe, Yasunori Sato, Tatsutoshi Nakahata, Yasuo Mori, Akio Koizumi, Kazuwa Nakao, Shinya Yamanaka, Kenji Osafune

SCIENTIFIC REPORTS ( NATURE PUBLISHING GROUP )  6   30013   2016年07月

Cardiovascular complications are the leading cause of death in autosomal dominant polycystic kidney disease (ADPKD), and intracranial aneurysm (ICA) causing subarachnoid hemorrhage is among the most serious complications. The diagnostic and therapeutic strategies for ICAs in ADPKD have not been fully established. We here generated induced pluripotent stem cells (iPSCs) from seven ADPKD patients, including four with ICAs. The vascular cells differentiated from ADPKD-iPSCs showed altered Ca2+ entry and gene expression profiles compared with those of iPSCs from non-ADPKD subjects. We found that the expression level of a metalloenzyme gene, matrix metalloproteinase (MMP) 1, was specifically elevated in iPSC-derived endothelia from ADPKD patients with ICAs. Furthermore, we confirmed the correlation between the serum MMP1 levels and the development of ICAs in 354 ADPKD patients, indicating that high serum MMP1 levels may be a novel risk factor. These results suggest that cellular disease models with ADPKD-specific iPSCs can be used to study the disease mechanisms and to identify novel disease-related molecules or risk factors.

DOI PubMed

Ablation of the N-type calcium channel ameliorates diabetic nephropathy with improved glycemic control and reduced blood pressure

Shoko Ohno, Hideki Yokoi, Kiyoshi Mori, Masato Kasahara, Koichiro Kuwahara, Junji Fujikura, Masaki Naito, Takashige Kuwabara, Hirotaka Imamaki, Akira Ishii, Moin A. Saleem, Tomohiro Numata, Yasuo Mori, Kazuwa Nakao, Motoko Yanagita, Masashi Mukoyama

SCIENTIFIC REPORTS ( NATURE PUBLISHING GROUP )  6   27192   2016年06月

Pharmacological blockade of the N- and L-type calcium channel lessens renal injury in kidney disease patients. The significance of specific blockade of alpha 1 subunit of N-type calcium channel, Ca(v)2.2, in diabetic nephropathy, however, remains to be clarified. To examine functional roles, we mated Ca(v)2.2(-/-) mice with db/db (diabetic) mice on the C57BLKS background. Ca(v)2.2 was localized in glomeruli including podocytes and in distal tubular cells. Diabetic Ca(v)2.2(-/-) mice significantly reduced urinary albumin excretion, glomerular hyperfiltration, blood glucose levels, histological deterioration and systolic blood pressure (SBP) with decreased urinary catecholamine compared to diabetic Ca(v)2.2(+/+) mice. Interestingly, diabetic heterozygous Ca(v)2.2(+/-) mice also decreased albuminuria, although they exhibited comparable systolic blood pressure, sympathetic nerve activity and creatinine clearance to diabetic Ca(v)2.2(+/+) mice. Consistently, diabetic mice with cilnidipine, an N-/L-type calcium channel blocker, showed a reduction in albuminuria and improvement of glomerular changes compared to diabetic mice with nitrendipine. In cultured podocytes, depolarization-dependent calcium responses were decreased by.-conotoxin, a Ca(v)2.2-specific inhibitor. Furthermore, reduction of nephrin by transforming growth factor-beta (TGF-beta) in podocytes was abolished with.-conotoxin, cilnidipine or mitogen-activated protein kinase kinase inhibitor. In conclusion, Ca(v)2.2 inhibition exerts renoprotective effects against the progression of diabetic nephropathy, partly by protecting podocytes.

DOI PubMed

"TRP inflammation" relationship in cardiovascular system

Tomohiro Numata, Kiriko Takahashi, Ryuji Inoue

SEMINARS IN IMMUNOPATHOLOGY ( SPRINGER HEIDELBERG )  38 ( 3 ) 339 - 356   2016年05月

Despite considerable advances in the research and treatment, the precise relationship between inflammation and cardiovascular (CV) disease remains incompletely understood. Therefore, understanding the immunoinflammatory processes underlying the initiation, progression, and exacerbation of many cardiovascular diseases is of prime importance. The innate immune system has an ancient origin and is well conserved across species. Its activation occurs in response to pathogens or tissue injury. Recent studies suggest that altered ionic balance, and production of noxious gaseous mediators link to immune and inflammatory responses with altered ion channel expression and function. Among plausible candidates for this are transient receptor potential (TRP) channels that function as polymodal sensors and scaffolding proteins involved in many physiological and pathological processes. In this review, we will first focus on the relevance of TRP channel to both exogenous and endogenous factors related to innate immune response and transcription factors related to sustained inflammatory status. The emerging role of inflammasome to regulate innate immunity and its possible connection to TRP channels will also be discussed. Secondly, we will discuss about the linkage of TRP channels to inflammatory CV diseases, from a viewpoint of inflammation in a general sense which is not restricted to the innate immunity. These knowledge may serve to provide new insights into the pathogenesis of various inflammatory CV diseases and their novel therapeutic strategies.

DOI PubMed

Distinct pharmacological and molecular properties of the acid-sensitive outwardly rectifying (ASOR) anion channel from those of the volume-sensitive outwardly rectifying (VSOR) anion channel

Kaori Sato-Numata, Tomohiro Numata, Ryuji Inoue, Yasunobu Okada

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY ( SPRINGER )  468 ( 5 ) 795 - 803   2016年05月

Expressed by many cell types, acid-sensitive outwardly rectifying (ASOR) anion channels are known to be activated by extracellular acidification and involved in acidotoxic necrotic cell death. In contrast, ubiquitously expressed volume-sensitive outwardly rectifying (VSOR) anion channels are activated by osmotic cell swelling and involved in cell volume regulation and apoptotic cell death. Distinct inhibitors to distinguish ASOR from VSOR anion channels have not been identified. Although leucine-rich repeats containing 8A (LRRC8A) was recently found to be an essential component of VSOR anion channels, the possibility of an LRRC8 family member serving as a component of ASOR anion channels has not been examined. In this study, we explored the effects of 12 known VSOR channel inhibitors and small interfering RNA (siRNA)-mediated knockdown of LRRC8 family members on ASOR and VSOR currents in HeLa cells. Among these inhibitors, eight putative VSOR blockers, including 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), were totally ineffective at blocking ASOR channel activity, whereas suramin, R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] acetic acid (DIOA), arachidonic acid, and niflumic acid were found to be effective ASOR anion channel antagonists. In addition, gene-silencing studies showed that no LRRC8 family members are essentially involved in ASOR anion channel activity, whereas LRRC8A is involved in VSOR anion channel activity in HeLa cells.

DOI PubMed

ADPKD特異的iPS細胞を用いた脳動脈瘤合併に関与する新規リスク因子の同定

天久 朝廷, 田浦 大輔, 曽根 正勝, 沼田 朋大, 塩田 文彦, 豊田 太郎, 佐藤 泰憲, 浅香 勲, 武曾 惠理, 深津 敦司, 乳原 善文, 小泉 昭夫, 森 泰生, 中尾 一和, 長船 健二

日本腎臓学会誌 ( (一社)日本腎臓学会 )  58 ( 3 ) 284 - 284   2016年05月

Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line

Heba Badr, Daisuke Kozai, Reiko Sakaguchi, Tomohiro Numata, Yasuo Mori

FRONTIERS IN PHARMACOLOGY ( FRONTIERS MEDIA SA )  7 ( FEB ) 19   2016年02月

Acetaminophen (APAP) is a safe analgesic antipyretic drug at prescribed doses. Its overdose, however, can cause life-threatening liver damage. Though, involvement of oxidative stress is widely acknowledged in APAP-induced hepatocellular death, the mechanism of this increased oxidative stress and the associated alterations in Ca2+ homeostasis are still unclear. Among members of transient receptor potential (TRP) channels activated in response to oxidative stress, we here identify that redox-sensitive TRPV1, TRPC1, TRPM2, and TRPM7 channels underlie Ca2+ entry and downstream cellular damages induced by APAP in human hepatoma (HepG2) cells. Our data indicate that APAP treatment of HepG2 cells resulted in increased reactive oxygen species (ROS) production, glutathione (GSH) depletion, and Ca2+ entry leading to increased apoptotic cell death. These responses were significantly suppressed by pretreatment with the ROS scavengers N-acetyl-L-cysteine (NAC) and 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt monohydrate (Tiron), and also by preincubation of cells with the glutathione inducer Dimethylfumarate (DMF). TRP subtype targeted pharmacological blockers and siRNAs strategy revealed that suppression of either TRPV1, TRPC1, TRPM2, or TRPM7 reduced APAP-induced ROS formation, Ca2+ influx, and cell death; the effects of suppression of TRPV1 or TRPC1, known to be activated by oxidative cysteine modifications, were stronger than those of TRPM2 or TRPM7. Interestingly, TRPV1 and TRPC1 were labeled by the cysteine-selective modification reagent, 5,5'-dithiobis (2-nitrobenzoic acid)-2-biotin (DTNB-2Bio), and this was attenuated by pretreatment with APAP, suggesting that APAP and/or its oxidized metabolites act directly on the modification target cysteine residues of TRPV1 and TRPC1 proteins. In human liver tissue, TRPV1, TRPC1 TRPM2, and TRPM7 channels transcripts were localized mainly to hepatocytes and Kupffer cells. Our findings strongly suggest that APAP-induced Ca2+ entry and subsequent hepatocellular death are regulated by multiple redox-activated cation channels, among which TRPV1 and TRPC1 play a prominent role.

DOI PubMed

TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation

Ryo Yonezawa, Shinichiro Yamamoto, Miki Takenaka, Yukiko Kage, Takaharu Negoro, Takahiro Toda, Masayuki Ohbayashi, Tomohiro Numata, Yasuko Nakano, Toshinori Yamamoto, Yasuo Mori, Masakazu Ishii, Shunichi Shimizu

FREE RADICAL BIOLOGY AND MEDICINE ( ELSEVIER SCIENCE INC )  90   101 - 113   2016年01月

Lung inflammation is a major adverse effect of therapy with the antitumor drug bleomycin (BLM). Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable channel that is activated by oxidative stress through the production of ADP-ribose. We herein investigated whether TRPM2 channels contributed to BLM-induced lung inflammation. The intratracheal instillation of BLM into wild-type (WT) mice increased the number of polymorphonuclear leukocytes (PMNs) and inflammatory cytokine levels in the lung. Increases in inflammatory markers in WT mice were markedly reduced in trpm2 knockout (KO) mice, which demonstrated that the activation of TRPM2 channels was involved in BLM-induced lung inflammation. The expression of TRPM2 mRNA was observed in alveolar macrophages, alveolar epithelial cells, and lung fibroblasts. Actually, TRPM2 protein was expressed in lung tissues. Of these, TRPM2 channels in epithelial cells were activated by the addition of H2O2 following a BLM pretreatment, resulting in the secretion of macrophage inflammatory protein-2 (MIP-2). The H2O2-induced activation of TRPM2 by the BLM pretreatment was blocked by the poly(ADP-ribose) polymerase (PARP) inhibitors PJ34 and 3-aminobenzamide. The accumulation of poly(ADP-ribose) in the nucleus, a marker for ADP-ribose production, was strongly induced by H2O2 following the BLM pretreatment. Furthermore, administration of PRAP inhibitors into WT mice markedly reduced recruitment of inflammatory cells and MIP-2 secretion induced by BLM instillation. These results suggest that the induction of MIP-2 secretion through the activation of TRPM2 channels in alveolar epithelial cells is an important mechanism in BLM-induced lung inflammation, and the TRPM2 activation is likely to be mediated by ADP-ribose production via PARP pathway. TRPM2 channels may be new therapeutic target for BLM-induced lung inflammation. (C) 2015 Elsevier Inc. All rights reserved.

DOI PubMed

Allosteric activation of glutamate receptors by on-cell coordination chemistry

Kiyonaka, Ryou Kubota, Yukiko Michibata, Masayoshi Sakakura, Hideo Takahashi, Tomohiro Numata, Ryuji Inoue, Michisuke Yuzaki, Itaru Hamachi

Nature Chemistry   8 ( 10 ) 958 - 967   2016年

DOI

Optical control of neuronal firing via photo-induced electron transfer in donor–acceptor conjugates.

Yuta Takano, Tomohiro Numata, Kazuto Fujishima, Kazuaki Miyake, Kazuya Nakao, Wesley David Grove, Ryuji Inoue, Mineko Kengaku, Shigeyoshi Sakaki, Yasuo Mori, Tatsuya Murakami, Hiroshi Imahori

Chemical Science ( ROYAL SOC CHEMISTRY )  7 ( 5 ) 3331 - 3337   2016年

A series of porphyrin-fullerene linked molecules has been synthesized to evaluate the effects of substituents and molecular structures on their charge-separation yield and the lifetime of a final charge-separated state in various hydrophilic environments. The selected high-performance molecule effectively achieved depolarization in a plasma cell membrane by visible light as well as two-photon excitation using a near-infrared light laser. Moreover, it was revealed that the depolarization can trigger neuronal firing in rat hippocampal neurons, demonstrating the potential and versatility for controlling cell functions using light.

DOI PubMed

アセトアミノフェンに誘導されるヒト肝ガン細胞死における酸化還元感受性TRPチャネルの役割

ヘバ バドゥー, 香西大輔, 坂口怜子, 沼田朋大, 森泰生

日本薬理学会近畿部会プログラム・要旨集   130th   2016年

J-GLOBAL

Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe2+ in human monocytic U937 cells

Shunichi Shimizu, Ryo Yonezawa, Takaharu Negoro, Shinichiro Yamamoto, Tomohiro Numata, Masakazu Ishii, Yasuo Mori, Takahiro Toda

INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY ( PERGAMON-ELSEVIER SCIENCE LTD )  68   119 - 127   2015年11月

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca2+-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30 degrees C to 37 degrees C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca2+ ([Ca2+](i)) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe2+-accumulation following pretreatment with FeSO4. Thus intracellular Fe2+-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe2+-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe2+-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37 degrees C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe2*-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe2+-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2 channels by Fe2+ may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe2+ is released by heme degradation under intracerebral hemorrhage. (C) 2015 Elsevier Ltd. All rights reserved.

DOI PubMed

Thermosensitive Ion Channel Activation in Single Neuronal Cells by Using Surface-Engineered Plasmonic Nanoparticles

Hirotaka Nakatsuji, Tomohiro Numata, Nobuhiro Morone, Shuji Kaneko, Yasuo Mori, Hiroshi Imahori, Tatsuya Murakami

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION ( WILEY-V C H VERLAG GMBH )  54 ( 40 ) 11725 - 11729   2015年09月

Controlling cell functions using external photo-responsive nanomaterials has enormous potential for the development of cell-engineering technologies and intractable disease therapies, but the former currently requires genetic modification of the target cells. We present a method using plasma-membrane-targeted gold nanorods (pm-AuNRs) prepared with a cationic protein/lipid complex to activate a thermosensitive cation channel, TRPV1, in intact neuronal cells. Highly localized photothermal heat generation mediated by the pm-AuNRs induced Ca2+ influx solely by TRPV1 activation. In contrast, the use of previously reported cationic AuNRs that are coated with a conventional synthetic polymer also led to photoinduced Ca2+ influx, but this influx resulted from membrane damage. Our method provides an optogenetic platform without the need for prior genetic engineering of the target cells and might be useful for novel TRPV1-targeted phototherapeutic approaches.

DOI PubMed

A screening of TRPC channels activators identifies novel neurotrophic compounds

Sawamura Seishiro, Hatano Masahiko, Sasaki Hajime, Takada Yoshinori, Kuwamura Takashi, Numata Tomohiro, Kiyonaka Shigeki, Hino Kyosuke, Kawamura Tetsuya, Tanikawa Jun, Nakagawa Hiroshi, Nagata Ryu, Inoue Ryuji, Mori Yasuo

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC )  128 ( 3 ) S90 - S90   2015年07月

Characterization of redox-sensitive TRP channels in acetaminophen-induced death of human hepatoma cell line

Elsaid Heba A, Numata Tomohiro, Mori Yasuo

JOURNAL OF PHARMACOLOGICAL SCIENCES   128 ( 3 ) S178   2015年07月

Involvement of TRPA1 activation through distinct modifications in oxaliplatin-induced acute peripheral neuropathy

Nakamura Saki, Miyake Takahito, Zhao Meng, Hamano Satoshi, Inoue Keisuke, Takahashi Nobuaki, Numata Tomohiro, Shirakawa Hisashi, Mori Yasuo, Nakagawa Takayuki, Kaneko Shuji

JOURNAL OF PHARMACOLOGICAL SCIENCES   128 ( 3 ) S232   2015年07月

Compromised maturation of GABAergic inhibition underlies abnormal network activity in the hippocampus of epileptic Ca2+ channel mutant mice, tottering

Akito Nakao, Takafumi Miki, Ken Shimono, Hiroaki Oka, Tomohiro Numata, Shigeki Kiyonaka, Kaori Matsushita, Hiroo Ogura, Tetsuhiro Niidome, Jeffrey L. Noebels, Minoru Wakamori, Keiji Imoto, Yasuo Mori

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY ( SPRINGER )  467 ( 4 ) 737 - 752   2015年04月

Cholinergically induced network activity is a useful analogue of theta rhythms involved in memory processing or epileptiform activity in the hippocampus, providing a powerful tool to elucidate the mechanisms of synchrony in neuronal networks. In absence epilepsy, although its association with cognitive impairments has been reported, the mechanisms underlying hippocampal synchrony remain poorly investigated. Here we simultaneously recorded electrical activities from 64 sites in hippocampal slices of Ca(V)2.1 Ca2+ channel mutant tottering (tg) mice, a well-established mouse model of spontaneous absence epilepsy, to analyze the spatiotemporal pattern of cholinergically induced hippocampal network activity. The cholinergic agonist carbachol induced oscillatory discharges originating from the CA3 region. In tg/tg mice, this hippocampal network activity was characterized by enhanced occupancy of discharges of relatively high frequency (6-10 Hz) compared to the wild type. Pharmacological analyses of slices, patch clamp electrophysiological characterization of isolated neurons, and altered patterns of hippocampal GABA(A) receptor subunit and Cl- transporter messenger RNA (mRNA) transcript levels revealed that this abnormality is attributable to a developmental retardation of GABAergic inhibition caused by immature intracellular Cl- regulation. These results suggest that the inherited Ca(V)2.1 Ca2+ channel mutation leads to developmental abnormalities in Cl- transporter expression and GABA(A) receptor compositions in hippocampal neurons and that compromised maturation of GABAergic inhibition contributes to the abnormal synchrony in the hippocampus of tg absence epileptic mice.

DOI PubMed

糖尿病性腎症におけるN型Ca²⁺チャネル阻害の腎保護効果

大野祥子, 横井秀基, 笠原正登, 森潔, 桑原宏一郎, 桑原孝成, 今牧博貴, 石井輝, 古賀健一, 沼田朋大, 菅原照, 森泰生, 中尾一和, 柳田素子, 向山政志

高血圧関連疾患モデル学会学術総会抄録集   51st   2015年

J-GLOBAL

オキサリプラチン誘発急性末梢神経におけるTRPA1の関与

三宅崇仁, 中村彩希, 趙萌, 浜野智, 井上圭亮, 沼田朋大, 高橋重成, 白川久志, 森泰生, 中川貴之, 中川貴之, 金子周司

日本薬学会年会要旨集(CD-ROM)   135th   2015年

J-GLOBAL

Transient Receptor Potential Melastatin 2 Protects Mice against Polymicrobial Sepsis by Enhancing Bacterial Clearance

XiaoWei Qian, Tomohiro Numata, Kai Zhang, CaiXia Li, JinChao Hou, Yasuo Mori, XiangMing Fang

ANESTHESIOLOGY ( LIPPINCOTT WILLIAMS & WILKINS )  121 ( 2 ) 336 - 351   2014年08月

Background: Recent studies suggest that the transient receptor potential melastatin 2 (TRPM2) channel plays an important role in inflammation and immune response. However, the role and mechanism of TRPM2 in polymicrobial sepsis remain unclear.
Methods: The authors explored the effects of genetic disruption of TRPM2 on mortality (n = 15), bacterial clearance (n = 6), organ injury, and systemic inflammation during cecal ligation and puncture-induced sepsis. Electrophysiology, immunoblot, bacterial clearance experiment, and quantitative real-time polymerase chain reaction were used to explore the role and mechanism of TRPM2 in sepsis.
Results: After cecal ligation and puncture, Trpm2-knockout mice had increased mortality compared with wild-type mice (73.3 vs. 40%, P = 0.0289). The increased mortality was associated with increased bacterial burden, organ injury, and systemic inflammation. TRPM2-mediated Ca2+ influx plays an important role in lipopolysaccharide or cecal ligation and puncture-induced heme oxygenase-1 (HO-1) expression in macrophage. HO-1 up-regulation decreased bacterial burden both in wildtype bone marrow-derived macrophages and in cecal ligation and puncture-induced septic wild-type mice. Disruption of TRPM2 decreased HO-1 expression and increased bacterial burden in bone marrow-derived macrophages. Pretreatment of Trpm2-knockout bone marrow-derived macrophages with HO-1 inducer markedly increased HO-1 expression and decreased bacterial burden. Pretreatment of Trpm2-knockout mice with HO-1 inducer reversed the susceptibility of Trpm2-knockout mice to sepsis by enhancing the bacterial clearance. In addition, septic patients with lower monocytic TRPM2 and HO-1 messenger RNA levels had a worse outcome compared with septic patients with normal monocytic TRPM2 and HO-1 messenger RNA levels. TRPM2 levels correlated with HO-1 levels in septic patients (r = 0.675, P = 0.001).
Conclusion: The study data demonstrate a protective role of TRPM2 in controlling bacterial clearance during polymicrobial sepsis possibly by regulating HO-1 expression.

DOI PubMed

Temperature sensitivity of acid-sensitive outwardly rectifying (ASOR) anion channels in cortical neurons is involved in hypothermic neuroprotection against acidotoxic necrosis

Kaori Sato-Numata, Tomohiro Numata, Yasunobu Okada

CHANNELS ( LANDES BIOSCIENCE )  8 ( 3 ) 278 - 283   2014年05月

The acid-sensitive outwardly rectifying (ASOR) anion channel has been found in non-neuronal cell types and was shown to be involved in acidotoxic death of epithelial cells. We have recently shown that the ASOR channel is sensitive to temperature. Here, we extend those results to show that temperature-sensitive ASOR anion channels are expressed in cortical neurons and involved in acidotoxic neuronal cell death. In cultured mouse cortical neurons, reduction of extracellular pH activated anionic currents exhibiting phenotypic properties of the ASOR anion channel. The neuronal ASOR currents recorded at pH 5.25 were augmented by warm temperature, with a threshold temperature of 26 degrees C and the Q(10) value of 5.6. After 1 h exposure to acidic solution at 37 degrees C, a large population of neurons suffered from necrotic cell death which was largely protected not only by ASOR channel blockers but also by reduction of temperature to 25 degrees C. Thus, it is suggested that high temperature sensitivity of the neuronal ASOR anion channel provides, at least in part, a basis for hypothermic neuroprotection under acidotoxic situations associated with a number of pathological brain states.

DOI PubMed

糖尿病性腎症におけるN型Ca2+チャネル阻害の意義

大野 祥子, 横井 秀基, 笠原 正登, 森 潔, 桑原 宏一郎, 藤倉 純二, 内藤 雅喜, 桑原 孝成, 今牧 博貴, 石井 輝, 古賀 健一, 沼田 朋大, 菅原 照, 森 泰生, 柳田 素子, 中尾 一和, 向山 政志

糖尿病 ( (一社)日本糖尿病学会 )  57 ( Suppl.1 ) S - 266   2014年04月

Transnitrosylation Directs TRPA1 Selectivity in N-Nitrosamine Activators

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

MOLECULAR PHARMACOLOGY ( AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS )  85 ( 1 ) 175 - 185   2014年01月

S-Nitrosylation, the addition of a nitrosyl group to cysteine thiols, regulates various protein functions to mediate nitric oxide (NO) bioactivity. Recent studies have demonstrated that selectivity in protein S-nitrosylation signaling pathways is conferred through transnitrosylation, a transfer of the NO group, between proteins via interaction. We previously demonstrated that sensitivity to activation by synthetic NO-releasing agents via S-nitrosylation is a common feature of members of the transient receptor potential (TRP) family of Ca2+-permeable cation channels. However, strategies to confer subtype selectivity to nitrosylating agents targeted to TRP channels are yet to be developed. Here, we show selective activation of TRPA1 channels by novel NO donors derived from the ABBH (7-azabenzobicyclo[2.2.1]heptane) N-nitrosamines, which exhibit transnitrosylation reactivity to thiols without releasing NO. The NNO-ABBH1 (N-nitroso-2-exo, 3-exo-ditrifluoromethyl-7-azabenzobicyclo[2.2.1]heptane) elicits S-nitrosylation of TRPA1 proteins, and dose-dependently induces robust Ca2+ influx via both recombinant and native TRPA1 channels, but not via other NO-activated TRP channels. TRPA1 activation by NNO-ABBH1 is suppressed by specific cysteine mutations but not by NO scavenging, suggesting that cysteine transnitrosylation underlies the activation of TRPA1 by NNO-ABBH1. This is supported by the correlation of N-NO bond reactivity and TRPA1-activating potency in a congeneric series of ABBH N-nitrosamines. Interestingly, nonelectrophilic derivatives of ABBH also activate TRPA1 selectively, but less potently, compared with NNO-ABBH1. Thus, ABBH N-nitrosamines confer subtype selectivity on S-nitrosylation in TRP channels through synergetic effects of two chemical processes: cysteine transnitrosylation and molecular recognition of the nonelectrophilic moiety.

DOI PubMed

Design Methodology of Tri-Gate Poly-Si MOSFETs with 10nm Nanowire Channel to Enhance Short-Channel Performance and Reduce V-th & I-d Variability

Masumi Saitoh, Kensuke Ota, Chika Tanaka, Toshinori Numata

2014 SYMPOSIUM ON VLSI TECHNOLOGY (VLSI-TECHNOLOGY): DIGEST OF TECHNICAL PAPERS ( IEEE )    2014年

We present the optimum design of tri-gate poly-Si nanowire transistors (NW Tr.) based on the systematic performance and variability analysis for various NW width (WNW) and thickness (TSi) down to 10nm. Ion difference between poly-Si and crystalline-Si Tr. at short L (down to 25nm) is much smaller than long L due to poly-Si defect-barrier lowering by high lateral field. Ion of poly-Si pFETs is close to nFETs due to smaller interface defects. Both WNW and TSi scaling reduces S factor, SCE and Avt (Vth & Id variations) caused by random grain placement. Avt of thin poly-Si Tr. falls even below doped bulk Tr. Since short-NW Tr. suffers from high RSD and low mu, narrow and tall poly-Si NW Tr. is the best for 3D CMOS.

Development of novel activators for TRPC channels aiming to treatment of neurodegenerative disorders

Seishiro Sawamura, Masahiko Hatano, Yoshinori Takada, Takashi Kuwamura, Tomohiro Numata, Shigeki Kiyonaka, Kyosuke Hino, Tetsuya Kawamura, Jun Tanikawa, Hiroshi Nakagawa, Ryu Nagata, Ryuji Inoue, Yasuo Mori

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC )  124   181P - 181P   2014年

Novel HCN2 Mutation Contributes to Febrile Seizures by Shifting the Channel's Kinetics in a Temperature-Dependent Manner

Yuki Nakamura, Xiuyu Shi, Tomohiro Numata, Yasuo Mori, Ryuji Inoue, Christoph Lossin, Tallie Z. Baram, Shinichi Hirose

PLOS ONE ( PUBLIC LIBRARY SCIENCE )  8 ( 12 ) e80376   2013年12月

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as I-h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p. S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of I-h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of Ih during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.

DOI PubMed

Acid-sensitive outwardly rectifying (ASOR) anion channels in human epithelial cells are highly sensitive to temperature and independent of ClC-3

Kaori Sato-Numata, Tomohiro Numata, Toshiaki Okada, Yasunobu Okada

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY ( SPRINGER )  465 ( 11 ) 1535 - 1543   2013年11月

A novel type of anion channel activated by extracellular acidification, called acid-sensitive outwardly rectifying (ASOR) anion channel, was shown to be involved in acidotoxic necrotic death in human epithelial cells. However, its biophysical property and molecular identity have remained elusive. In human epithelial HeLa cells, here, whole-cell currents of ASOR anion channel were found to be augmented by warm temperature, with a threshold temperature of 32 A degrees C. Temperature sensitivity of the conductance was found to be high (with Q (10) of 8.8) in the range of body temperature, suggesting a possible involvement of a non-diffusion-limited process such as a transporter-mediated conduction. In this regard, it is interesting that a Cl-/H+ antiporter ClC-3 has recently been proposed as a candidate for the ASOR channel. However, siRNA-mediated knockdown of hClC-3 failed to suppress ASOR currents in HeLa cells. Also, endogenous ASOR currents in HEK293T cells were not affected by overexpression of human or mouse ClC-3. Furthermore, functional expression of the ASOR channel was virtually absent in the cisplatin-resistant human cancer KCP-4 cell line despite the fact that molecular expression of ClC-3 was indistinguishable between KCP-4 cells and parental cisplatin-sensitive KB-3-1 cells which endogenously exhibit high activity of ASOR anion channels. These results indicate that the ASOR anion channel is highly sensitive to temperature and independent of ClC-3.

DOI PubMed

N型Caチャネルノックアウトマウスを用いた糖尿病性腎症におけるN型カルシウムチャネルの意義

大野 祥子, 横井 秀基, 笠原 正登, 森 潔, 桑原 宏一郎, 藤倉 純二, 内藤 雅喜, 桑原 孝成, 今牧 博貴, 古賀 健一, 沼田 朋大, 菅原 照, 森 泰生, 中尾 一和, 向山 政志

日本高血圧学会総会プログラム・抄録集 ( (NPO)日本高血圧学会 )  36回   282 - 282   2013年10月

TRP channels as sensors of oxygen availability

Tomohiro Numata, Nozomi Ogawa, Nobuaki Takahashi, Yasuo Mori

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY ( SPRINGER )  465 ( 8 ) 1075 - 1085   2013年08月

An ability to adapt to changes in oxygen availability is essential for survival in both prokaryotic and eukaryotic organisms. Recently, cation channels encoded by the transient receptor potential (trp) gene superfamily have been recognized as multimodal sensors of a wide variety of factors inside the cells and in the extracellular environment and also as transducers of electrical and chemical signals mediated by ions such as Ca2+. The functional features of TRP channels enable the body to react and adapt to different forms of environmental changes, including oxygen levels. A subclass of TRP channels regulates various cellular processes in response to fluctuations in oxygen. In this article, we describe the physiological and pathological significance of the oxygen-sensitive TRP channels, which are heterogeneous in the cellular responses to acute changes in oxygen, by contrasting their oxygen monitoring function with that of other ion channels, transporters, and enzymes. We also discuss the physiological relevance of oxygen-sensitive TRP channels as a novel class of target proteins for pharmaceutical therapeutics.

DOI PubMed

糖尿病性腎症におけるN型カルシウムチャネルの意義

大野 祥子, 向山 政志, 横井 秀基, 笠原 正登, 森 潔, 藤倉 純二, 桑原 宏一郎, 内藤 雅喜, 桑原 孝成, 今牧 博貴, 石井 輝, 古賀 健一, 沼田 朋大, 菅原 照, 森 泰生, 中尾 一和

日本腎臓学会誌 ( (一社)日本腎臓学会 )  55 ( 3 ) 349 - 349   2013年04月

Targeting TRPs in Neurodegenerative Disorders

Yoshinori Takada, Tomohiro Numata, Yasuo Mori

CURRENT TOPICS IN MEDICINAL CHEMISTRY ( BENTHAM SCIENCE PUBL LTD )  13 ( 3 ) 322 - 334   2013年02月

Neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis present a significant medical challenge in the modern world. Recent evidence indicates that perturbation of neuronal Ca2+ homeostasis is involved in the pathogenesis of these neurodegenerative disorders. Transient receptor potential (TRP) channels are non-selective cation channels that are expressed in various cell types and tissues, and play an important role in regulating Ca2+ signaling in both non-neuronal and neuronal cells. TRP channels are related to the onset or progression of several diseases, and defects in the genes encoding TRP channels (so-called "TRP channelopathies") underlie certain neurodegenerative disorders due to their abnormal Ca2+ signaling properties. In this article, we review recent findings regarding the relationship between TRPs and neurodegenerative disorders, and discuss the therapeutic potential of targeting TRP channels pharmacologically.

DOI PubMed

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年

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年

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年

J-GLOBAL

Involvements of the ABC protein ABCF2 and α-actinin-4 in regulation of cell volume and anion channels in human epithelial cells.

Ando-Akatsuka Y, Shimizu T, Numata T, Okada Y

Journal of cellular physiology ( WILEY-BLACKWELL )  227 ( 10 ) 3498 - 3510   2012年10月

After osmotic swelling, cell volume is regulated by a process called regulatory volume decrease (RVD). Although actin cytoskeletons are known to play a regulatory role in RVD, it is not clear how actin-binding proteins are involved in the RVD process. In the present study, an involvement of an actin-binding protein, a-actinin-4 (ACTN4), in RVD was examined in human epithelial HEK293T cells. Overexpression of ACTN4 significantly facilitated RVD, whereas siRNA-mediated downregulation of endogenous ACTN4 suppressed RVD. When the cells were subjected to hypotonic stress, the content of ACTN4 increased in a 100,000 x g pellet, which was sensitive to cytochalasin D pretreatment. Protein overlay assays revealed that ABCF2, a cytosolic member of the ABC transporter superfamily, is a binding partner of ACTN4. The ACTN4-ABCF2 interaction was markedly enhanced by hypotonic stimulation and required the NH2-terminal region of ABCF2. Overexpression of ABCF2 suppressed RVD, whereas downregulation of ABCF2 facilitated RVD. We then tested whether ABCF2 has a suppressive effect on the activity of volume-sensitive outwardly rectifying anion channel (VSOR), which is known to mediate Cl- efflux involved in RVD, because another ABC transporter member, CFTR, was shown to suppress VSOR activity. Whole-cell VSOR currents were largely reduced by overexpression of ABCF2 and markedly enhanced by siRNA-mediated depletion of ABCF2. Thus, the present study indicates that ACTN4 acts as an enhancer of RVD, whereas ABCF2 acts as a suppressor of VSOR and RVD, and suggests that a swelling-induced interaction between ACTN4 and ABCF2 prevents ABCF2 from suppressing VSOR activity in the human epithelial cells. J. Cell. Physiol. 227: 34983510, 2012. (C) 2012 Wiley Periodicals, Inc.

DOI PubMed

Active Zone Protein Bassoon Co-Localizes with Presynaptic Calcium Channel, Modifies Channel Function, and Recovers from Aging Related Loss by Exercise

Hiroshi Nishimune, Tomohiro Numata, Jie Chen, Yudai Aoki, Yonghong Wang, Miranda P. Starr, Yasuo Mori, John A. Stanford

PLOS ONE ( PUBLIC LIBRARY SCIENCE )  7 ( 6 ) e38029   2012年06月

The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca2+ influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise.

DOI PubMed

Utilization of Photoinduced Charge-Separated State of Donor-Acceptor-Linked Molecules for Regulation of Cell Membrane Potential and Ion Transport

Tomohiro Numata, Tatsuya Murakami, Fumiaki Kawashima, Nobuhiro Morone, John E. Heuser, Yuta Takano, Kei Ohkubo, Shunichi Fukuzumi, Yasuo Mori, Hiroshi Imahori

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY ( AMER CHEMICAL SOC )  134 ( 14 ) 6092 - 6095   2012年04月

The control of ion transport across cell membranes by light is an attractive strategy that allows targeted, fast control of precisely defined events in the biological membrane, Here we report a novel general strategy for the control of membrane potential and ion transport by using charge-separation molecules and light. Delivery of charge-separation molecules to the plasma membrane of PC12 cells by a membranous nanocarrier and subsequent light irradiation led to depolarization of the membrane potential as well as inhibition of the potassium ion flow across the membrane. Photoregulation of the cell membrane potential and ion transport by using charge-separation molecules is highly promising for control of cell functions.

DOI PubMed

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

TRPM1: a vertebrate TRP channel responsible for retinal ON bipolar function.

Koike Chieko, Numata Tomohiro, Ueda Hiroshi, Mori Yasuo, Furukawa Takahisa

Cell calcium   48 ( 2-3 ) 95 - 101   2010年

The transient receptor potential (TRP) channels affect essential functions widely in sensory systems ofvarious species,both invertebratesand vertebrates. The channel protein encodedby the trp gene, the first identified TRP superfamily molecule, is known to mediate the Drosophila light response. A vertebrate TRP channel playing a crucial rolein the visual system has not yet been discovered, although numerous studies have revealed primal functions of TRP superfamily molecules in various sensory systems other than vision. In the retina, which is the entry tissue in the vertebrate visual pathway, the transduction cation channel in ON bipolar 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) subfamily to be discovered, is a visual transduction channel in retinal ON bipolar cells. This review covers the significant discoveries on the physiological function and regulatory mechanism of the TRPM1 channel in retinal ON bipolar cells and the association of human TRPM1 mutations with congenital stationary night blindness.

DOI PubMed

A Pathogenic C Terminus-truncated Polycystin-2 Mutant Enhances Receptor-activated Ca2+ Entry via Association with TRPC3 and TRPC7

Kyoko Miyagi, Shigeki Kiyonaka, Kazunori Yamada, Takafumi Miki, Emiko Mori, Kenta Kato, Tomohiro Numata, Yuichi Sawaguchi, Takuro Numaga, Toru Kimura, Yoshikatsu Kanai, Mitsuhiro Kawano, Minoru Wakamori, Hideki Nomura, Ichiro Koni, Masakazu Yamagishi, Yasuo Mori

JOURNAL OF BIOLOGICAL CHEMISTRY ( AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC )  284 ( 49 ) 34400 - 34412   2009年12月

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca2+ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca2+ influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca2+ influx that may lead to dysregulated cell growth in ADPKD.

DOI PubMed

AFTERNOON TEA

沼田 朋大

日本生理学雜誌   71 ( 6 ) 232 - 232   2009年06月

Pathophysiology and puzzles of the volume-sensitive outwardly rectifying anion channel

Yasunobu Okada, Kaori Sato, Tomohiro Numata

JOURNAL OF PHYSIOLOGY-LONDON ( WILEY-BLACKWELL )  587 ( 10 ) 2141 - 2149   2009年05月

Cell swelling activates or upregulates a number of anion channels. Of the volume-activated or -regulated anion channels (VAACs or VRACs), the volume-sensitive outwardly rectifying anion channel (VSOR) is most prominently activated and ubiquitously expressed. This channel is known to be involved in a variety of physiological processes including cell volume regulation, cell proliferation, differentiation and cell migration as well as cell turnover involving apoptosis. Recent studies have shown that VSOR activity is also involved in a number of pathophysiological processes including the acquisition of cisplatin resistance by cancer cells, ischaemia-reperfusion-induced death of cardiomyocytes and hippocampal neurons, glial necrosis under lactacidosis as well as neuronal necrosis under excitotoxicity. Moreover, VSOR serves as the pathway for glutamate release from astrocytes under ischaemic conditions and when stimulated by bradykinin, an initial mediator of inflammation. So far, many signalling molecules including the EGF receptor, PI3K, Src, PLC gamma and Rho/Rho kinase have been implicated in the regulation of VSOR activity. However, our pharmacological studies suggest that these signals are not essential components of the swelling-induced VSOR activation mechanism even though some of these signals may play permissive or modulatory roles. Molecular identification of VSOR is required to address the question of how cells sense volume expansion and activate VSOR.

DOI PubMed

Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound

Shigeki Kiyonaka, Kenta Kato, Motohiro Nishida, Kazuhiro Mio, Takuro Numaga, Yuichi Sawaguchi, Takashi Yoshida, Minoru Wakamori, Emiko Mori, Tomohiro Numata, Masakazu Ishii, Hiroki Takemoto, Akio Ojida, Kenta Watanabe, Aya Uemura, Hitoshi Kurose, Takashi Morii, Tsutomu Kobayashi, Yoji Sato, Chikara Sato, Itaru Hamachi, Yasuo Mori

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA ( NATL ACAD SCIENCES )  106 ( 13 ) 5400 - 5405   2009年03月

Canonical transient receptor potential (TRPC) channels control influxes of Ca(2+) and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca(2+) influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced Ca(2+) response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca(2+)-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.

DOI PubMed

EXPRESSION OF TRPM7 CHANNELS SWITCHES ACIDOSIS-INDUCED CELL DEATH FROM APOPTOSIS TO NECROSIS IN HUMAN EPITHELIAL CELLS

Tomohiro Numata, Yasunobu Okada

JOURNAL OF PHYSIOLOGICAL SCIENCES ( SPRINGER TOKYO )  59   153 - 153   2009年

REGULATORY ROLE OF THE INTERACTION BETWEEN ACTN4 AND ABCF2 IN CELL VOLEME REGULATION AND VOLUME-SENSITIVE ANION CHANNEL

Yuhko Ando-Akatsuka, Takahiro Shimizu, Tomohiro Numata, Yasuo Mori, Yasunobu Okada

JOURNAL OF PHYSIOLOGICAL SCIENCES ( SPRINGER TOKYO )  59   392 - 392   2009年

Selective and direct inhibition of TRPC3 channels underlies biological activities of the novel pyrazole compound

Kenta Kato, Shigeki Kiyonaka, Motohiro Nishida, Kazuhiro Mio, Tomohiro Numata, Yuichi Sawaguchi, Emiko Mori, Chikara Sato, Itaru Hamachi, Yasuo Mori

JOURNAL OF PHARMACOLOGICAL SCIENCES ( JAPANESE PHARMACOLOGICAL SOC )  109   55P - 55P   2009年

THE AUTOCRINE ROLE OF ARGININE-VASOPRESSIN RELEASED FROM OSMOSENSORY NEURONS UNDER HYPOOSMOTIC CONDITIONS

Kaori Sato, Tomohiro Numata, Yoichi Ueta, Yasunobu Okasa

JOURNAL OF PHYSIOLOGICAL SCIENCES ( SPRINGER TOKYO )  59   300 - 300   2009年

TRPチャネルの構造と多様な機能

沼田 朋大, 香西 大輔, 高橋 重成, 加藤 賢太, 瓜生 幸嗣, 山本 伸一郎, 金子 雄, 眞本 達生, 森 泰生

生化學 ( 日本生化学会 )  81 ( 11 ) 962 - 983   2009年

PubMed

新規TRPC3チャネル阻害剤の開発及び作用機序の解明

清中茂樹, 清中茂樹, 加藤賢太, 西田基宏, 三尾和弘, 澤口諭一, 沼田朋大, 佐藤主税, 浜地格, 浜地格, 森泰生, 森泰生

生化学     2009年

J-GLOBAL

Hypertonicity-induced cation channels rescue cells from staurosporine-elicited apoptosis

Tomohiro Numata, Kaori Sato, Yasunobu Okada, Frank Wehner

APOPTOSIS ( SPRINGER )  13 ( 7 ) 895 - 903   2008年07月

Cell shrinkage is one of the earliest events during apoptosis. Cell shrinkage also occurs upon hypertonic stress, and previous work has shown that hypertonicity-induced cation channels (HICCs) underlie a highly efficient mechanism of recovery from cell shrinkage, called the regulatory volume increase (RVI), in many cell types. Here, the effects of HICC activation on staurosporine-induced apoptotic volume decrease (AVD) and apoptosis were studied in HeLa cells by means of electronic cell sizing and whole-cell patch-clamp recording. It was found that hypertonic stress reduces staurosporine-induced AVD and cell death (associated with caspase-3/7 activation and DNA fragmentation), and that this effect was actually due to activation of the HICC. On the other hand, staurosporine was found to significantly reduce osmotic HICC activation. It is concluded that AVD and RVI reflect two fundamentally distinct functional modes in terms of the activity and role of the HICC, in a shrunken cell. Our results also demonstrate, for the first time, the ability of the HICC to rescue cells from the process of programmed cell death.

DOI PubMed

Molecular determinants of sensitivity and conductivity of human TRPM7 to Mg2+ and Ca2+

Tomohiro Numata, Yasunobu Okada

CHANNELS ( LANDES BIOSCIENCE )  2 ( 4 ) 283 - 286   2008年07月

It is known that extracellular Mg2+ and Ca2+ can permeate TRPM7 and at the same time block the permeation by monovalent cations. In the present study, we examined the molecular basis for the conductivity and sensitivity of human TRPM7 to these divalent cations. Extracellular acidification to pH 4.0 markedly reduced the blocking effects of Mg2+ and Ca2+ on the Cs+ currents, decreasing their binding affinities: their IC50 values increased 510-and 447-fold, respectively. We examined the effects of neutralizing each of four negatively charged amino acid residues, Glu-1047, Glu-1052, Asp-1054 and Asp-1059, within the putative pore-forming region of human TRPM7. Mutating Glu-1047 to alanine (E1047A) resulted in non-functional channels, whereas mutating any of the other residues resulted in functionally expressed channels. Cs+ currents through D1054A and E1052A were less sensitive to block by divalent cations; the IC50 values were increased 5.5- and 3.9-fold, respectively, for Mg2+ and 10.5- and 6.7-fold, respectively, for Ca2+. D1059A also had a significant reduction, though less marked compared to the reductions seen for D1054A and E1052A, in sensitivity to Mg2+ (1.7-fold) and Ca2+ (3-9-fold). The D1054A mutation largely abolished inward currents conveyed by Mg2+ and Ca2+. In the E1052A and D1059A mutants, inward Mg2+ and Ca2+ currents were sizable but significantly diminished. Thus, it is concluded that in human TRPM7, (1) both Asp-1054 and Glu-1052, which are located near the narrowest portion in the pore's selectivity filter, may provide the binding sites for Mg2+ and Ca2+, (2) Asp-1054 is an essential determinant of Mg2+ and Ca2+ conductivitv, and (3) Glu-1052 and Asp-1059 facilitate the conduction of divalent cations.

DOI PubMed

Proton conductivity through the human TRPM7 channel and its molecular determinants

Tomohiro Numata, Yasunobu Okada

JOURNAL OF BIOLOGICAL CHEMISTRY ( AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC )  283 ( 22 ) 15097 - 15103   2008年05月

TRPM7 is a divalent cation-permeable channel that is ubiquitously expressed. Recently, mouse TRPM7 has been shown to be sensitive to, and even permeable to, protons when heterologously expressed. Here we have demonstrated that human TRPM7 expressed either heterologously or endogenously also exhibits proton conductivity. The gene silencing of TRPM7 by small interfering RNA suppressed H(+) currents in human cervical epithelial HeLa cells. In HEK293T cells transfected with human TRPM7, the inward proton conductance was suppressed by extracellular Mg(2+) or Ca(2+) with IC(50) values of 0.5 and 1.9 mm, respectively. Anomalous mole fraction behavior of H(+) currents in the presence of Mg(2+) or Ca(2+) indicated that these divalent cations compete with protons for binding sites. Systematic mutation of negatively charged amino acid residues within the putative pore-forming region of human TRPM7 into the neutral amino acid alanine was tested. E1047A resulted in non-functional channels, and D1054A abolished proton conductance, whereas E1052A and D1059A only partially reduced proton conductivity. Thus, it is concluded that Asp-1054 is an essential determinant of the proton conductivity, whereas Glu-1047 might be required for channel formation, and the remaining negatively charged amino acids in the pore region (Glu-1052 and Asp- 1059) may play a facilitating role in the proton conductivity of human TRPM7. It is suggested that proton conductivity of endogenous human TRPM7 plays a role in physiologically/ pathologically acidic situations.

DOI PubMed

メカノセンサーとしてのTRPM7チャネルとその生理的役割

沼田 朋大

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 )  2008   009 - 009   2008年

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>

DOI CiNii Research

高浸透圧誘導性カチオンチャネル活性化によるヒト上皮細胞アポトーシスの救済

岡田 泰伸, ベーナー フランク, 沼田 朋大

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 )  2008   209 - 209   2008年

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>

DOI CiNii Research

A novel inhibitor of hypertonicity-induced cation channels in HeLa cells

Tomohiro Numata, Frank Wehner, Yasunobu Okada

JOURNAL OF PHYSIOLOGICAL SCIENCES ( PHYSIOLOGICAL SOC JAPAN )  57 ( 4 ) 249 - 252   2007年08月

Despite the paramount significance of hypertonicity-induced cation channels (HICCs) in cell proliferation and apoptosis, a detailed analysis of these processes is hindered by the very limited number of HICC blockers available. Here, 2-amino-ethoxydiphenyl borate (2-APB) is introduced as a novel and effective inhibitor of the HICC in HeLa cells. Its efficiency is defined with reference to flufenamic acid (as the most potent blocker so far), both, in whole-cell patch-clamp recordings and in measurements of cell volume regulation.

DOI PubMed

Impaired activity of volume-sensitive Cl- channel is involved in cisplatin resistance of cancer cells

Elbert L. Lee, Takahiro Shimizu, Tomoko Ise, Tomohiro Numata, Kimitoshi Kohno, Yasunobu Okada

JOURNAL OF CELLULAR PHYSIOLOGY ( WILEY-LISS )  211 ( 2 ) 513 - 521   2007年05月

The platinum-based drug cisplatin is a widely used anticancer drug which acts by causing the induction of apoptosis. However, resistance to the drug is a major problem. In this study we show that the KCP-4 human epidermoid cancer cell line, which serves as a model of acquired resistance to cisplatin, has virtually no volume-sensitive, outwardly rectifying (VSOR) chloride channel activity. The VSOR chloride channel's molecular identity has not yet been determined, and semi-quantitative RT-PCR experiments in this study suggested that the channel corresponds to none of three candidate genes. However, because it is known that the channel current plays an essential role in apoptosis, we hypothesized that lack of the current contributes to cisplatin resistance in these cells and that its restoration would reduce resistance. To test this hypothesis, we attempted to restore VSOR chloride current in KCP-4 cells. It was found that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, caused VSOR chloride channel function to be partially restored. Treatment of the cells with both TSA and cisplatin resulted in an increase in caspase-3 activity at 24 h and a decrease in cell viability at 48 h. These effects were blocked by simultaneous treatment of the cells with a VSOR chloride channel blocker. These results indicate that restoration of the channel's functional expression by TSA treatment leads to a decrease in the cisplatin resistance of KCP-4 cells. We thus conclude that impaired activity of the VSOR chloride channel is involved in the cisplatin resistance of KCP-4 cancer cells.

DOI PubMed

Role of acid-sensitive outwardly rectifying anion channels in acidosis-induced cell death in human epithelial cells

Hai-Yan Wang, Takahiro Shimizu, Tomohiro Numata, Yasunobu Okada

PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY ( SPRINGER )  454 ( 2 ) 223 - 233   2007年05月

Recently, a novel type of anion channel activated by extracellular acidification has been found in a variety of mammalian cell types. However, the role of this acid-sensitive outwardly rectifying (ASOR) anion channel is not known. In human epithelial HeLa cells, reduction in extracellular pH below 5 rapidly activated anion-selective whole-cell currents. The currents exhibited strong outward rectification, activation kinetics at positive potentials, low-field anion selectivity, and sensitivity to 4,4'-diisothiocya-natostilbene-2,2'-disulfonic acid (DIDS) and phloretin. When outside-out patches were exposed to acidic bathing solution, single-channel events of the anion channel could be observed. The unitary conductance was 4.8 pS in the voltage range between -80 and +80 mV. The single-channel activity prominently increased with depolarization and was suppressed by DIDS or phloretin. After 1h incubation in acidic solution (pH 4.5), a significant population of HeLa cells suffered from necrotic cell injury characterized by stainability with propidium iodide and lack of caspase-3 activation. Upon exposure to acidic solution, HeLa cells exhibited immediate, persistent swelling. Both the necrotic volume increase and cell injury induced by extracellular acidification were inhibited by DIDS or phloretin. Therefore, it is concluded that the ASOR anion channel is involved in the genesis of necrotic cell injury induced by acidosis in human epithelial cells.

DOI PubMed

TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells

Tomohiro Numata, Takahiro Shimizu, Yasunobu Okada

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY ( AMER PHYSIOLOGICAL SOC )  292 ( 1 ) C460 - C467   2007年01月

Stretch- and swelling-activated cation (SSAC) channels play essential roles not only in sensing and transducing external mechanical stresses but also in regulating cell volume in living cells. However, the molecular nature of the SSAC channel has not been clarified. In human epithelial HeLa cells, single-channel recordings in cell-attached and inside-out patches revealed expression of a Mg(2+)- and Gd(3+)-sensitive nonselective cation channel that is exquisitely sensitive to membrane stretch. Whole cell recordings revealed that the macroscopic cationic currents exhibit transient receptor potential (TRP) melastatin (TRPM)7-like properties such as outward rectification and sensitivity to Mg(2+) and Gd(3+). The whole cell cation current was augmented by osmotic cell swelling. RT-PCR and Western blotting demonstrated molecular expression of TRPM7 in HeLa cells. Treatment with small interfering RNA ( siRNA) targeted against TRPM7 led to abolition of single stretch-activated cation channel currents and of swelling-activated, whole cell cation currents in HeLa cells. The silencing of TRPM7 by siRNA reduced the rate of cell volume recovery after osmotic swelling. A similar inhibition of regulatory volume decrease was also observed when extracellular Ca(2+) was removed or Gd(3+) was applied. It is thus concluded that TRPM7 represents the SSAC channel endogenously expressed in HeLa cells and that, by serving as a swelling-induced Ca(2+) influx pathway, it plays an important role in cell volume regulation.

DOI PubMed

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 &lt;= 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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