研究者詳細 - 沼田　朋大

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

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