研究等業績 - その他 - 沼田 朋大
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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.
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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.
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AFTERNOON TEA
沼田 朋大
日本生理学雜誌 71 ( 6 ) 232 - 232 2009年06月
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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.
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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.
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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
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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
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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
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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
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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
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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
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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.
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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.
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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.
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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>
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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>
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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.
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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.
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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.
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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.