Research Achievements - Original paper -
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A Newly Cloned ClC-3 Isoform, ClC-3d, as well as ClC-3a Mediates Cd2+- Sensitive Outwardly Rectifying Anion Currents
Toshiaki Okada, Tenpei Akita, Kaori Sato-Numata, Md Rafiqul Islam, Yasunobu Okada
CELLULAR PHYSIOLOGY AND BIOCHEMISTRY ( KARGER ) 33 ( 3 ) 539 - 556 2014 [Refereed]
Research paper (journal) International Co-author
Background: ClC-3, a member of the ClC family, is predicted to have six isoforms, ClC-3a to -3f, with distinct N- and C-terminal amino acid sequences. There have been conflicting reports on the properties of ClC-3a (also known as the N- terminal short form of ClC-3) and ClC-3b (the N- terminal long form of ClC-3) as plasmalemmal Cl-channels. Meanwhile, little is known about other isoforms. The amino acid sequence of ClC-3d (a C-terminal variant of the short form) listed in the NCBI database was derived from the genomic sequence, but there has been no experimental evidence for the mRNA. Methods: PCR-cloning was made to obtain the full coding region of ClC-3d from mouse liver. Its molecular expression on the plasma membrane was microscopically examined in HEK293T cells transfected with GFP-tagged ClC3d. Its functional plasmalemmal expression and the properties of currents were studies by whole-cell recordings in the cells transfected with ClC-3d. Results: The cloned ClC-3d was found to be the only isoform which has an N- terminal amino acid sequence identical to ClC3a. When introduced into HEK293T cells, a minor fraction of exogenous ClC-3d proteins was detected at the plasma membrane, and activation of anion currents was observed at neutral pH under normotonic conditions. The properties of ClC-3d currents were found to be shared by ClC-3a-mediated currents. Also, both ClC-3d and -3a currents were found to be sensitive to Cd2+. ClC-3d overexpression never affected the endogenous activity of acid- or swelling-activated anion channels. Conclusion: We thus conclude that plasmalemmal ClC-3d, like ClC3a, mediates Cd2+- sensitive outwardly rectifying anion currents and that ClC-3d is distinct from the molecular entities of acid- and volume-sensitive anion channels.
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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 2013.11 [Refereed]
Research paper (journal) Domestic Co-author
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.
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TMEM16F is a component of a Ca2+-activated Cl- channel but not a volume-sensitive outwardly rectifying Cl- channel
Takahiro Shimizu, Takahiro Iehara, Kaori Sato, Takuto Fujii, Hideki Sakai, Yasunobu Okada
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY ( AMER PHYSIOLOGICAL SOC ) 304 ( 8 ) C748 - C759 2013.04 [Refereed]
Research paper (journal) Domestic Co-author
Shimizu T, Iehara T, Sato K, Fujii T, Sakai H, Okada Y. TMEM16F is a component of a Ca2+-activated Cl- channel but not a volume-sensitive outwardly rectifying Cl- channel. Am J Physiol Cell Physiol 304: C748-C759, 2013. First published February 20, 2013; doi:10.1152/ajpcell.00228.2012.-TMEM16 (transmembrane protein 16) proteins, which possess eight putative transmembrane domains with intracellular NH2- and COOH-terminal tails, are thought to comprise a Cl- channel family. The function of TMEM16F, a member of the TMEM16 family, has been greatly controversial. In the present study, we performed whole cell patch-clamp recordings to investigate the function of human TMEM16F. In TMEM16F-transfected HEK293T cells but not TMEM16K- and mock-transfected cells, activation of membrane currents with strong outward rectification was found to be induced by application of a Ca2+ ionophore, ionomycin, or by an increase in the intracellular free Ca2+ concentration. The free Ca2+ concentration for half-maximal activation of TMEM16F currents was 9.6 mu M, which is distinctly higher than that for TMEM16A/B currents. The outwardly rectifying current-voltage relationship for TMEM16F currents was not changed by an increase in the intracellular Ca2+ level, in contrast to TMEM16A/B currents. The Ca2+-activated TMEM16F currents were anion selective, because replacing Cl- with aspartate(-) in the bathing solution without changing cation concentrations caused a positive shift of the reversal potential. The anion selectivity sequence of the TMEM16F channel was I- > Br- > Cl- > F- > aspartate(-). Niflumic acid, a Ca2+-activated Cl- channel blocker, inhibited the TMEM16F-dependent Cl- currents. Neither overexpression nor knockdown of TMEM16F affected volume-sensitive outwardly rectifying Cl- channel (VSOR) currents activated by osmotic swelling or apoptotic stimulation. These results demonstrate that human TMEM16F is an essential component of a Ca2+-activated Cl- channel with a Ca2+ sensitivity that is distinct from that of TMEM16A/B and that it is not related to VSOR activity.
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Early-phase occurrence of K+ and Cl- efflux in addition to Ca2+ mobilization is a prerequisite to apoptosis in HeLa cells
Katsuya Dezaki, Emi Maeno, Kaori Sato, Tenpei Akita, Yasunobu Okada
APOPTOSIS ( SPRINGER ) 17 ( 8 ) 821 - 831 2012.08 [Refereed]
Research paper (journal) Domestic Co-author
Sustained rise in cytosolic Ca2+ and cell shrinkage mainly caused by K+ and Cl- efflux are known to be prerequisites to apoptotic cell death. Here, we investigated how the efflux of K+ and Cl- as well as the rise in cytosolic Ca2+ occur prior to caspase activation and are coupled to each other in apoptotic human epithelial HeLa cells. Caspase-3 activation and DNA laddering induced by staurosporine were abolished by blockers of K+ and Cl- channels or cytosolic Ca2+ chelation. Staurosporine induced decreases in the intracellular free K+ and Cl- concentrations ([K+](i) and [Cl-](i)) in an early stage prior to caspase-3 activation. Staurosporine also induced a long-lasting rise in the cytosolic free Ca2+ concentration. The early-phase decreases in [K+](i) and [Cl-](i) were completely prevented by a blocker of K+ or Cl- channel, but were not affected by cytosolic Ca2+ chelation. By contrast, the Ca2+ response was abolished by a blocker of K+ or Cl- channel. Strong hypertonic stress promptly induced a cytosolic Ca2+ increase lasting > 50 min together with sustained shrinkage and thereafter caspase-3 activation after 4 h. The hypertonic stress induced slight increases in [K+](i) and [Cl-](i) in the first 50 min, but these increases were much less than the effect of shrinkage-induced condensation, indicating that K+ and Cl- efflux took place. Hypertonicity induced caspase-3 activation that was prevented not only by cytosolic Ca2+ chelation but also by K+ and Cl- channel blockers. Thus, it is concluded that not only Ca2+ mobilization but early-phase efflux of K+ and Cl- are required for caspase activation, and Ca2+ mobilization is a downstream and resultant event of cell shrinkage in both staurosporine- and hypertonicity-induced apoptosis.
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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 2012.02 [Refereed]
Research paper (journal) International Co-author
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.
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Endogenous arginine vasopressin-positive retinal cells in arginine vasopressin-eGFP transgenic rats identified by immunohistochemistry and reverse transcriptase-polymerase chain reaction
Satoru Moritoh, Kaori Sato, Yasunobu Okada, Amane Koizumi
MOLECULAR VISION ( MOLECULAR VISION ) 17 ( 349-53 ) 3254 - 3261 2011.12 [Refereed]
Research paper (journal) Domestic Co-author
Purpose: Recently, arginine vasopressin (AVP) has been revealed to have diverse functional roles in nervous tissues beyond that of a vasoconstrictor. Several previous studies have indicated the existence of AVP in the retina, but the source of AVP has not been determined. The objective of the present study was to address the question of whether retinal cells have the ability to synthesize endogenous AVP to act in a paracrine or autocrine manner.
Methods: We used AVP-eGFP transgenic rats to find endogenous AVP-positive cells in the retina by immunohistochemistry with an AVP antibody and a GFP antibody. We also examined AVP mRNA and AVP receptor genes by reverse transcriptase (RT)-PCR of dissociated GFP-positive cells and whole retinas.
Results: Endogenous AVP-positive cells were found in the ganglion cell layer and inner nuclear layer of the retina of AVP-eGFP transgenic rats by immunohistochemistry. As indicated by the results of RT-PCR of dissociated GFP-positive cells, these cells have the ability to synthesize endogenous AVP, as well as transgenic AVP-eGFP. In addition, the V1a and V1b AVP receptors were found in the wild-type rat retina by whole retina RT-PCR, but the V2 receptor was not detectable. In dissociated AVP-eGFP-positive cells, no AVP receptor was detected by RT-PCR. Moreover, AVP secretion was not detected by stimulation with a high potassium (50 mM) solution.
Conclusions: In the rat retina, we found retinal cells that have the ability to synthesize endogenous AVP, and that the retina possesses V1a and V1b AVP receptors. Taken together, these results suggest that the retina has an intrinsic AVP-synthesizing and -receiving mechanism that can operate in a paracrine manner via V1a and V1b receptors. -
V2 Receptor-Mediated Autocrine Role of Somatodendritic Release of AVP in Rat Vasopressin Neurons Under Hypo-Osmotic Conditions
Kaori Sato, Tomohiro Numata, Takeshi Saito, Yoichi Ueta, Yasunobu Okada
SCIENCE SIGNALING ( AMER ASSOC ADVANCEMENT SCIENCE ) 4 ( 157 ) ra5 2011.01 [Refereed]
Research paper (journal) Domestic Co-author
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 V-2-type vasopressin receptor. The V-2 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 V-2 receptor as an autocrine signal to enhance volume-sensitive anion channel activity and thereby facilitate cell volume regulation.
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Acid-sensing ion channels in rat hypothalamic vasopressin neurons of the supraoptic nucleus
Toyoaki Ohbuchi, Kaori Sato, Hideaki Suzuki, Yasunobu Okada, Govindan Dayanithi, David Murphy, Yoichi Ueta
JOURNAL OF PHYSIOLOGY-LONDON ( WILEY-BLACKWELL ) 588 ( 12 ) 2147 - 2162 2010.06 [Refereed]
Research paper (journal) International Co-author
Body fluid balance requires the release of arginine vasopressin (AVP) from the neurohypophysis. The hypothalamic supraoptic nucleus (SON) is a major site of AVP synthesis, and AVP release is controlled somatodendritically or at the level of nerve terminals by electrical activities of magnocellular neurosecretory cells (MNCs). Acid-sensing ion channels (ASICs) are neuronal voltage-insensitive cationic channels that are activated by extracellular acidification. Although ASICs are widely expressed in the central nervous system, functional ASICs have not been assessed in AVP neurons. ASICs are modulated by lactate (La-), which reduces the extracellular calcium ion concentration. We hypothesize that ASICs modify neuronal function through La- that is generated during local hypoxia resulting from osmotic stimulation in the SON. In the present study, we used the whole-cell patch-clamp technique to show that acid-induced ASIC current is enhanced by La- in isolated rat SON MNCs that express an AVP-enhanced green fluorescent protein (eGFP) transgene. Immunohistochemistry and multi-cell reverse transcriptase-polymerase chain reaction experiments revealed that these neurons express the ASIC1a and ASIC2a subunits. In addition, increased La- production was specifically observed in the SON after osmotic stress. These results suggest that interaction between ASICs and La- in the SON plays an important role in the regulatory mechanism of body fluid homeostasis.
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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 2009.05 [Refereed]
Research paper (journal) Domestic Co-author
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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Octopaminergic modulation of the single Ca2+ channel currents in Kenyon cells isolated from the mushroom body of the cricket brain
K. Kosakai, K. Satoh, M. Yoshino
JOURNAL OF INSECT PHYSIOLOGY ( PERGAMON-ELSEVIER SCIENCE LTD ) 54 ( 12 ) 1479 - 1486 2008.12 [Refereed]
Research paper (journal) Domestic Co-author
Octopamine plays an important role in mediating reward signals in olfactory learning and memory formation in insect. However, its target molecules and signaling pathways are still unknown. In this study, we investigated the effects of octopamine on the voltage-activated Ca2+ channels expressed in native Kenyon cells isolated from the mushroom body of the cricket(Gryllus bimaculatus) brain. The cell-attached patch clamp recordings with 100 niM Ba2+ outside showed the presence of dihydropyridine (DHP) sensitive L-type Ca2+ channels with a single channel conductance of approximately 21 +/- 2 pS (n = 12). The open probability (NPo) of single Ca2+ channel currents decreased by about 29 +/- 7% (n = 6) by bath application of 10 mu M octopamine. Octopamine-induced decrease in Po was imitated by bath application of 8-Br-cAMP, a membrane-permeable cAMP analog. Pre-treatment of Kenyon cells with the octoparnine receptor antagonist phentolamine blocked the inhibitory effect of octoparnine on Ca2+ channels. Pretreatment of Kenyon cells with H-89, a selective inhibitor of cAMP-dependent protein kinase (PKA) attenuated the inhibitory effect of bath applied octoparnine on Ca2+ channels. These results indicate that DHP-sensitive L-type Ca2+ channel is a target protein for octoparnine and its modulation is mediated via cAMP and PKA-dependent signaling pathways in freshly isolated Kenyon cell in the cricket G. bimaculatus. (C) 2008 Elsevier Ltd. All rights reserved.
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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 2008.07 [Refereed]
Research paper (journal) International Co-author
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.