一般演題（ポスター）

一般演題（ポスター） Ion Channels・Receptors

1P-01 The 5-HT3 receptor is essential for exercise-induced hippocampal neurogenesis and antidepressant effects Kondo Makoto，Nakamura Yukiko，Ishida Yusuke，Shimada Shoichi Department of Neuroscience and Cell Biology, Osaka University Exercise has a variety of beneficial effects on brain structure and function, such as hippocampal neurogenesis, mood and memory. Previous studies have shown that exercise enhances hippocampal neurogenesis, induces antidepressant effects, and improves learning behavior. Brain serotonin（5-hydroxytryptamine, 5-HT）levels increase following exercise, and the 5-HT system has been suggested to play an important role in these exercise-induced neuronal effects. However, the precise mechanism remains unclear. In this study, analysis of the 5-HT type 3A receptor subunit-deficient（htr3a-/-）mice revealed that lack of the 5-HT type 3（5-HT3）receptor resulted in loss of exercise-induced hippocampal neurogenesis and antidepressant effects, but not of learning enhancement. Furthermore, stimulation of the 5-HT3 receptor promoted neurogenesis. These findings demonstrate that the 5-HT3 receptor is the critical target of 5-HT action in the brain following exercise, and is indispensable for hippocampal neurogenesis and antidepressant effects induced by exercise. This is the first report of a pivotal 5-HT receptor subtype that plays a fundamental role in exercise-induced morphological changes and psychological effects.		1P-02 Involvement of calcium-activated potassium channel in the inhibitory network oscillation in the rat basolateral amygdala Hashizume Miki，Shinozaki Rina，Murakoshi Takayuki Department of Biochemistry, Faculty of Medicine, Saitama Medical University Basolateral amygdaloid complex（BLA）is deeply involved in emotional processing and is sensitive to chronic stress. Its abnormality is related to several psychiatric disorders. In the BLA, sensory information from the cortex and the thalamus are evaluated in terms of emotional valence and these signals are transmitted to central nucleus of amygdala. Previously, we showed that projection neurons in BLA receive rhythmic inhibitory inputs which are evoked by synchronous firings of interneurons. Such neurons were mostly distributed in ventral part of BLA, and the inhibitory oscillation requires the glutamatergic transmission within BLA, suggesting that local network activities are essential for this phenomenon. Moreover, we demonstrated that gap-junctions, several receptors and ion-channels including the low-threshold Ca channel are involved in the generation and/or maintenance of the oscillation, also suggesting the potential role of the calcium-activated potassium channels. In fact, SK channel is involved in amygdala function, suggested by the study that an injection of its activator contributed to recovery from electrophysiological and behavioral alterations by chronic restraint stress. In the present study, we examined the effect of modulators of the calcium-activated potassium channel on inhibitory network oscillation in rat BLA. In BLA projection neurons in vitro, the power of the low-frequency（0.1-3 Hz）oscillation was enhanced by an SK channel blocker, apamin, and was attenuated by an SK channel activator, 1-EBIO, respectively. On the other hand, the oscillation power was insensitive to a BK channel blocker, iberiotoxin. These results suggest that the slow network oscillation is regulated by SK channel activity.

1P-03 Regulation of IP3 Receptor by Transglutaminase Hamada Kozo¹，Terauchi Akiko¹，Nakamura Kyoko²，Higo Takayashu¹，Nukina Nobuyuki²，Matsumoto Nagisa¹，Hisatsune Chihiro¹，Nakamura Takeshi²，Mikoshiba Katsuhiko¹ ¹RIKEN，²Juntendo University Reversible and repetitive structural changes are essential for ligand-gated ion channels to mediate biological signaling. Here we show a new mode of posttranslational modification that chronically controls the structural changes in the ligand-gated ion channels. The inositol 1,4,5-trisphosphate receptor（IP3R）in the endoplasmic reticulum assembles ligand-gated ion channels that mediate calcium signaling. IP3Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP3 at a distance. Defective allostery in IP3R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. We demonstrate that a pleiotropic enzyme transglutaminase type 2（TG2）targets the allosteric coupling domain of IP3R type 1（IP3R1）and negatively regulates IP3R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of Gln2746 residue which TG2 tethers to the adjacent subunit. Modification of Gln2746 and IP3R1 function was observed in Huntington’s disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels which relate to disease states. This is the first demonstration of transglutaminase-catalyzed posttranslational modification in ligand-gated ion channel allostery and provides a new framework for enzymatic regulation of ligand-gated ion channels.		1P-04 Roles of acid-sensing ion channel-1a in hippocampal adult neurogenesis Kumamoto Natsuko，Hoshikawa Mariko，Shibata Yasuhiro，Ueda Takashi，Ugawa Shinya Dept. of Neurobio. and Anat. Grad. Sch. of Med. Sci., Nagoya City Univ. ASIC1a（acid-sensing ion channel-1a）is a neuronal acid-activated cation channel located in the postsynaptic membrane. The channel receives synaptic protons, contributing to synaptic plasticity, learning and memory. It is well known that adult neurogenesis is enhanced after ischemic brain injury accompanied with local tissue acidosis. However, the relationship between adult neurogenesis and ASIC1a remains to be elucidated. To examine the potential roles of ASIC1a in mouse adult hippocampal neurogenesis, we used an onco-retrovirus-mediated approach to genetically label and manipulate newborn dentate granule cells（DGCs）in vivo. Three-dimensional reconstruction of confocal Z-stucks was applied to morphologically characterize the dendritic arborization and spine formation of ASIC1a-deficient newborn DGCs labeled with EGFP. We found that newborn DGCs of ASIC1a KO mouse at 28dpi（days post injection）had significantly shorter dendrites and smaller spine heads. Furthermore, retroviral shRNA knockdown of ASIC1a in newborn DGCs induced the same phenotype as the ASIC1a KO mouse. Our results indicate that ASIC1a is necessary for dendritic development and synaptic organization of mouse hippocampal newborn neurons.

1P-05 Study about the mechanisms of DHA-induced enhancement of glial excitatory amino-acid transporter EAAT2 function Hoshikawa Kazue，Takahashi Kanako，Irie Tomohiko，Sekino Yuko，Saito Kaoru Div Pharmacol, NIHS EAAT2 is a predominant astrocytic L-Glutamate（L-Glu）transporter in the forebrain. EAAT2 removes L-Glu from synaptic cleft and maintain efficient synaptic transmissions. Recent studies have clarified that poly-unsaturated fatty acids（PUFAs）regulate the functions of the membrane proteins of neural cells. Omega-3 fatty acid docosahexanoic acid（DHA；C22：6）is a major constituent of astrocyte membrane phospholipids and is released after L-Glu stimulation. However, its effects on EAAT2 is largely unknown. In this study, we investigated the effects of DHA on EAAT2 currents using two-electrode voltage clamp technique in Xenopus oocytes expressing EAAT2 isoform 1. Exogenously-applied DHA（30-300 microM）increased the amplitude of L-Glu-induced currents of EAAT2 but not of EAAT1 in a dose-dependent manner and these effects were reversible. Exogenously-applied DHA-CoA, a membrane impermeable DHA-analog, increased EAAT2 currents, suggesting that DHA regulate EAAT2 from extracellular side of membrane. PUFAs are also reported to regulate membrane protein functions by changing the elasticity of the lipid bilayer. However, transient application of Triton X-100（200 microM）, which increases membrane fluidity, had little effects on EAAT2 currents, suggesting that the DHA-induced enhancement of EAAT2 currents is independent of the membrane elasticity. The enhancement of EAAT2 was not prevented by the inhibitors of cyclooxygenase or lipoxygenase, suggesting that DHA may exert the effects through direct interaction with EAAT2 not through signal transduction pathways. DHA has a carboxyl group that is protonated（uncharged）, or deprotonated（negatively charged）in a pH dependent manner. When the extracellular pH increased, the enhancement of EAATs by DHA was increased, suggesting that the charge of the DHA carboxyl group is important for the effects of DHA. In accordance with this, DHA-methyl ester（200 microM）, an uncharged DHA-analog, had little effects on EAAT2 currents. Currently, we are identifying which structure or domain of EAAT2 is important for the enhancement of EAAT2 currents using various chimeras of EAAT2 and EAAT1.		1P-06 Phosphorylation of serotonin 1A receptor（5HT1AR）by Cdk5 activity. Takahashi Miyuki，Saito Taro，Asada Akiko，Hisanaga Shinichi Dept. of Biol. Sci., Tokyo Met. Univ. Mental disorders including depression are one of urgent issues to be addressed. To prevent the onset and develop the treatment, it is important to understand a mechanism of diseases at molecular level. It is generally considered that dysregulation of neuronal activities is an underlying mechanism. Cyclin-dependent kinase 5（Cdk5）is a neuron-specific Ser/Thr kinase, which is activated by regulatory subunit p35 or p39. Recent reports suggest its function in synaptic activity and association with anxiety and depression. I investigated here a role of Cdk5-p35 in mental disorders by focusing on serotonin 1A receptor（5HTlAR）. 5HTlAR is expressed highly in central nervous system and is thought to be involved in psychiatric activity. 5HT1AR is a seven transmembrane G-protein-coupled receptor, which binds to Gi or Go of trimeric G proteins to inhibit adenylyl cyclase or open K+ channels in neurons. Dysfunction of the serotonin signal is considered as the cause of many mental diseases. So, 5HT1AR has been a target of drug development for anxiety and depression. It is not fully known, however, how 5HTlAR is regulated. There are three possible Cdk5 phosphorylation sites in 5HT1AR. We examined phosphorylation of 5HT1AR by Cdk5-p35. Expression level of 5HT1AR was decreased by co-transfection with Cdk5-p35, but not with kinase negative Cdk5-p35, in COS-7 cells. 5HT1AR was indeed phosphorylated by Cdk5-p35. We constructed non-phosphorylatable Ala mutants, T149A, S245A, and T314A, and examined their phosphorylation. Thr314 was identified as a phosphorylation site in 5HT1AR. These results suggest that Cdk5 controls the serotonin signal through phosphorylation-dependent down regulation of 5HT1AR.

1P-07 Melanin-concentrating hormone-mediated signaling induces reduction of the primary cilium length Yamato Shogo，Hamamoto Akie，Kobayashi Yuki，Saito Yumiko Graduate School of Integrated Arts and Sciences, Hiroshima University Primary cilia are microtubule-based organelles present on nearly every cell in the mammalian body. The cilium has an important chemosensory function in many types of cells and ciliary dysfunction is associated with ciliopathies such as polycystic kidney disease and obesity. Although the ciliary membrane is contiguous with the plasma membrane, ciliary localization of protein is tightly regulated and only certain molecules are permitted to traffic there. Melanin-concentrating hormone（MCH）is the natural peptide ligand for two G-protein-coupled receptors（GPCR）, MCHR1 and MCHR2. The MCH-MCHR1 system has been implicated in the regulation of feeding and emotional processing in rodents. Recently, MCHR1 expression was detected in primary cilia of the central nervous system. However, the underlying function and signaling pathway via MCHR1 located in primary cilia is unclear. Here we show that treatment of MCH significantly reduces cilia length in hTERT-RPE1 epithelial cells（hRPE1）transfected with Flag-MCHR1. Quantitative analysis indicated that the rate of MCH-induced cilia shorting progressed in time-dependent manner during the first 3 hour with an EC50 value of 1.6nM, and the process was significantly inhibited by pretreatment with Gi/o-selective inhibitor pertussis toxin. In addition, a series of receptor mutagenesis experiment showed that distinct amino acid residues in the second intracellular loop were responsible for MCH-mediated shorting of receptor-positive cilia. These data suggests that MCH-MCHR1 governs the sensitivity by controlling the length of the cell’s sensory organelle. Further characterization of MCHR1 as a ciliary GPCR provides a potential molecular mechanism to link defects in cilia with obesity.		1P-08 Identification and appreciation of novel antagonists of GPR173 Yanai Toshihiro¹，Anton N. Shuvaev¹，Konno Ayumu²，Hirai Hirokazu²，Kurosawa Aya¹，Saito Tamio³，Takeda Shigeki¹ ¹Faculty of Sci. and Tech., Gunma Univ.，²Dept. Med., Gunma Univ.，³RIKEN G protein-coupled receptors（GPCRs）are integral membrane proteins in the cell surface, and are known to be targets of approximately 60% of the present drugs. Thus, identification of novel synthetic ligands for GPCRs, especially for orphan GPCRs of which endogenous ligands are unknown, is important for not only to our understanding of human physiology but also to the development of novel drugs. We have been developed the ligand screening system for GPCRs using receptor-Gα fusion proteins and[35S]GTPγS binding assay. One of the orphan GPCR subfamily, Super conserved Receptor Expressed in Brain（SREB）, is consisted of GPR27, GPR86, and GPR173, and specifically express in central nervous system. To examine SREB physiological functions, we performed screening of chemical compound using a RIKEN chemical library containing approximately 12,000 compounds. The binding of[35S]GTPγS to GPR173-Gsα fusion proteins expressed in Sf9 cells was measured, and resulted a few potential candidates of a GPR173 antagonist. These compounds are also antagonist for other SREBs, GPR27 and GPR85. These results also indicated that GPR173 was possible to couple Gs type G proteins, and had significant constitutive activity. These results also indicated that GPR173 was possible to couple Gs type G proteins, and had significant constitutive activity. Then, physiological significance of these compounds on the synaptic transmission was examined by whole cell patch clamp technique. Bath application of these compounds significantly increased the amplitude of EPSC elicited in cerebellar Purkinje cells after electrical stimulation to parallel fibers. In addition, the ratio of paired pulse facilitation of the parallel fiber-mediated EPSC was significantly decreased. These results suggest that constitutive active SREBs regulate the synaptic transmission at parallel fiber to Purkinje cells by modulating the glutamate release from parallel fiber terminals.

1P-09 Functional characterization of the phosphorylation sites of rat melanin-concentrating hormone receptor 1 Mitsuyama Seika，Hamamoto Akie，Kobayashi Yuki，Saito Yumiko Graduate School of Integrated Arts and Sciences, Hiroshima University Melanin-concentrating hormone（MCH）receptor 1 belongs to the rhodopsin family of G protein-coupled receptors（GPCR）. One of the main pharmacological interests of MCH-MCHR1 system resides in its ability to regulate feeding and energy homeostasis. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating GPCR activity. However, until now, only little information concerning the role of MCHR1 phosphorylation is available. In this study, we performed a comprehensive site-directed mutagenesis to analyze the predicted phosphorylation site of the rat MCHR1 in receptor expression, signaling, internalization, and trafficking in a HEK293T cell line. We identified the phosphorylation sites responsible for internalization（S151 and S158）at the second intracellular loop of the receptor, and additional sites involved in internalization（S246 and T251）at the third intracellular loop of the receptor. Although these four amino acid residues of MCHR1 play a critical role for promoting optimal internalization, they are not essential for signal transduction in calcium mobilization. A further goal of our research will be to reveal the coordinated biochemical mechanism involving sequential and hierarchical multisite phosphorylation of the receptor.