イオンチャンネル、興奮性膜 Ion Channels and Excitable Membranes
P1-1-1 一次聴覚皮質第6層でのアセチルコリンによる神経細胞種特異的な活動電位制御 Acetylcholine differentially regulates action potentials of layer 6 neurons in a cell type-specific manner in primary auditory cortex ○三枝拓矢1, 川井秀樹1 ○Takuya Mieda1, Hideki D Kawai1 創価大学大学院　工学研究科　生命情報工学専攻1 Department of Bioinformatics, Graduate school of Engineering, Soka University1 Layer 6 neurons of primary auditory cortex (A1) control cortical gain of auditory information by providing feedback to primary thalamic neurons in the ventral division of medial geniculate nucleus (MGv) and by regulating thalamo-recipient neurons in the middle layer of A1. Since cholinergic inputs to A1 regulate receptive fields of MGv neurons, cholinergic modulation of cortical neurons in layer 6 will be critical for gating auditory information. Little is known, however, about cellular mechanisms of cholinergic regulation in layer 6 neurons. Here we investigate if and how tonic application of ACh regulates neuronal excitability in layer 6 of mouse A1 using the whole-cell patch-clamp recording technique. Based on action potential (spike) firing properties in response to square pulse current injections and a post-recording immunofluorescent staining against GAD67, an inhibitory neuron marker, we divided layer 6 neurons into three types: regular spiking (RS), inhibitory RS (iRS), and fast spiking (FS) neurons. In all cell types, tonic application of ACh (<100 μM, ~5 min) increased resting membrane potentials, input resistance, and spike number while decreasing the onset latency of initial spikes. Cell type-specific effects were also observed. In RS neurons, ACh decreased both initial spike threshold and fast afterhyperpolarization (fAHP) amplitudes, but had little effects on peak amplitudes and spike half-width. In iRS neurons, ACh decreased spike threshold and peak amplitudes but increased fAHP amplitudes and spike half-width. In FS neurons, ACh had little effects on spike threshold, peak amplitudes, and spike half-width, but decreased fAHP amplitudes. Overall, ACh increased the excitability of layer 6 neurons by increasing resting membrane potentials and input resistance, but its modulation of spike properties differs depending on neuronal types.	P1-1-2 The Persistent Sodium Current in Rat Locus Coeruleus Neurons ○Chin-Lin Chen1,2, Ming-Yuan Min1,2, Hsiu-Wen Yang3 Graduate Institute of Brain and Mind Sciences, National Taiwan University1, Institute of Zoology, National Taiwan University2 Locus coeruleus (LC) neurons provide norepinephrine to many brain area, which is important in many cognitive functions, including sleep-awake cycle, alertness attention, pain, etc. Thus, investigating the regulation of LC neurons is essential to understand how LC-NE system are regulated. Here we reported the persistent voltage-gated sodium current (INaP) could be a major player for generating spontaneous firing in LC neurons. We recorded membrane currents responded to a voltage-ramp from 30mV to -100mV (slope=10 mV/s) in control and in the presence of 1μM tetrodotoxin (TTX); subtracting recording in TTX from that in control condition yielded inward INaP, the activation threshold and peak amplitude of which were about -50 mV and -144±22 pA (n=18). The activation curve of INaP was fitted with Boltzmann's equation with estimated activation slope and half-activation voltage being 7.8±0.6 and -21±1.7 mV, respectively. By using noninvasive membrane potential (Vm) measurement, we estimated resting Vm of LC neurons without interrupting ion compositions of the recorded neurons. Averaged Vm from 17 neurons was -51±1.5 mV. These results show that Vm and activation threshold of INaP are about the same value. These features together with the high activation slope of INaP could allow the self-generation of AP in LC neurons. In current clamp recording, most LC neurons showed sub-threshold voltage-oscillation, which was blocked by carbenoxolone, showing the activity was generated by action currents of discharging neighboring cells through gap junctions. This voltage-oscillation could further enhance activation of INaP and the generation of AP in recorded LC neuron.
P1-1-3 側坐核中型有棘ニューロンのスパイク発火に対するコリン作動性調節 Cholinergic interneurons suppress action potential initiation of medium spiny neurons in rat nucleus accumbens shell ○鰕原賀子1,2, 山本清文1, 植田耕一郎2, 越川憲明1, 小林真之1 ○Katsuko Ebihara1,2, Kiyofumi Yamamoto1, Koichiro Ueda2, Noriaki Koshikawa1, Masayuki Kobayashi1 日本大学歯学部薬理学教室1, 日本大学歯学部摂食機能療法学講座2 Department of Pharmacology, Nihon University School of Dentistry, Tokyo1, Department of Dysphagia Rehabilitation, Nihon University School of Dentistry, Tokyo2 Acetylcholine plays a crucial role in the regulation of neural functions in the nucleus accumbens (NAc) shell. It is not clear how intrinsic acetylcholine released from NAc cholinergic interneurons regulates the neural activity of MS neurons. To explore the cholinergic effects on the subthreshold responses and action potential properties of MS neurons in the NAc shell, we first tested the effects of carbachol (CCh), a non-selective cholinergic agonist, on MS neuronal activity. Then, we tested the effects of the activation of cholinergic interneurons on the electrophysiological properties of MS neurons via multiple whole-cell patch-clamp recordings. Bath application of CCh induced resting membrane potential depolarization accompanied by an increase in the voltage response to negative current injection. The pre-application of M1 muscarinic receptor antagonist blocked these effects. Repetitive spike firing of a cholinergic interneuron following positive current injection induced a similar increase in the rheobase, which delayed the action potential initiation in 38.9% MS neurons. In contrast to the bath application of CCh, cholinergic interneuronal stimulation had little effect on the resting membrane potential in MS neurons. These results suggest that the acetylcholine released from a cholinergic interneuron is sufficient to suppress the repetitive spike firing of the adjacent MS neurons, although the depolarization of the resting membrane potential may require simultaneous activation of multiple cholinergic interneurons.	P1-1-4 一次聴覚皮質第3／4層錐体細胞におけるコリン作動性反応の視覚剥奪による下降調節 Visual deprivation downregulates cholinergic responsiveness in layer III/IV pyramidal neurons of primary auditory cortex ○鈴木俊雄1, 川井秀樹1 ○Toshio Suzuki1, Hideki D Kawai1 創価大学大学院　工学研究科　生命情報工学専攻1 Department of Bioinformatics, Graduate School of Engineering, Soka University1 Blind individuals who lost eye sight at birth or during infancy have highly developed abilities of sound perception such as better sound localization and longer audible distance. Paradoxically, however, neuronal activities in auditory cortex in response to pure tones or frequency-modulated sounds are much reduced in the blind than in sighted individuals. The reduced activities could involve changes in neuronal properties in primary auditory cortex (A1). Here, we investigated neuronal excitability and its regulation by acetylcholine (ACh) in pyramidal neurons of layer III/IV , thalamocortical input layers, in a mouse model of early blindness. We enucleated both eyes at the time of eye opening (postnatal days 14-15). Control mice received sham surgery except enucleation. After raising animals for 8-12 days, we prepared auditory thalamocortical slices and recorded neuronal membrane potentials in response to square pulses in the current-clamp mode using the whole-cell patch-clamp recording technique. Comparison of intrinsic membrane properties between control and blind mice showed little difference in resting membrane potentials (RMPs), input resistance and membrane time constants. No difference in onset latency for initial spikes, spike threshold, peak amplitudes, and spike half-width was observed. Tonic application of ACh (<100 μM) on these parameters revealed striking differences in cholinergic regulation of neuronal excitability. ACh increased input resistance in control mice, but not in blind mice. This difference was apparent in ACh-induced elevation in RMPs, where acute elevation of RMPs associated with increased input resistance was not observed in blind mice. ACh reduced peak amplitudes and widened spike width in control, but not in blind mice. ACh affected other parameters, but no difference was observed between animals. Thus, visual deprivation affects some aspects of cholinergic regulatory mechanisms in primary auditory cortex.
P1-1-5 アロマ精油成分は化学構造特異的に蛙坐骨神経の複合活動電位を抑制する Frog sciatic nerve compound action potential inhibition by aroma-oil compounds in a manner specific to their chemical structures ○大坪瀬奈1, 藤田亜美1, 蒋昌宇1, 羅清甜1, 康欽1, 松下晋大1, 熊本栄一1 ○Sena Ohtsubo1, Tsugumi Fujita1, Chang-Yu Jiang1, Qing-Tian Luo1, Qin Kang1, Akitomo Matsushita1, Eiichi Kumamoto1 佐賀大・医・生体構造機能学1 Dept Physiol, Saga Med Sch, Saga, Japan1 We have previously revealed that fast-conducting and Na+-channel blocker tetrodotoxin-sensitive compound action potentials (CAPs) are inhibited by opioids and adrenoceptor agonists in a manner dependent on their chemical structures. Similar actions were seen for transient receptor potential (TRP) V1 agonist capsaicin-related chemicals and also for TRPM8 agonist menthol-related chemicals; these actions were not mediated by TRP activation. Some of aroma-oil compounds have an ability to activate TRP channels. The present study examined whether many kinds of aroma-oil compound have a similar action and if so which structures of them are important for nerve conduction inhibition. The experiments were performed by recording CAPs from the frog sciatic nerve by use of the air-gap method. Citral and citronellal, which have -CHO group (aldehydes), reduced the peak amplitude of the CAP with the IC50 values of 0.48 mM and 0.50 mM, respectively. Similar inhibitions were seen by geraniol and citronellol (having -OH group; alcohols) and also by geranyl acetate (having -COO- group; esters); they had the IC50 values of 0.53, 0.38 and 0.51 mM, respectively. On the other hand, aroma-oil compound having hydrocarbon group (myrcene) and that having -CO- group (camphor; ketones) at a high concentration such as 5 mM reduced CAP peak amplitudes by only 10 and 30 %, respectively. Taking into consideration previously-reported data, an efficacy sequence of aroma-oil compounds for CAP inhibition was phenols (IC50:thymol, 0.42 mM; carbacrol, 0.35 mM; eugenol, 0.81 mM) > alcohols (IC50: geraniol, citronellol, menthol, 0.93-1.1 mM) > aldehydes (citral; citronellal) > esters (geranyl acetate) > ketones (IC50: camphor, carbone, 1.4-1.6 mM; menthone, 1.5-2.3 mM; plegone, 1.4 mM) > oxides (IC50:cineole, 6.6-7.5 mM) >> hydrocarbons (myrcene; limonene: 10 % inhibition at 10 mM). It is suggested that aroma-oil compounds inhibit nerve conduction in a specific manner to their chemical structures.	P1-1-6 カイニン酸受容体サブユニットGluK2ノックアウトマウスでは、運動障害がみられる Kainate receptor GluK2 subunit-deficient mouse shows motor dis-coordination ○渡辺和泉1,2, 明石馨1,2, 阿部学1,2, 夏目里恵1,2, 渡辺雅彦2,3, 崎村建司1,2 ○Izumi Watanabe1,2, Kaori Akashi1,2, Manabu Abe1,2, Rie Natsume1,2, Masahiko Watanabe2,3, Kenji Sakimura1,2 新潟大・脳研・細胞神経生物1, 戦略的創造研究推進事業2, 北海道大院・医・解剖3 Dept Cell Neurobiol, Brain Res Inst, Niigata Univ, Niigata, Japan1, JST, CREST, Tokyo, Japan2, Dept Anatomy, Grad Sch Med, Hokkaido Univ, Sapporo, Japan3 A kainate receptor (KAR) is a member of the ionotropic glutamate receptor family, which plays various roles in the central nervous system. KARs are tetramers composed of combinations of low-affinity GluK1-GluK3 (GluR5-GluR7) and high-affinity GluK4-GluK5 (KA1-KA2) subunits. GluK1-3 subunits can form functional homomeric receptors on their own, while GluK4 & 5 require GluK1-3 to form functional receptors. Although it is very important to know the quantity of each subunit in various brain regions for the understanding of KAR function, no quantitative analysis has been made so far. We newly developed a quantitative Western blot method to determine the amount of KAR subunits. Analytical western blots showed that the amounts of four KAR subunits, GluK2-GluK5, were different in each brain region. In the crude membrane fractions (P2), there were abundant GluK2 subunits in the hippocampus (K2 : K3 : K4 : K5 = 1.0 : 0.38 : 0.08 : 0.22), whereas GluK2 and GluK3 subunits were abundant in the cerebellum (K2 : K3 : K4 : K5 = 1.0 : 0.78 : 0.08 : 0.13). Interestingly, relative amounts of low affinity subunits GluK2 and GluK3 were larger than those of high affinity GluK4 and GluK5. This finding shows a possibility that low affinity subunits play a role in motor regulation, because they, especially GluK2, are largely expressed in cerebellum which regulates motor function. Thus we confirmed the association between motor function and KAR low affinity subunits, comparing GluK2, GluK4 and GluK2/GluK4 double knockout mice. As a result, an open field test showed significantly decreased moving activity in GluK2 and GluK2/GluK4 double, but there was no change in GluK4 knockout mice. In addition, a balance beam test which measures walking balance revealed severe motor deficits in GluK2 and GluK2/GluK4 double knockout mice. These results suggest that a low affinity KAR subunit GluK2 but not high affinity GluK4 is related to motor regulation.
P1-1-7 P2X2受容体のチャネル機能に対する細胞内ドメインの役割 The role of the intracellular domain of the P2X2 receptor on the channel functions ○右田啓介1, 山田順子1, 二階堂義和1, 上野伸哉1 ○Keisuke Migita1, Junko Yamada1, Yoshikazu Nikaido1, Shinya Ueno1 弘前大院・医・脳神経生理1 Dept Neurophysiol, Univ of Hirosaki, Hirosaki1 The P2X receptors are a family of ligand-gated ion channels composed of seven subunits (P2X1-P2X7). P2X receptors contain two putative pore-forming transmembrane segments, a large cysteine-rich ligand-binding extracellular domain, and intracellularly located N and C termini. The receptors operate as a nonselective cationic channel. Several P2X receptor subtypes, including P2X7 receptor, have the property of changing their ion selectivity during prolonged exposure to their natural agonist ATP, which results in pore dilation of the channel and the development of permeability to molecules as large as 800 Da. P2X7 receptors are activated by high concentrations of ATP, unlike other P2X receptors are activated by low doses of ATP. Our previous data showed that P2X2 and P2X3 receptors in dorsal root ganglion were involved in chronic pain associated with sciatic nerve damage or diabetes. On the other hand, P2X7 receptors are known to be localized and functional in microglial cells, and the receptor activity has been demonstrated to be involved in chronic pain associated with inflammatory diseases. However, it is still poorly known the role of the intracellular domain of P2X receptor on the channel functions. We thus generate a chimera consisting of P2X2 receptor with the intracellular domain replaced with that of P2X7 receptor. Replacement of both the N- and C-terminal domain of P2X2 receptor with that of P2X7 receptor had decreased sensitivity to ATP. On the other hand, the chimera consisting of P2X7 receptor with the intracellular domain replaced with that of P2X2 receptor had increased sensitivity to ATP. Replacement of the C-terminal domain of P2X7 receptor with that of P2X2 had decreased the current density.These results suggest that the intracellular domain plays an important role in the agonist operation and the channel function of P2X2 receptor.	P1-1-8 抗癌剤による5-HT3受容体への直接作用 Direct modulation of 5-HT3 receptor by anti-cancer drugs ○中村雪子1, 石田雄介1, 近藤誠1, 山田貴博1, 島田昌一1 ○Yukiko Nakamura1, Yusuke Ishida1, Makoto Kondo1, Takahiro Yamada1, Shoichi Shimada1 阪大・医・神経細胞生物1 Neuroscience and Cell Biology, Univ of Osaka, Osaka1 It has been shown that anti-cancer drug induces secretion of serotonin (5-HT) from small intestine which activates serotonin type 3 (5-HT3) receptor to cause nausea and vomiting. In general, antagonist for 5-HT3 receptor is used as anti-emetics during chemotherapy. However, we found that many anti-cancer drug themself modulate 5-HT-gated current through 5-HT3 receptor. Several anti-cancer drugs have different effect depending on the subunit of 5-HT3 receptor. These result shows that some anticancer drugs themself could affect the degree of nausea and vomiting.
P1-1-9 脊髄小脳変性症にみられる変異型Kv3.3チャネルは、培養小脳プルキンエ細胞において細胞死と興奮性変化を引き起こす Kv3.3 channels harboring a missense mutation of spinocerebellar ataxia type 13 alter neuronal excitability and induce cell death in cultured cerebellar Purkinje cells of mice ○入江智彦1, 松崎泰教2, 関野祐子1, 平井宏和2 ○Tomohiko Irie1, Yasunori Matsuzaki2, Yuko Sekino1, Hirokazu Hirai2 国立医薬品食品衛生研薬理1, 群馬大院医神経生理2 Div Pharmacol, Natl Inst Health Sci, Tokyo, Japan1, Dept. of Neurophysiol., Grad. Sch. of Med., Gunma Univ., Maebashi, Japan2 The cerebellum plays a role in the sensorimotor functions. The output from cerebellar cortex is transmitted by Purkinje cells (PCs), whose impairments cause cerebellar ataxia. Recently, studies have shown that mutations in the voltage-gated potassium channel Kv3.3 are responsible for spinocerebellar ataxia type 13 (SCA13). SCA13 is an autosomal dominant disease and the patients exhibit cerebellar atrophy. In rodent brain, Kv3.3 mRNAs are expressed most strongly in PCs, implying that the mutations severely affect PCs of SCA13 patients. Nevertheless, how the mutant channels alter cell morphology and electrophysiological properties of cerebellar neurons remain unclear. To investigate these issues, Kv3.3 gene with a missense mutation found in a SCA13 pedigree was expressed in a primary culture of mouse cerebellum. The effects were examined by immunohistochemistry and patch-clamp recording. Whereas PCs expressing GFP or wild-type Kv3.3 showed normal dendritic development at 10 days in vitro (DIV), those expressing mutant Kv3.3 displayed defective dendritic development and cell death. Moreover, patch-clamp analysis revealed that PCs expressing mutant Kv3.3 showed decreased outward current, broadened action potentials and altered firing properties at 8-10 DIV. Thus we established a neuronal culture model of SCA13, which would be useful for examining the pathophysiology and drug screening for SCA13.	P1-1-10 神経興奮におけるCdk5の役割―カルシウムイメージング法による解析 The role of Cdk5 in neuronal excitation, a study using calcium imaging and PC-12 cells ○古澤孝太郎1, 斎藤太郎1, 淺田明子1, 久永眞市1 ○Kotaro Furusawa1, Taro Saito1, Akiko Asada1, Shin-ichi Hisanaga1 首都大院・理工・生命1 Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachiohji, Tokyo, Japan1 Cyclin-dependent protein kinase 5 (Cdk5) is activated by p35 regulatory subunit. Cdk5-p35 plays an important role in a variety of neuronal functions. Cdk5-p35 is also involved in regulation of synaptic activity. Excitatory neurotransmitter glutamate downregulates Cdk5 activity by stimulating p35 degradation and long-term potentiation was induced easily in p35-lacking mouse brain hippocampus slices (Wei et al ., 2005). These results suggested that Cdk5-p35 keeps the threshold for excitation. In the presynaptic region, Cdk5-p35 is shown to inhibit P/Q type Ca2+ channel by phosphorylation and in the postsynaptic region, Cdk5-p35 is suggeststed to suppress the activation of CaMKII. However, it is not clear yet how Cdk5 regulates the synaptic activity. We wanted to examine the role of Cdk5 activity in synaptic excitation more directly. Neuronal excitation is manifested by Ca2+ influx into the cytoplasm, which can be visualized with fluorescent Ca2+ indicators. We examined in this study whether Cdk5 indeed regulates excitation by using yellow cameleon-Nano (YC-Nano) as a Ca2+ indicator (Horikawa et al ., 2010). We used PC-12 cells, which are differentiated to neuron-like cells by NGF treatment and excited by exposing to high K+ medium. When PC-12 cells were transfected with YC-Nano and then treated with high K+ solution, transient increase in FRET fluorescence of YC-Nano was observed. FRET was considered to reflect Ca2+ entry through plasma membrane because FRET was abolished by chelating Ca2+ in the medium with EGTA. Next, we compared Ca2+ influx in PC-12 cells expressing Cdk5 or kinase negative (kn) Cdk5 in the presence of p35 and YC-Nano. Ca2+ influx was suppressed by expression of Cdk5 but not knCdk5. Those results suggest that the Cdk5 activity reduces neuronal excitability, consistent with our hypothesis that Cdk5-p35 maintains the threshold of excitation. We are now studying the mechanism how Cdk5 suppresses the excitation more in detail.
P1-1-11 カドミウムは侵害受容性TRPA1チャネルの活性化によりマウスに急性痛を引き起こす Cadmium induces acute pain through the activation of nociceptive TRPA1 channel in mice ○太田利男1, 三浦冴子1, 高橋賢次1, 今川敏明2, 内田邦敏3, 齋藤茂3, 富永真琴3 ○Toshio Ohta1, Saeko Miura1, Kenji Takahashi1, Toshiaki Imagawa2, Kunitoshi Uchida3, Shigeru Saito3, Makoto Tominaga3 鳥取大・農・獣医薬理1, 北海道大・理・生物化学2, 自然科学研究機構・岡崎統合バイオ・細胞生理3 Dept Vet Pharmacol., Tottori Univ, Tottori1, Lab Biol Chem, Dept Chem, Fac Sci, Hokkaido Univ, Sapporo2, Div Cell Signaling, Okazaki Inst Natural Sci, Okazaki, Japan3 Cadmium (Cd) is an environmental pollutant and acute exposure to it causes symptoms related to pain and inflammation in the airway and gastrointestinal tract, but the underlying mechanisms are still unclear. Transient receptor potential ankyrin 1 (TRPA1) is a nonselective cation channel expressed in sensory neurons and acts as a nociceptive receptor. Some metal ions such as Ca, Mg, Ba and Zn are reported to modulate TRPA1 channel activity. In the present study, we investigated the effect of Cd on cultured mouse dorsal root ganglion neurons and a heterologous expression system to analyze the effect of Cd at the molecular level. In addition, we examined whether Cd caused acute pain in vivo. In wild-type mouse sensory neurons, Cd evoked an elevation of the intracellular Ca concentration ([Ca2+]i) that was inhibited by external Ca removal and TRPA1 blockers. Most of the Cd-sensitive neurons were also sensitive to cinnamaldehyde (a TRPA1 agonist) and [Ca2+]i responses to Cd were absent in TRPA1(-/-) mouse neurons. Heterologous expression of TRPA1 mutant channels that were less sensitive to Zn showed attenuation of Cd sensitivity. Intracellular Cd imaging revealed that Cd entered sensory neurons through TRPA1. The stimulatory effects of Cd were confirmed in TRPA1-expressing rat pancreatic cancer cells (RIN-14B). Intraplantar injection of Cd induced pain-related behaviors that were largely attenuated in TRPA1(-/-) mice. Cd excites sensory neurons via activation of TRPA1 and causes acute pain, the mechanism of which may be similar to that of Zn. The present results indicate that TRPA1 is involved in the nociceptive or inflammatory effects of Cd.	P1-1-12 大建中湯の生薬成分が蛙坐骨神経の複合活動電位に及ぼす作用 Actions of crude medicines contained in daikenchuto on compound action potentials in the frog sciatic nerve ○松下晋大1, 大坪瀬奈1, 藤田亜美1, 蒋昌宇1, 羅清甜1, 康欽1, 熊本栄一1 ○Akitomo Matsushita1, Sena Ohtsubo1, Tsugumi Fujita1, Chang-Yu Jiang1, Qing-Tian Luo1, Qin Kang1, Eiichi Kumamoto1 佐賀大学 医学部 生体構造機能学講座 神経生理学分野1 Dept Physiol, Saga Med Sch, Saga, Japan1 We have recently revealed that TRP channel agonists such as capsaicin, zingerone (each TRPV1 agonist) and (-)-menthol (TRPM8 agonist), which are contained in capsicum, ginger and peppermint, respectively, have an inhibitory action on nerve conduction without TRP channel activation. TRPA1 agonists (allyl isothiocyanate and cinnamaldehyde) also reduced frog sciatic nerve compound action potential (CAP) amplitudes with the IC50 values of 1.4 mM and 1.2 mM, respectively. A similar inhibitory action was seen by traditional Japanese medicine (Kampo medicine) containing many plant-derived chemicals; daikenchuto, rikkosan, kikyoto, rikkunshito and kakkonto reduced the peak amplitude of the CAP in a concentration-dependent manner. Daikenchuto had an IC50 value of 1.1 mg/ml. When compared at a concentration of 2 mg/ml, the extents of the reductions by daikenchuto, rikkosan, kikyoto, rikkunshito and kakkonto were 70, 30, 25, 15 and 12 %, respectively. Daikenchuto being the most effective in inhibiting CAPs is composed of three kinds of crude medicine, Japanese pepper, processed ginger and ginseng. The present study examined how the three crude medicines of daikenchuto and also hydroxy-α-sanshool (a TRPV1 and TRPA1 agonist) contained in daikenchuto affect CAPs recorded from the frog sciatic nerve by using the air-gap method. When each of the crude medicines at 2 mg/ml was tested, Japanese pepper and processed ginger reduced CAP peak amplitude by 70 and 30 %, respectively, while ginseng radix hardly affected CAPs. The inhibitory action of Japanese pepper had an IC50 value of 0.77 mg/ml. Hydroxy-α-sanshool reduced the amplitude with an IC50 value of 45 μM. These results indicate that the inhibitory actions of TRP agonists on CAPs could contribute to at least a part of the daikenchuto-mediated CAP inhibition. It is suggested that the pharmacological actions of Kampo medicine may be partly due to nerve conduction inhibition by TRP agonists contained in Kampo medicine.
P1-1-13 TASK1/3チャネルに依存したラット閉口筋α運動ニューロンの序列動員 Rank-ordered recruitment of jaw-closing α-motoneurons depending on the activities of TASK1/3 channels in the rat ○齋藤充1, 磯貝-森田由佳子1, 榎村徳仁1, 豊田博紀1, 佐藤元1, 姜英男1 ○Mitsuru Saito1, Yukako Isogai-Morita1, Norihito Emura1, Hiroki Toyoda1, Hajime Sato1, Youngnam Kang1 大阪大学大学院 歯学研究科 高次脳機能学1 Dept Neurosci & Oral Physiol, Osaka Univ Grad Sch Dent, Osaka1 The muscle tension is regulated by the two factors: the number of recruited motor units and the firing rate of the respective motor units. It is well established that motor units are recruited depending on the order of sizes or input resistances (IRs) of α-motoneurons (MNs), which is known as the size principle. We first examined the size distribution of jaw-closer αMNs in the rat. In contrast to the unimodal and relatively narrow-ranged size distribution of the spinal motoneurons in the mice, we found that the size distribution of jaw-closer αMNs was bimodal or skewed to the left, revealing the presence of many αMNs as small as γMNs. Such wide-ranged size distribution of αMNs might contribute to the precise regulation of muscle tension. We next examined the distributions of two-pore-domain acid-sensitive K+ (TASK) type 1/3 channels responsible for leak conductances in the jaw-closer MNs, and found that TASK1/3 channels were differentially distributed depending on the size of jaw-closer MNs. Since TASK1 and TASK3 channels were differentially modulated by cGMP-dependent protein kinases, such differential distribution of TASK1/3 in large and small jaw-closer MNs might contribute to the modification of rank-ordered recruitment of jaw-closer MNs for performing various jaw movements. We also examined the involvement of spindle Ia inputs onto jaw-closing MNs and TASK channels in the orderly recruitment of MNs in rat slice preparations using whole-cell patch-clamp recordings. Dual whole-cell current-clamp recordings obtained from two adjacent MNs revealed the IR-ordered recruitment of MNs in response to repetitive stimulation of the presumed spindle Ia inputs. Thus, the orderly recruitment of jaw-closing αMNs could be modulated depending on the activities of TASK1/3 channels.	P1-1-14 歯状回門苔状細胞のバースト活動を光で誘導する Optogenetic induction of bursting activity of hilar mossy cells ○三嶋孝知1,2, 八尾寛1,2, 石塚徹1,2 ○Takaaki Mishima1,2, Hiromu Yawo1,2, Toru Ishizuka1,2 東北大院・生命・脳機能解析1, 戦略的創造研究推進事業2 Dept Dev Biol and Neurosci, Grad Sch of Life Sci Tohoku Univ, Sendai1, JST, CREST. Tokyo, Japan2 In the dentate gyrus (DG) of hippocampus, a hilar mossy cell (HMC) receives excitatory inputs convergently from a group of granule cells (GCs) and sends excitatory outputs divergently to another group of GCs. Based on this anatomical feature GCs may have positive feedback association with other GCs through HMCs ("GC association" hypothesis). The activity of hippocampal neurons generally follows theta rhythm of 4-10 Hz. Here we tested whether the magnitude of GC association is dependent on the rhythmic activity using optogenetics. A slice of hippocampus was made from a transgenic rat which expresses channelrhodopsin-2 (ChR2) under regulation of thy1.2 promotor (W-TChR2V4, 4-5 weeks old). One of HMCs was identified under micoroscopy with its localization and morphology and served to the conventional whole-cell patch clamp. Its morphology was identified by the fluorescent image of Alexa-594-conjugated dextran included in the patch pipette. The dentate gyrus was optogenetically stimulated with a spatio-temporal pattern of irradiation generated by a projector-managing optical system (MiLSS). Under current clamp, spontaneous action potentials were observed at low frequency. However, bursts of high-frequency action potentials were often generated in synchronous with rhythmical irradiation of DG at 10 Hz. The bursting activity continued for 10-40 s after termination of irradiation. These bursting activities were almost negligible in the presence of D-AP5 (25 μM). It is suggested that the GC association of DG neurons were enhanced by the theta-rhythmic activity through a mechanism dependent on NMDA receptors.
P1-1-15 ジャンクトフィリン欠損マウスにおけるメタンフェタミン誘発行動逆耐性の形成異常 Aberrant behavioral sensitization by methamphetamine in junctophilin deficient mice ○森口茂樹1, 西美幸2, 竹島浩2, 福永浩司1 ○Shigeki Moriguchi1, Miyuki Nishi2, Hiroshi Takeshima2, Kohji Fukunaga1 東北大学・大学院薬学研究科・薬理学1, 京都大学・大学院薬学研究科・生体分子認識学2 Dept. Pharmacol., Grad. Sch. Pharmaceut. Sci., Tohoku Univ, Sendai1, Dept. Biol. Chem., Grad. Sch. Pharmaceut. Sci., Kyoto Univ, Kyoto2 Junctophilins (JPs) are expressed in the endoplasmic/sarcoplasmic reticulum (ER/SR) interacts with the plasma membrane, thereby constructing junctional membrane complexes (JMC). We here report that mutant (JP-DKO) mice lacking neuronaal two JPs subtypes exhibit an impairment of synaptic plasticity in the striatal slice and irregular behavioral sensitization following methamphetamine (MAP) treatment (7days after 6days consecutive treatment with MAP (1.0 mg/kg)). To address the mechanism of impairment, we first analyzed cortico-striatal glutamitergic plasticity by electrophysiological stimulation of white matter. In saline-treated mice, an obvious long-term depression (LTD) was observed in JP-DKO mice similar to control mice (JP-double hetero mice: JP-DHE). Interestingly, long-term potentiation (LTP) was conversely induced in JP-DHE mice but not in JP-DKO mice following chronic MAP treatment. Impairment of LTP in JP-DKO mice ameliorated by pre-treatment of FK506, calcineurin inhibitors. In immunoblot analyses, autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) was significantly increased in the striatal slice from JP-DHE mice following MAP treatment. However, CaMKII autophosphorylation did not affected by MAP treatment in JP-DKO mice. The dysfunction of CaMKII activity in the striatum of JP-DKO mice was correlated with lack of MAP-induced behavioral sensitization. Thus, JP-mediated Ca2+ mobilization from ER/SR reported previously (Moriguchi et al., PNAS, 2006) affect CaMKII autophosphorylation and in turn MAP-induced behavioral sensitization.	P1-1-16 ストア作動性カルシウムチャネルは自発カルシウム濃度変化に関与している Involvement of the store operated calcium channels in the long-lasting spontaneous calcium transients in the striatal GABAergic neuron ○菊田里美1,2, 柳川右千夫2,3, 森一生1, 小山内実1,2 ○Satomi Kikuta1,2, Yuchio Yanagawa2,3, Issei Mori1, Makoto Osanai1,2 東北大院・医・医用画像工学1, 群馬大院・医3 Tohoku Univ Grad Sch Med, Sendai, Japan1, JST, CREST, Tokyo, Japan2, Gunma Univ Grad Sch Med, Maebashi, Japan3 We previously reported that the long-lasting spontaneous Ca2+ transients (spontaneous [Ca2+]i transients), which lasted up to about 300 s, were observed in striatal cells. These Ca2+ transients were mainly caused by the Ca2+ release from the intracellular Ca2+ store (ER) via IP3 receptors. But, the mechanisms that underlie the spontaneous [Ca2+]i transients remains unclear. Store operated Ca2+ channel (SOCC) is thought to be activated by the depletion of Ca2+ in ER by the Ca2+ release from that. Thus, we investigated whether SOCC involved in the spontaneous [Ca2+]i transients or not.In the striatum, there are no reports concerning SOCC. Therefore, at first, we confirmed the presence of SOCC in the striatal neurons using Ca2+ imaging in the striatal slice preparations. In the striatum more than 95% neurons are GABAergic, thus, the GAD67-GFP knock-in mice were used for visualization of the GABAergic neurons. Since depletion of ER by thapsigargin induced the [Ca2+]i elevation, which was blocked by application of SOCC blocker, SKF96365, the striatal GABAergic neurons must have SOCC. Next, we investigated the contribution of SOCC in the spontaneous [Ca2+]i transients. To quantify the amount of the Ca2+ influx, we used the method of the Mn2+ quenching of the fluorescent Ca2+ indicator. The time constant of the quenching was faster in the neurons exhibiting the spontaneous [Ca2+]i transients compared with the neurons not exhibiting the [Ca2+]i transients in the condition of TTX administration (193 ± 4 s (n = 9) and 211 ± 4 s (n = 23), respectively (p < 0.01)). SKF96365 tended to decrease the peak amplitude and the frequency of the spontaneous [Ca2+]i transients. These results suggested that the spontaneous [Ca2+]i transients led to the opening of SOCC in the striatal cells, and that SOCC might contribute to the maintenance of the high [Ca2+]i levels during the spontaneous [Ca2+]i transients.
P1-1-17 膜電位イメージング法によるラット海馬スライスにおけるGABAA受容体を介するシャンティングと細胞外電場の相互作用の解析 Optical analysis of interactions between GABAergic shunting and extracellular electric fields in CA1 pyramidal neuron dendrites ○上田昭弘1, 中島奈津子1, 宮川博義1, 井上雅司1 ○Akihiro Ueda1, Natsuko Nakajima1, Hiroyoshi Miyakawa1, Masashi Inoue1 東京薬科大学大学院 生命科学研究科 生命科学専攻 脳神経機能学研究室1 Laboratory of Cellular Neurobiology, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Japan1 Extracellular electric fields are produced by synchronous activityes of neurons in the brain and they may have functional roles for non-synaptic comunication among the neurons. We have previously reported extracellular DC electric fields induce characteristic non-uniform membrane polarization in rat hippocampal CA1 pyramidal neurons. Here we examined interaction between postsynaptic potentials. (PSPs) and extracellular electric field using voltage-sensitive dye imaging. Rat hippocampal slices were stained with a voltage sensitive dye RH414 and a pair of electrodes were placed so as to apply electric fields parallel to the somato-dendritic axis of CA1 pyramidal neurons. Optical responses of CA1 region were monitored using a 16 x 16 square array of photodiodes. Synaptic inputs to distal apical dendrites of the CA1 pyramidal neurons were electrically activated under different conditions including presence or absence of small ± 40 mV mm-1 DC electric fields that alone produce only subthreshold polarizations in the neurons. We have examined spatial distribution of subthreshold PSPs along the dendrites with or without aplication of GABAA receptor antagonist bicuculline. Under the application of bicuculline, subthreshold glutamatergic excitatory PSPs (EPSPs) were linearly summated with direct membrane polarizations by the DC electric fields and time-courses and amplitudes of EPSPs in every regions along the dendrites were not affected by the DC fields However, without blocking GABAA receptors, the time-courses and the amplitudes of PSPs along the dendrites were non-linearly affected with the DC fields. These results suggest that GABAergic components of PSPs and shunting inhibition at the dendrites were more sensitive to the DC fields than glutamatergic EPSPs, maybe due to the fluctuations of membrane potentials of the neurons effectively increase or decrease driving forces of the ionic currents through the GABAA receptor channels.	P1-1-18 ゼブラフィッシュのマウスナー細胞の単発発火を構成するKv7/KCNQチャネルの発現とリン酸化による調節 Expression and phosphorylation of Kv7/KCNQ channels underlie single spiking of Mauthner cell in zebrafish ○島崎宇史1, 渡邉貴樹1, 小田洋一1 ○Takashi Shimazaki1, Takaki Watanabe1, Yoichi Oda1 名古屋大学大学院　理学研究科　生命理学1 Department of Biological Science, Graduate School of Science, Nagoya University1 Voltage-gated potassium (Kv) channels are crucial to determine various firing patterns of neurons. The Mauthner (M) cells, which are bilaterally paired giant reticulospinal neurons (RSNs) in teleost hindbrain and known to trigger fast escape, deliver a single spike at the onset of depolarizing input. By contrast, morphologically homologous RSNs show repetitive firing in response to depolarization. Interestingly, the unique firing property of zebrafish M-cell is acquired during larval development: immature M-cells exhibit burst firing. Our previous studies showed that coexpression of a low-Kv, Kv1.1, channels with Kvβ2 subunits was a key, but not enough, to produce single spike of M-cell. Here we show involvement of another low-Kv, Kv7/KCNQ, channels are involved in single spiking of M-cell. Pharmacological blocking both Kv1 and Kv7 channels resulted in repetitive firing of the mature M-cells as homologous RSNs exhibited. In situ hybridization showed that Kv7.4 was expressed exclusively in M-cells but not in the homologous RSNs, suggesting that specific expression of Kv7.4 in M-cell explains the unique firing of M-cell. Kv7.4, however, was already expressed in immature M-cells which fired with burst. Thus, the Kv7.4 channels may be inhibited at early stage and allowed to function in developed M-cell. Under application of a PKC activator, which was expected to phosphorylate Kv7.4 channels, in addition to Kv1 blocker, mature M-cells fired repetitively as immature M-cells. Kv7.4 channels expressed in Xenopus oocytes were activated above -60mV, and suppressed by the PKC activator. Taken together, these results suggest that Kv7.4 channels are expressed specifically in M-cells but suppressed at early developmental stage probably with phosphorylation by PKC and allowed to be active by dephosphorylation in mature M-cell. It may contribute to establishing the unique single-spiking property of M-cell during development.
P1-1-19 Human epilepsy mutants alter gating and channel-protein interactions in the Slack KNa channel ○Matthew R. Fleming1, Gulia Barcia2, Laurence Colleaux2, Rima Nabbout2, Leonard K. Kaczmarek1 Dept. of Pharmacology, Yale School of Medicine, New Haven, CT, USA1, Hospital Necker-Enfants Malades, Paris, France2 Na+-activated potassium (KNa) channels encoded by the Slack and Slick genes contribute to neuronal adaptation during sustained stimulation and regulate the accuracy of timing of action potentials. Activation of protein kinase C (PKC) increases the amplitude of Slack-B currents and slows their rate of activation. Mutations in Slack channels which result in constitutive channel activation by mimicking phosphorylation cause malignant migrating partial seizures of infancy (MMPSI), a rare epileptic encephalopathy of infancy that combines pharmacoresistant seizures with severe developmental delay. Slack protein is known to interact with a variety of cytoplasmic signaling molecules. Using resonance wavelength grating optical biosensors (the SRU Biosciences BIND system), we have determined that direct pharmacological activation ofSlack channels by bithionol produces a sustained decrease in mass distribution close to theplasma membrane, and that phosphorylation of Slack channels mimics this decrease in mass. Thevery C-terminal domain of Slack has been previously shown necessary for channel-proteininteractions, and deletion of this region abolished the observed signal. To determine whichproteins or signaling molecules are translocating from the plasma membrane upon channelactivation, an RNAi screen against probable channel binding partners was performed, and theProtein Phosphatase 1 (PP1) targeting protein Phactr1 was found to be necessary for thisdecrease in mass. We hypothesize that activation of Slack by either bithionol or phosphorylationleads to the dissociation of Phactr1 with PP1 from the channel complex, allowing the Slackchannel to remain in its phosphorylated and active state. Activation of PKC does not result in adecrease in mass in the human MMPSI mutants, possibly linking channel excitability todownstream signaling mechanisms which may result in developmental delay.	P1-1-20 乳酸脱水素酵素によるてんかん制御 Regulation of epilepsy by the metabolic enzyme, lactate dehydrogenase ○吉田渚1, 勝孝1, 井上剛1 ○Nagisa Yoshida1, Takashi Katsu1, Tsuyoshi Inoue1 岡山大院・医歯薬・生体分子解析学1 Dept Biophys chem, Okayama Univ, Okayama, Japan1 Epilepsy is characterized by hyperexcitable electrical activities in the brain, and diet control (e.g., ketogenic diets) is effective to cure intractable epilepsy. By the ketogenic diet, energy sources in the brain are switched from glucose to ketone bodies. In this study, we focused on the metabolic switch, and examined which molecules in metabolic pathways regulate membrane potentials in neurons and suppress epilepsy in vivo. We first recorded membrane potentials in subthalamic neurons of mice, using slice patch-clamp technique. The recording revealed that the membrane potentials were hyperpolarized by the metabolic switch to ketone bodies. Interestingly, the hyperpolarization was reversed by addition of lactate. These results indicated that the lactate regulates the hyperpolarization by the metabolic switch. Lactate is converted to pyruvate by lactate dehydrogenase (LDH). We therefore examined effects of LDH in neurons on their membrane potentials by intracellular application of oxamate, an LDH inhibitor. We found that the intracellular oxamate induced hyperpolarization, and decreased membrane excitability in neurons. Furthermore, this electrical regulation by LDH was observed in seizure-related brain regions, the basal ganglia and the hippocampus. In the hippocampus, only glutamatergic pyramidal cells were hyperpolarized, whereas GABAergic fast-spiking cells were not changed. We then recorded electroencephalogram in the cerebral cortex of a mouse model of generalized seizures in vivo. The in vivo recording revealed that LDH inhibition can suppress generalized epilepsy. These results indicated that LDH is a key enzyme regulating membrane potentials in neurons and suppressing epilepsy. To date, molecular targets of antiepileptic drugs are mostly ion channels, synaptic receptors and transporters. Thus, our results show that metabolic enzyme LDH could be a new target for developing antiepileptic drugs.
P1-1-21 電位依存性ナトリウムチャネルNav1.1ハプロ不全を原因とするてんかんの神経回路基盤の解明 Nav1.1 haploinsufficient excitatory and inhibitory neurons play distinct roles in epilepsy ○荻原郁夫1, 岩里琢治2,3,4, 宮本浩行5,6, 岩田亮平3,4, 山形哲司1, 眞崎恵美1, 柳川右千夫7,8, 玉巻伸章9, ヘンシュ貴雄5,10, 糸原重美2, 山川和弘1 ○Ikuo Ogiwara1, Takuji Iwasato2,3,4, Hiroyuki Miyamoto5,6, Ryohei Iwata3,4, Testushi Yamagata1, Emi Mazaki1, Yuchio Yanagawa7,8, Nobuaki Tamamaki9, Takao Hensch5,10, Shigeyoshi Itohara2, Kazuhiro Yamakawa1 理化学研究所　脳科学総合研究センター　神経遺伝研究チーム1, 理化学研究所　脳科学総合研究センター　行動遺伝学技術開発研究チーム2, 遺伝学研究所　形質遺伝研究部門3, 総合研究大学院大学　遺伝学専攻4, 理化学研究所　脳科学総合研究センター　神経回路発達研究チーム5, さきがけ6, 群馬大学大学院　医学系研究科 高次統御系 脳神経発達統御学講座7, 熊本大学大学院　生命科学研究部　脳回路構造学分野9, ハーバード大学　脳科学センター　分子細胞生物学科10 Laboratory for Neurogenetics, RIKEN Brain Science Institute, Wako, Saitama1, Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, Wako, Saitama2, Division of Neurogenetics, National Institute of Genetics, Mishima, Shizuoka3, Department of Genetics, the Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka4, Laboratory for Neuronal Circuit Development, RIKEN Brain Science Institute, Wako, Saitama5, PRESTO, Japan Science and Technology Agency, Tokyo6, Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi, Gunma7, CREST, Japan Science and Technology Agency, Tokyo8, Department of Morphological Neural Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto9, Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA, USA10 Heterozygous mutations of the SCN1A gene encoding a voltage-gated sodium channel Nav1.1 cause childhood epilepsy. We previously reported Nav1.1 localization to the axon initial segments of parvalbumin-positive (PV) inhibitory neurons, and the abnormal firing patterns of PV interneurons in heterozygous Scn1a knockout mice suffering epileptic seizures. We here investigated the physiological consequences of selective Nav1.1 elimination from all GABAergic inhibitory neurons or excitatory neurons by crossing floxed Scn1a mice with Vgat-Cre and Emx1-Cre driver lines, respectively. We found that Scn1a (flox/+);Vgat-Cre mice develop epileptic seizures with a significantly higher risk of sudden death than those seen in heterozygous Scn1a knockout mice. Scn1a (flox/flox); Emx1-Cre mice did not develop epileptic seizures. Instead, we find that Nav1.1 elimination from forebrain excitatory neurons reduces the mortality rate of Scn1a (flox/+);Vgat-Cre mice to a level close to that of heterozygous Scn1a knockout mice. These results indicate that Nav1.1 haploinsufficient inhibitory neurons contribute to the pathogenesis of epileptic seizures, while Nav1.1 haploinsufficient excitatory neurons provide a protective influence on the seizure pathology.	P1-1-22 初代培養ミクログリアにおける電位依存性プロトンチャネルの細胞内局在 Subcellular localization of voltage-gated proton channels in primary cultured microglia ○河合喬文1, 井村誉史雄2, 小泉修一2, 岡村康司1 ○Takafumi Kawai1, Yoshio Imura2, Schuichi Koizumi2, Yasushi Okamura1 大阪大 院医 統合生理1, 山梨大院医工薬理2 Lab. of Integr. Physiol., Grad. Sch. of Med., Osaka Univ.1, Dept Neuropharmacol, Univ of Yamanashi, Yamanashi, Japan2 Previously, we have identified a protein, VSOP (Hv1) which consists of voltage sensor domain lacking pore domain and functions as voltage-gated proton channels (Sasaki et al, Science. 2006). VSOP has a crucial role in the production of reactive oxygen species (ROS) by compensating for charge imbalance upon electron extrusion by NADPH oxidase in phagocyte such as neutrophil and macrophage. Although the previous study showed that expression of VSOP is specific to microglia in mouse brain, subcellular localization of VSOP in microglia and its dynamics remain elusive. In the present study, we examined the subcellular localization of VSOP by immunocytochemistry in primary cultured microglia using confocal microscopy. Without any chemical stimulation, numerous small VSOP-positive vesicles were observed in the intracellular compartments of microglia. These signals were not observed in microglia from VSOP knockout mice, suggesting that the signal derives from endogeneous VSOP in the microglia. Our preliminary data suggests that VSOP containing vesicles were not colocalized with gp91 (a component of NADPH oxidase complex), Rab5 (early endosome marker) and Rab11 (recycling endosome marker). Thus VSOP appears to be packed in the unconventional vesicles. When microglia was stimulated by ATP, a strong chemoattractant for microglia, VSOP containing vesicles were recruited to the cell membrane and well colocalized with β-actin fibers. LPS stimulation also slightly induced the recruitment of VSOP containing vesicles to the cell membrane. These results suggest that the chemical stimuli induce the recruitment of VSOP to the cell membrane, thereafter efficiently recruiting VSOP to phagosome in microglia.
P1-1-23 アンキリンG（イオンチャネルアンカー蛋白）の結晶構造解析 Crystal Structure of Ankyrin-G: the Ion Channel Anchoring Protein ○藤原祐一郎1, 岡村康司1 ○Yuichiro Fujiwara1, Yasushi Okamura1 大阪大学大学院　医学系研究科　統合生理学1 Integrative Physiology, Graduate School of Medicine, Osaka University, Suita1 Ankyrin-G configures the membrane-excitation platform by clustering various ion channels such as voltage-gated Na+ channels, voltage-gated K+ channels, Na+-K+ ATPase and CNG channels at the initial segment and the nodes of Ranvier of neuronal axons, at the intercalated disk of cardiomyocytes or at the outer segment of retinal rod cells. Disorder of the clustering induces channelopathy, such as the Brugada syndrome and the periodic paralysis, and Ankyrin-G itself involved in the bipolar disorder. Although to clarify the molecular mechanism of the channel clustering on the membrane, thus, will bring significant merits, no structural analysis of Ankyrin-G has been performed. Ankyrin-G consists of 24 tandem ankyrin repeats, and the channels are known to bind somewhere in the repeats; and Ankyrin-G itself associates to the plasma membrane via the s-palmitoylation of Cys in the 1st repeat. We succeeded in expressing a large amount of ankyrin repeat protein (24 repeats) of rat Ankyrin-G in E.coil. Solubility of the purified proteins was high, and reversible precipitations were induced by changing the salt concentration. Circular Dichroism spectrum of the purified proteins indicated a high α-helical content of structure in solution, which corresponds to the secondary structure prediction. Dynamic light scattering showed that the size distribution of protein particles exhibited a mono-dispersity. These results suggest that the purified proteins fold properly and stably in solution, implying the good protein materials for crystallization. We succeeded in determining the high resolution (1.6 Å) crystal structure of the initial 5 ankyrin repeats, which uncovers the structural basis of membrane association of Ankyrin-G.	P1-1-24 海馬CA1領域錐体細胞先端樹状突起における自発的な活動の位相は微弱な細胞外交流電場によって調節される Weak extracellular AC electric field modulates timing and synchrony of spontaneous Ca transients in CA1 pyramidal neuron of rat hippocampal slices ○前田佳主馬1, 丸山隆一2, 諸田元2, 青西亨2, 井上雅司1, 宮川博義1 ○Kazuma Maeda1, Ryuichi Maruyama2, Hajime Moroda2, Toru Aonishi2, Masashi Inoue1, Hiroyoshi Miyakawa1 東京薬科大学大学院 生命科学研究科 脳神経機能学研究室1, 東工大・総理工2 Lab. Cellular Neurobiology, Sch. Life Sci., Tokyo Univ. Pharm. Life Sci.,Japan1, Tokyo Tech.2 Field potential recording from freely moving rat revealed that hippocampal pyramidal neurons are exposed to extracellular electric field during the theta activity. Although the theta activity is thought to reflect activity of neural population, there is a possibility that the field during theta affect the neural activity. In other words, neurons may interact ephaptically during theta activity. We are interested in this type of non-synaptic interaction. We postulate that the architecture of dendrites makes it easy for the neurons to generate and detect extracellular electric field. While dendrites in hippocampal CA1 region are bipolar and lie parallel each other to form an open field arrangement suitable to generate field potential, the arrangement is suitable to detect the field as well. Moreover, along the dendrite, there are many kinds of voltage-gated channels suitable to amplify subtle changes in membrane potential and provide current source to generate field potential. However, it is not known how subthreshold electric field modulates the activities of the dendrite. Ca imaging techniques has been used to observe Ca events such as Ca release, Ca spikes, and Ca transients associated with Na spikes. Recently, we devised a method to separately monitor spontaneous Ca transients in the dendrites of population of neurons. Here we apply this method to examine the effects of weak AC electric field to the timing and synchrony of dendritic activities. As the intensity of the field was increased from 1 to 20 mV/mm, the frequency of Ca transients did not change significantly. On the other hand, the timing of local transient was phase-locked to 1-8Hz extracellular sinusoidal field at an intensity even as weak as 1 mV/mm. Surprisingly, the extent of phase-lock modulation did not increase significantly with applying field intensity. Our results show that weak extracellular electric field indeed modulates the timing and synchrony of dendritic spontaneous local Ca transient.
P1-1-25 脳組織の誘電率を考慮に入れるとより精確な電流源密度 (CSD)が推定できる： コンパートメントモデルによる解析 Considering the permittivity of brain tissue give rise to more precise estimation of current-source density: compartmental model analysis ○毛内拡1,2,3, 宮川博義4, 青西亨1 ○Hiromu Monai1,2,3, Hiroyoshi Miyakawa4, Toru Aonishi1 東工大 総理工1, 学振特別研究員2, 理研BSI3, 東薬大・生命4 Tokyo Tech., Kanagawa, Japan1, JSPS Research Fellow, Tokyo, Japan2, RIKEN BSI, Saitama, Japan3, Tokyo Univ. of Pharm. and Life Sci., Tokyo, Japan4 The current-source density (CSD) analysis was developed for analyzing the local field potential (LFP). It has been used to estimate the location and timing of the source of the membrane current that generates the field potentials. In conventional CSD analysis, the extracellular space is assumed to be purely resistive. However, it is known that the permittivity of brain tissue exhibits strong frequency dependence (dispersion) and that the permittivity is very large in the low-frequency region. Recently, we have calculated the extracellular permittivity by using the brain tissue modeled by bundles of cables existing within a purely resistive extracellular medium and showed that the extracellular medium exhibits a large effective permittivity in the low-frequency region due to the electrically long cables and the contribution of the capacitive current in the low-frequency region is not negligible (Monai et al., 2012). Here, we estimate the distribution of membrane current by using the general formula for the CSD analysis by which CSD in biological tissue with frequency dependent permittivity and conductivity can be obtained from the extracellular potential (Miyakawa and Aonishi, 2012). In the numerical simulation by using the compartment model, we show that the proposed CSD analysis can estimate the distribution of membrane current more precisely than that of conventional method. We should not hastily ignore extracellular capacitive current and need to take the permittivity into account in the CSD analysis.	P1-1-26 hERGチャネルのゲーティングの環状ヌクレオチド結合相同ドメインによる制御機構の解析 Analyses of the regulation mechanisms of the gating of hERG channel by cyclic nucleotide binding homology domain ○粂慎一郎1,2, 中條浩一1,2, 久保義弘1,2 ○Shinichiro Kume1,2, Koichi Nakajo1,2, Yoshihiro Kubo1,2 生理研・神経機能素子1, 総研大・生理科学2 Div. of Biophysics and Neurobiology, National Institute for Physiological Sciences, Aichi1, Dept. of Physiological Sciences, The Graduate University for Advanced Studies, Aichi2 Human Ether-a-go-go Related Gene (hERG) channel is a member of the voltage-gated K+ channel subfamily H (KCNH), and is expressed in the heart, smooth muscle cells, and various brain regions. This channel is known for its characteristic biophysical properties, such as slow activation and deactivation. It has been suggested previously that the cytoplasmic domains of hERG channel contribute to these properties, in particular slow deactivation. KCNH members have a cyclic nucleotide binding homology domain (CNBHD) in its cytoplasmic C-terminus, which lacks affinity to cyclic nucleotides. Therefore, this subfamily is not regulated by cyclic nucleotides. Brelidze et al. (2012) recently reported the crystal structure of CNBHD of zebrafish KCNH subfamily member, zELK channel. They showed that Tyr740 in CNBHD of zELK channel fills the cyclic nucleotide binding pocket of CNBHD, possibly mimicking cyclic nucleotide as an endogenous ligand. We performed homology modeling of CNBHD of hERG channel by SWISS-MODEL server based on the crystal structure of zELK channel. We observed a high similarity of the structure of CNBHD. We found that Tyr740 of zELK channel is substituted with phenylalanine in hERG channel (Phe860), and there is another phenylalanine (Phe805) in the nucleotide binding pocket of CNBHD which possibly interacts with Phe860 according to the homology modeling. We mutated Phe860 and Phe805 of hERG channel to Alanine (F860A and F805A, respectively), and analyzed their electrophysiological properties using Xenopus oocytes under two electrode voltage clamp. We observed that (1) the V1/2 value of F805A is positively shifted compared to that of WT, (2) the speed of activation of F805A is slower than that of WT, and (3) the speed of deactivation of F860A and F805A is faster than that of WT. These results suggest that both Phe860 and Phe805 in CNBHD of hERG channel play critical roles in the slow deactivation and that Phe805 plays a role for the activation process as well.

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