Functional analysis of neuronal Ca^<2+> channel domains as targets of therapeutics

作为治疗靶标的神经元 Ca^2 通道域的功能分析

• 批准号: 13672278
• 负责人: KANEKO Shuji
• 金额: $ 2.3万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672278/
• 关键词: calcium channels; voltage-dependent; TRP; Alzheimer's disease; alternative splicing; Xenopus oocytes; neuropathic pain; neuronal death; TRPチャネル; アンチセンスDNA; 免疫染色; RT-PCR; 初代培養ニューロン; 不活性化; アフリカツメガエル卵母細胞; スプライシングバリアント; GTP結合タンパク; FK960; PMP

What we found were (1) The interaction of N terminal region of Ga_O and C-terminal domain of α_<1A> subunit causes the voltage-resistant inhibition of P/Q-type channel current. (2) Cytosolic Ca^<2+> is required for the opening of store-operated TRP4 channels, whereas the activation of inositol-1,4,5-trisphosphate receptor (IP_3-R) is required and sufficient for the opening of receptor-activated TRP5 channels. (3) Two cDNAs encoding RNA splicing variants of human Ca^<2+> channel α1B subunit were isolated from human brain cDNA libralies. These variants were RNA splicing variants by genomic analysis. One of the variants lacking the II III linker region formed Ca^<2+>-permeable channel having lowωconotoxin sensitivity, as revealed by exogenous expression in human embryonic kidney (HER) cells. (4) An insect peptide PMP-D2 selectively inhibited R-type Cav2.3 channels. (5) A novel antiamneasic drug, FK960, selectively potentiated N-type Cav2.2 channel current in a protein kinase C-dependent manner. (6) A novel analgesic, ONO-2921, selectively inhibited N- and R- type channel currents due to its specific action on inactivated channels. (7) The store-operated and receptoractivated Ca^<2+> entry in rat cerebral cortical neurons in culture by Fura-2 fluorometry. By RT-PCR analysis, we found the presence of rat TRP1, 3, 5 and 6 mRNAs in neurons. Immunostaninig results also suggest that TRP1 and 5 proteins are expressed in the cultured neurons.

结果表明：（1）Ga_O的N端区域与α_亚基的C端区域相互作用<1A>，引起P/Q型通道电流的电压抵抗性抑制。(2)胞浆Ca^&lt;2+&gt;是钙库操纵的TRP 4通道开放所必需的，而1，4，5-三磷酸肌醇受体（IP_3-R）的激活是受体激活的TRP 5通道开放所必需和充分的。(3)从人脑cDNA文库中分离到两个编码人Ca^2+通道α 1 B亚基RNA剪接变体的cDNA。通过基因组分析，这些变体是RNA剪接变体。其中一种缺乏II/III连接区的变体形成了具有低ω芋螺毒素敏感性的Ca^2+渗透通道，这一点在人胚肾（HER）细胞中的外源表达中得到了揭示。(4)昆虫肽PMP-D2选择性抑制R型Cav2.3通道。(5)一种新型的抗健忘症药物FK 960以蛋白激酶C依赖的方式选择性增强N型Cav2.2通道电流。(6)ONO-2921是一种新型的镇痛剂，由于其对失活通道的特异性作用，选择性地抑制N-和R-型通道电流。(7)用Fura-2荧光法研究大鼠大脑皮层神经元钙池操纵和受体激活的Ca^2+内流通过RT-PCR分析，我们发现大鼠TRP 1，3，5和6的mRNA在神经元中的存在。免疫组化结果也提示TRP 1和5蛋白在培养的神经元中表达。

项目成果

金子周司: "Ca2+チャネル遺伝子の選択的スプライシングとその機能的意義"日本薬理学雑誌. 121. 233-240 (2003)

Shuji Kaneko：“Ca2+通道基因的选择性剪接及其功能意义”日本药理学杂志 121. 233-240 (2003)。

Hiroyuki Tabata, Satoshi Tanaka, Yukihiko Sugimoto, Hideaki Kanki, Shuji Kaneko and Atsushi Ichikawa: "Possible coupling of prostaglandin E receptor EP_1 to TRP5 expressed in Xenopus oocytes"Biochem. Biophys. Res. Commun.. 298. 398-402 (2002)

Hiroyuki Tabata、Satoshi Tanaka、Yukihiko Sugimoto、Hideaki Kanki、Shuji Kaneko 和 Atsushi Ichikawa：“前列腺素 E 受体 EP_1 与爪蟾卵母细胞中表达的 TRP5 的可能偶联”Biochem。

Kume T.et al.: "Isolation of a diprenoid substance with potent neuroprotective activity from fetal calf serum"Proceedings of the National Academy of Sciences USA. 99. 3288-3293 (2002)

Kume T.等人：“从胎牛血清中分离出具有有效神经保护活性的二烯类物质”美国国家科学院院刊。

Kinoshita, M. et al.: "Binding of G α o is responsible for the voltage-resistant inhibition of α_<1A>1A(P/Q-type, Cav2.1 ) Ca^<2+> channels"The Journal of Biological Chemistry. 276. 28731-28738 (2001)

Kinoshita, M. 等人：“G α o 的结合负责 α_<1A>1A（P/Q 型，Cav2.1 ）Ca^<2+> 通道的耐电压抑制”The Journal of生物化学。276。28731-28738（2001）

Kaneko, S. et al.: "Identification and characterization of novel human Cav2.2 calcium channel variants lacking the synaptic protein interaction site"The Journal of Neuroscience. 22. 82-92 (2002)

Kaneko, S. 等人：“缺乏突触蛋白相互作用位点的新型人类 Cav2.2 钙通道变体的鉴定和表征”神经科学杂志。