A study on the role of two different Ca^<2+> channel families in neurons

两种不同Ca^2通道家族在神经元中作用的研究

• 批准号: 11672168
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672168/
• 关键词: calcium channels; voltage-dependent; TRP; store-operated; receptor-activated; Xenopus oocytes; cerebral neurons; Fura-2 fluorescence; trp; 卵母細胞; HEK細胞; 大脳皮質; Fura-2

(1) We have isolated two cDNAs encoding RNA splicing variants of human Ca^<2+> channel a1B subunit from human brain cDNA libralies. These variants were proved to be the RNA splicing variants by genomic analysis. One of the variants lacking the II-III linker region formed Ca^<2+>-permeable channel having low ω-conotoxin sensitivity, as revealed by exogenous expression in human embryonic kidney (HEK) cells. (2) We have analyzed the G-protein-mediated modulation of P/Q-type, α_<1A> channels biochemically and electrophygiologically, and found that the interaction of N-terminal region of Gα_o and C-terminal domain of a_<1A> subunit causes the voltage-resistant inhibition of P/Q-type channel current. (3) We have found, using Xenopus oocyte expression system, that cytosolic Ca^<2+> is required for the opening of store-operated TRP4 channels, and that the activation of inositol-1, 4, 5-trisphosphate receptor (IP_3-R) is required and sufficient for the opening of receptor-activated TRP5 channels. (4) We have found the store-operated and receptor-activated 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.

(1)我们从人脑cDNA文库中分离出两个编码人类Ca^<2+>通道a1B亚基RNA剪接变体的cDNA。通过基因组分析证实这些变异是RNA剪接变异。在人胚胎肾（HEK）细胞中的外源表达显示，缺乏II-III连接区的一种变体形成了Ca^<2+>-渗透性通道，具有低ω- concontoxin敏感性。(2)对g蛋白介导的P/ q型、α_<1A>通道的生化和电生理调控进行了分析，发现Gα_o的n端区域和α_<1A>亚基的c端区域相互作用导致了P/ q型通道电流的耐压抑制。(3)我们利用爪蟾卵母细胞表达系统发现，储存型TRP4通道的开启需要胞质Ca^<2+>，而受体激活的TRP5通道的开启需要肌醇- 1,4,5 -三磷酸受体（IP_3-R）的激活。(4)我们用Fura-2荧光法在培养的大鼠大脑皮质神经元中发现了储存操作和受体激活的Ca^<2+>进入。通过RT-PCR分析，我们发现神经元中存在大鼠TRP1、3、5和6 mrna。免疫染色结果也表明TRP1和5蛋白在培养的神经元中有表达。

项目成果

Hideaki Kanki et al.: "Opening of mouse TRP5 channels is primarily dependent on activation of IP_3 receptors"Neuroscience Research. Suppl.24. S27 (2000)

Hideaki Kanki 等人：“小鼠 TRP5 通道的开放主要依赖于 IP_3 受体的激活”神经科学研究。

Mariko Kinoshita et al.: "Effects of anti-Gα_o-antiserum on G-protein-mediated inhibition of α_<1A> and α_<1B> Ca^<2+> channels"Neuroscience Research. Suppl.24. S27 (2000)

Mariko Kinoshita 等人：“抗 Gα_o 抗血清对 G 蛋白介导的 α_<1A> 和 α_<1B> Ca^<2+> 通道的抑制的影响”神经科学研究 Suppl.24。

Kiyoshi Mizukami et al.: "Some physiological and pharmacological properties of slow depolarization of substantia gelatinosa neurons by repetitive stimulation of C-fibers of dorsal root in adult rat spinal cord"Neuroscience Letters. 274. 49-52 (1999)

Kiyoshi Mizukami 等人：“通过重复刺激成年大鼠脊髓背根 C 纤维，缓慢去极化凝胶质神经元的一些生理学和药理学特性”《神经科学快报》。

Kiyoshi Mizukami: "Some physiological properties of slow depolarization of substantia gelatinosa neurons by repetitive stimulation of C-fibers"Neunosci.Lett.. 274. 49-52 (1999)

Kiyoshi Mizukami：“通过重复刺激 C 纤维，凝胶质神经元缓慢去极化的一些生理特性”Neunosci.Lett.. 274. 49-52 (1999)