Mechanism underlying polarized subcellular distribution of Ca channels and its neurobiological significance.

Ca 通道极化亚细胞分布的机制及其神经生物学意义。

• 批准号: 14380362
• 负责人: MORI Yasuo
• 金额: $ 7.36万
• 依托单位: KYOTO UNIVERSITY (2003)Okazaki National Research Institutes (2002)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380362/
• 关键词: receptor activated channel; Ca2+ signal; smooth muscle; endoplasmic reticulum; 薬理学; ノックアウト

Two mechanisms that link presynaptic defects to postsynaptic aspects of neuronal differentiation and maturation have been revealed. Firstly, in N-type, Ca^<2+> channel-deficient mice we created, positive, regulation by automic nervous system was ablated. This is reasonable, considering that noradrenalin release from presynaptic terminal is predominantly controlled by N-type channels in wild-type mice. However, interestingly, cardiac contraction, vascular constriction, and sensitivity of receptors to their agonists were upregulated in the mutant mice. Secondly, we compared fundamental properties of excitatory synaptic transmission in the cerebellum and roles of Ca^<2+> channel subtypes among wild-type control, tottering (tg) and rolling Nagoya (tg^<rol>) that carry mutations in the P/Q-type Ca^<2+> channel α_<1A> (Cav2.l) subunit gene. The EPSC amplitude of the climbing fiber-Purkinje cell (CF-PC) synapses was preserved in tg, and it was even increased in tg^<rol>, which was associated … More with altered properties of the postsynaptic glutamate receptors. The climbing fiber mediated EPSC was more dependent on other Ca^<2+> channel subtypes in mutant mice, suggesting that such compensatory mechanisms contribute to maintaining the CF-PC synaptic transmission virtually intact. Thus, presynaptic Ca^<2+> channels control postsynaptic excitability and function and thereby regulate differentiation and maturation synapses. We showed that G protein-coupled, metabotropic glutamate receptor subtype 1 (mGluR1) and P/Q-type Cav2.1 are colocalized at dendrites of cerebellar Purkinje neurons and form the heteromeric assembly in both the brain and heterologously expressing COS-7 cells. mGluR1 inhibited Cav2.1-mediated [Ca^<2+>]i increases and Ba2+ currents in HEK 293 cells expressing Cav2.1 with auxiliary alpha2/delta and beta1 subunits, respectively, in a ligand-independent manner and was enhanced by pre activation of mGluR1 in a ligand-dependent manner. Furthermore, in dihdropyridine (DHP)-sensitive L-type Ca^<2+> channel Cav1.2 (α1c), Ser1142 was the recognition site for both the DHP agonist BAYk8644 and Phosphatase inhibited by okadaic acid. This suggests that upregulation of L-type channels by DHP agonist and that by Ser phophorylation are mediated by a common mechanism involving Ser1142. Finally, we carried out two-hybrid screening using the β4 subunit as a bate to isolate a clone encoding the protein regulating fusion of synaptic vesicles. Functional and molecular characterization of the protein is under way. Less

已经揭示了将突触前缺陷与神经元分化和成熟的突触后方面联系起来的两种机制。首先，在我们建立的N型Ca^<2+>通道缺陷小鼠中，自主神经系统的正性调节被消除。这是合理的，因为在野生型小鼠中，突触前末梢的去甲肾上腺素释放主要由N型通道控制。然而，有趣的是，心脏收缩，血管收缩和受体对其激动剂的敏感性在突变小鼠中上调。其次，我们比较了小脑兴奋性突触传递的基本特性以及携带P/Q型Ca^&lt;2 +&gt;通道α_（Cav2.1）亚基基因突变的野生型对照、蹒跚型（tg）和滚动型名古屋型（tg^）中Ca^&lt;2 +&gt;通道亚型的作用<rol><1A>。爬纤维-浦肯野细胞（CF-PC）突触的EPSC振幅在tg时保持不变，甚至在tg^时增加<rol>，这与 ...更多信息 突触后谷氨酸受体的特性发生了改变在突变小鼠中，攀爬纤维介导的EPSC更依赖于其他Ca^&lt;2+&gt;通道亚型，这表明这种代偿机制有助于维持CF-PC突触传递几乎完整。因此，突触前Ca^2+通道控制突触后兴奋性和功能，从而调节突触的分化和成熟。我们发现，G蛋白偶联，代谢型谷氨酸受体亚型1（mGluR 1）和P/Q型Cav2.1共定位在小脑浦肯野神经元的树突，并形成在大脑和异源表达COS-7细胞的异聚体组装。在分别表达Cav2.1和辅助α 2/δ和β 1亚基的HEK 293细胞中，mGluR 1以配体非依赖性方式抑制Cav2.1介导的[Ca^&lt;2+&gt;]i增加和Ba 2+电流，并通过mGluR 1的预激活以配体依赖性方式增强。此外，在二氢吡啶（DHP）敏感的L型Ca^2+通道Cav1.2（α1c）中，Ser 1142是DHP激动剂BAYk 8644和冈田酸抑制的磷酸酶的识别位点。这表明DHP激动剂和Ser磷酸化对L型通道的上调是由涉及Ser 1142的共同机制介导的。最后，我们以β4亚基为底物进行双杂交筛选，分离到一个编码突触囊泡融合调节蛋白的克隆。目前正在对该蛋白质进行功能和分子鉴定。少

项目成果

西田基宏, 森泰生: "カルシウムチャネル拮抗剤"分子生物学・免疫学キーワード辞典 第2版(医学書院). (in press). (2002)

Motohiro Nishida、Yasuo Mori：“钙通道拮抗剂”分子生物学/免疫学关键词词典第二版（Igaku Shoin）（印刷中）。

森泰生, 西田基宏: "カルシウムチャネル"分子生物学・免疫学キーワード辞典 第2版(医学書院). 2. 242-243 (2003)

Yasuo Mori，Motohiro Nishida：“钙通道”分子生物学/免疫学关键词词典第二版（医学书院）。

Dos Santos RG, Van Renterghem C, Martin-Moutot N, Mansuel P, Sampieri F, Diniz C, Mori Y, De Lima M-E, Seagar M: "・Phoneutria nigriventer IIA toxin blocks the Ca_v2 family of calcium channels and interacts with・conotoxin binding sites"J. Biol. Chem.. 277.

Dos Santos RG、Van Renterghem C、Martin-Moutot N、Mansuel P、Sampieri F、Diniz C、Mori Y、De Lima M-E、Seagar M：“·Phoneutria nigriventer IIA 毒素阻断钙通道 Ca_v2 家族并与·芋螺毒素相互作用结合位点”J. Biol. Chem. 277。

Nishida M, Sugimoto K, Hara Y, Mori E, Morii T, Kurosaki T, Mori Y: "Amplification of receptor signaling by Ca^<2+> entry-mediated translocation and activation of phospholipase C・2 in B lymphocytes."EMBO J.. 22. 4677-4688 (2003)

Nishida M、Sugimoto K、Hara Y、Mori E、Morii T、Kurosaki T、Mori Y：“B 淋巴细胞中 Ca^2+ 进入介导的易位和磷脂酶 C·2 的激活放大受体信号传导。”EMBO J.. 22. 4677-4688 (2003)

Erxleben C, Allegria-Gomez C, Darden, Mori Y, Birnbaumer L, Armstrong DL: "Modulation of cardiac Ca_v 2.1 channels by BAYk8644 and Okadaic acid requires S1142 in the domain III pore loop."Proc.Natl.Acad.Sci.USA. 100. 2929-2934 (2003)

Erxleben C、Allegria-Gomez C、Darden、Mori Y、Birnbaumer L、Armstrong DL：“BAYk8644 和冈田酸对心脏 Ca_v 2.1 通道的调节需要域 III 孔环中的 S1142。”Proc.Natl.Acad.Sci.USA