Analysis of the molecular mechanisms of GABA_A receptor assembly and trafficking.

GABA_A受体组装和运输的分子机制分析。

• 批准号: 15591969
• 负责人: KANEMATSU Takashi
• 金额: $ 2.37万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591969/
• 关键词: Ins(1,4,5)P_3 binding protein; GABARAP; PRIP; GABA_A receptor; GABARAP; PRIP-1; GABA_A 受容体; NSF

PRIP-1 [phospholipase C(PLC)-related inactive protein type 1], a novel D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P_3] binding protein, is a molecule similar to PLC-δ1 but catalytically inactive and expresses predominantly in the brain tissues. PRIP-1 has a number of binding partners, including Ins(1,4,5)P_3, catalytic subunit of protein phosphatase 1α (PP1c), GABA_A receptor-associated protein(GABARAP) and GABA_A receptor β-subunits. These findings prompted us to examine the possible roles of PRIP in GABA_A receptor signaling as well as Ins(1,4,5)P_3-mediated Ca^<2+> signaling. Recently, we reported that the mice lacking PRIP-1 gene (PRIP-1 KO) exhibited altered GABA_A receptor pharmacology and behavior, and phospho-dependent modulation of GABA_A receptors in response to cAMP-dependent protein kinase A(PKA) activation was also altered. An isoform, PRIP-2,has later been identified, and the presence of this molecule is relatively ubiquitous, including brain tissues. PRIP-2 also exhib … More its the binding activities to both PP1c and GABARAP. Hence, the generation of PRIP-1 and -2 double knockout(PRIP-DKO) mice are absolutely required for analyzing further the roles of PRIP in brain tissues regarding GABA_A receptor function. We have generated the PRIP-DKO mice and analyzed the GABA_A receptor functions at biochemical, pharmacological and behavioral aspects. Ligand binding assays using [^3H]muscimol, a GABA agonist, and [^3H]Ro15-1788,a diazepam antagonist, showed that the cell surface expression levels of GABA binding site (α/β) were increased, but the numbers of diazepam binding site (α/γ2) were reduced in the PRIP-DKO mice, compared to WT mice. Diazepam sensitivity in electrophysiological and behavioral analysis was reduced in PRIP-DKO mice. These findings indicate that PRIP are involved in trafficking of GABA_A receptors to cell surface membrane, probably by competing with GABARAP for γ-subunit of GABA_A receptors. Furthermore, we elucidate that the possible involvement of PRIP in the modulation of postsynaptic GABA_A receptor by BDNF. The exposure to BDNF reduced the GABA-evoked inhibitory current (/_<GABA >) in cultured hippocampal neurons of wild type(WT) mice, whereas a little potentiation was observed in the PRIP-DKO mice, corresponding to the surface expression of GABA_A receptor number. The direct interaction of PRIP to β-subunits of GABA_A receptor was important for the GABA_A receptor internalization, indicating the PRIP is essential for the GABA_A receptor endocytosis and controls the surface expression of GABA_A receptor. Less

PRIP-1 [磷脂酶 C(PLC) 相关失活蛋白 1 型] 是一种新型 D-肌醇 1,4,5-三磷酸 [Ins(1,4,5)P_3] 结合蛋白，是一种类似于PLC-δ1 但催化失活并主要在脑组织中表达 PRIP-1 有许多结合伙伴，包括 Ins(1,4,5)P_3，蛋白磷酸酶 1α (PP1c) 催化亚基、GABA_A 受体相关蛋白 (GABARAP) 和 GABA_A 受体 β 亚基这些发现促使我们研究 PRIP 在 GABA_A 受体信号传导以及 Ins(1,4,5) 中的可能作用。 )P_3介导的Ca^2+信号传导最近，我们报道缺乏PRIP-1基因(PRIP-1 KO)的小鼠表现出改变。 GABA_A 受体的药理学和行为，以及 GABA_A 受体响应 cAMP 依赖性蛋白激酶 A (PKA) 激活的磷酸依赖性调节也发生了改变，后来发现了同种型 PRIP-2，并且该分子的存在已被确定。 PRIP-2 相对普遍存在，包括脑组织，也表现出与 PP1c 和 GABARAP 的结合活性，因此 PRIP-1 和 -2 的产生加倍。进一步分析 PRIP 在脑组织中 GABA_A 受体功能的作用绝对需要敲除 (PRIP-DKO) 小鼠，我们已经生成了 PRIP-DKO 小鼠，并在生化、药理学和行为方面分析了 GABA_A 受体功能。使用 GABA 激动剂 [^3H]muscimol 和地西泮拮抗剂 [^3H]Ro15-1788 显示细胞表面 GABA 结合位点的表达水平与 WT 小鼠相比，PRIP-DKO 小鼠的地西泮 (α/β) 增加，但地西泮结合位点 (α/γ2) 的数量减少。表明 PRIP 可能通过与 GABARAP 竞争 GABA_A 受体的 γ 亚基参与 GABA_A 受体向细胞表面膜的运输。此外，我们还阐明了可能的参与。 PRIP 在 BDNF 调节突触后 GABA_A 受体中的作用 BDNF 暴露降低了野生型 (WT) 小鼠培养的海马神经元中 GABA 诱发的抑制电流 (/_<GABA >)，而在野生型 (WT) 小鼠中观察到了一点增强作用。 PRIP-DKO 小鼠，对应于 GABA_A 受体的表面表达 PRIP 与 GABA_A 受体 β 亚基的直接相互作用对于 GABA_A 很重要。受体内化，表明 PRIP 对于 GABA_A 受体内吞作用至关重要，并控制 GABA_A 受体的表面表达。

项目成果

Mizoguchi Y: "A rapid increase in the total number of cell-surface functional GABA_A receptors induced by BDNF in rat visual cortex"J Biol Chem.. 278(45). 44097-44102 (2003)

Mizoguchi Y：“大鼠视觉皮层中 BDNF 诱导的细胞表面功能性 GABA_A 受体总数快速增加”J Biol Chem.. 278(45)。

Hidaka K: "The importance to chondrocyte differentiation of changes in expression of multiple polyphosphate phosphatase"Exp Cell Res.. 290(2). 254-264 (2003)

Hidaka K：“多种多磷酸磷酸酶表达变化对软骨细胞分化的重要性”Exp Cell Res.. 290(2)。

The importance to chondrocyte differentiation of changes in expression of multiple polyphosphate phosphatase.

多种多聚磷酸酶表达变化对软骨细胞分化的重要性。

• 发表时间: 2003
• 期刊: Exp Cell Res. 290(2)
• 作者: Sato;F.;Kawamoto;T.;Fujimoto;K.;Noshiro;M.;Honda;K.;Honma;S.;Honma;K.;Kato;Y.;Ohishi M.;Kenichi Ishibashi et al.;Hidaka K et al.
• 通讯作者: Hidaka K et al.

Hypersensitivity to pentylenetetrazol-induced convulsion in mice lacking the PLC-related inactive protein-1

缺乏 PLC 相关失活蛋白 1 的小鼠对戊四氮引起的惊厥过敏

• 发表时间: 2004
• 期刊: Brain Res. 1025
• 作者: Yamaguchi;T.et al.
• 通讯作者: T.et al.

GABA_A receptor phospho-dependent modulation is regulated by phospholipase C-related inactive protein type 1, a novel protein phosphatase

GABA_A 受体磷酸依赖性调节受磷脂酶 C 相关无活性蛋白 1 型（一种新型蛋白磷酸酶）的调节

• 发表时间: 2004
• 期刊: J Neurosci. 111
• 作者: Abe N.;et al.;Terunuma M et al.
• 通讯作者: Terunuma M et al.