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）相关的非活性蛋白1型]，一种新型的D-myo-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的分子，类似于PLC-δ1，但催化性非活性，表达了脑部脑组织的主要表达。 PRIP-1具有许多结合伙伴，包括INS（1,4,5）P_3，蛋白质磷酸酶1α（PP1C）的催化亚基，GABA_A受体相关蛋白（GABARAP）和GABA_A_A受体受体β-亚基蛋白。这些发现促使我们检查了PRIP在GABA_A受体信号传导以及INS（1,4,5）P_3介导的CA^<2+>信号传导中的可能作用。最近，我们报道说，缺乏PRIP-1基因（PRIP-1 KO）暴露的小鼠对GABA_A受体的磷酸依赖性调节剂量响应于CAMP依赖性蛋白激酶A（PKA）激活的GABA_A受体的磷酸依赖性调节。同工型，PRIP-2，后来被鉴定出来，该分子的存在相对无处不在，包括脑组织。 PRIP-2还展示了其与PP1C和Gabarap的结合活动更多。因此，在进一步分析PRIP在gaba_a受体功能方面的PRIP-1和-2双敲除（PRIP-DKO）小鼠的产生是绝对必需的。我们已经生成了PRIP-DKO小鼠，并分析了在生化，药物和行为方面的GABA_A受体功能。 Ligand binding assays using [^3H]muscimol, a GABA agonist, and [^3H]Ro15-1788,a diazepam antagonist, shown 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.在PRIP-DKO小鼠中，电生理和行为分析中的地西ep敏感性降低。这些发现表明，PRIP参与将GABA_A受体运输到细胞表面膜上，可能是通过与Gabarap竞争GABA_A受体的γ-亚基。此外，我们阐明BDNF可能参与Prip的调节后GABA_A受体的调节。暴露于BDNF可降低GABA诱发的抑制电流（/_ <GABA>）在培养的野生型（WT）小鼠的海马神经元中，而PRIP-DKO小鼠中观察到了一点增强，与GABA_A受体数的表面表达相对应。 PRIP与GABA_A受体的β-亚基的直接相互作用对于GABA_A受体内在化很重要，这表明PRIP对于GABA_A受体内吞作用至关重要，并且控制了GABA_A接收器的表面表达。较少的

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.