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

磷脂酶C（PLC）相关失活蛋白1（PLC-related inactive protein type 1，PRIP-1）是一种新的D-肌肌醇1，4，5-三磷酸（Ins（1，4，5）P_3）结合蛋白，与PLC-δ1相似，但无催化活性，主要在脑组织中表达。PRIP-1具有多个结合伙伴，包括Ins（1，4，5）P_3、蛋白磷酸酶1α催化亚基（PP 1c）、GABA_A受体相关蛋白（GABARAP）和GABA_A受体β亚基。这些结果促使我们研究PRIP在GABA_A受体信号以及Ins（1，4，5）P_3介导的Ca^<2+>信号中的可能作用。最近，我们报道了PRIP-1基因缺失小鼠（PRIP-1 KO）表现出GABA_A受体药理学和行为学的改变，并且GABA_A受体对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。后来发现了一种亚型PRIP-2，这种分子的存在相对普遍，包括脑组织。PRIP-2也是 ...更多信息 其对PP 1c和GABARAP的结合活性。因此，产生PRIP-1和-2双敲除（PRIP-DKO）小鼠是绝对必要的，以进一步分析PRIP在脑组织中关于GABA_A受体功能的作用。本研究建立了PRIP-DKO小鼠模型，并从生物化学、药理学和行为学等方面对GABA_A受体的功能进行了研究。使用GABA激动剂[^3H]蝇蕈醇和地西泮拮抗剂[^3H] Ro 15 -1788进行的配体结合试验表明，与WT小鼠相比，PRIP-DKO小鼠中GABA结合位点的细胞表面表达水平（α/β）增加，但地西泮结合位点的数量（α/γ2）减少。PRIP-DKO小鼠在电生理和行为分析中的地西泮敏感性降低。这些结果表明PRIP可能通过与GABARAP竞争GABA_A受体的γ-亚基而参与GABA_A受体向细胞膜表面的运输。进一步阐明PRIP可能参与BDNF对突触后GABA_A受体的调节。BDNF使<GABA >野生型（WT）小鼠海马神经元GABA诱发的抑制电流（I_）降低，而PRIP-DKO小鼠海马神经元GABA_A受体数目的表达有所增强。PRIP与GABA_A受体β亚基的直接相互作用对GABA_A受体的内化起重要作用，表明PRIP是GABA_A受体内吞的关键，并控制着GABA_A受体的表面表达。少

项目成果

Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated, Ca2+ signaling

• DOI: 10.1002/jcp.20136
• 发表时间: 2005-02-01
• 期刊: JOURNAL OF CELLULAR PHYSIOLOGY
• 影响因子: 5.6
• 作者: Harada, K;Takeuchi, H;Hirata, M
• 通讯作者: Hirata, M

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)。

ブレインサイエンスレビュー2004(伊藤正男, 川合述史編)

脑科学评论 2004（伊藤正雄、河合正司编辑）

• 发表时间: 2004
• 作者: 五十嵐 道弘

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.

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)。