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

δ-1是一种新的D-肌醇-1，4，5-三磷酸结合蛋白，是一种与磷脂酶C相关的失活蛋白1，4，5-三磷酸[INS(1，4，5)P_3]结合的蛋白，是一个类似于磷脂酶C相关失活蛋白1的分子，但不具有催化活性，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)P3、蛋白磷酸酶1催化亚基α(PP1c)、GABBA_A受体相关蛋白(GABARAP)和GABBA_A受体β-亚基。这些发现促使我们研究了PRIP在GABA_A受体信号转导以及INS(1，4，5)P_3介导的Ca~(2+)和Gt；信号转导中的可能作用。最近，我们报道了缺乏PRIP-1基因(PRIP-1 KO)的小鼠表现出GABA_A受体药理和行为的改变，以及对cAMP依赖的蛋白激酶A(PKA)激活反应的GABA_A受体的磷酸化调节也发生了改变。后来发现了一种名为PRIP-2的异构体，这种分子的存在相对普遍，包括脑组织。PRIP-2还展出了…更重要的是，它与PP1c和GABARAP具有结合活性。因此，PRIP-1和PRIP-2双基因敲除(PRIP-DKO)小鼠的产生是进一步分析PRIP在脑组织中与GABA_A受体功能相关的作用所必需的。我们已经建立了PRIP-DKO小鼠，并从生化、药理和行为方面分析了GABA_A受体的功能。用γ-氨基丁酸激动剂[^3 H]蝇草酚和安定拮抗剂[^3 H]Ro15-1788进行的配体结合分析表明，与WT小鼠相比，PRIP-DKO小鼠细胞表面γ-氨基丁酸结合位点(α/β)的表达水平增加，而安定结合位点(α/γ2)的数目减少。在PRIP-DKO小鼠中，安定在电生理和行为分析中的敏感性降低。这些结果表明，PRIP可能通过与GABARAP竞争GABBA_A受体的γ亚基，参与了GABBA_A受体向细胞表膜的转运。此外，我们还阐明了PRIP可能参与了BDNF对突触后GABA_A受体的调控。暴露于BDNF后，野生型(WT)小鼠海马神经元GABA诱发的抑制电流(/_&lt；GABA&gt；)降低，而PRIP-DKO小鼠海马神经元GABA_A受体数量的表面表达略有增强。PrIP与GABAA受体β亚基的直接相互作用对GABAA受体内化起重要作用，提示PRIP对GABAA受体内吞作用是必需的，并控制GABAA受体的表面表达。较少

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