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

PRIP-1[磷脂酶C（PLC）相关无活性蛋白1型]是一种新型的d -肌醇1,4,5-三磷酸[Ins(1,4,5)P_3]结合蛋白，与PLC-δ1分子相似，但催化无活性，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催化亚基（PP1c）、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，我们报道了缺乏PRIP-1基因（PRIP-1 KO）的小鼠表现出GABA_A受体药理学和行为的改变，并且GABA_A受体对camp依赖性蛋白激酶A（PKA）激活的磷酸化依赖性调节也发生了改变。一种异构体，rip -2，后来被鉴定出来，这种分子的存在相对普遍，包括脑组织。prp -2对PP1c和GABARAP均有明显的结合活性。因此，为了进一步分析PRIP在脑组织中对GABA_A受体功能的作用，绝对需要产生PRIP-1和-2双敲除（PRIP- dko）小鼠。我们制备了ripp - dko小鼠，并从生化、药理学和行为学方面分析了GABA_A受体的功能。用GABA激动剂[^3H]muscimol和地西泮拮抗剂[^3H]Ro15-1788进行配体结合实验，结果表明，与WT小鼠相比，PRIP-DKO小鼠细胞表面GABA结合位点（α/β）的表达量增加，而地西泮结合位点（α/γ2）的表达量减少。在电生理和行为分析中，PRIP-DKO小鼠对地西泮的敏感性降低。这些发现表明，PRIP可能通过与GABARAP竞争GABA_A受体的γ-亚基，参与了GABA_A受体向细胞膜的运输。此外，我们阐明了PRIP可能参与BDNF对突触后GABA_A受体的调节。BDNF使野生型（WT）小鼠海马神经元GABA诱发抑制电流（/_<GABA >）降低，而PRIP-DKO小鼠海马神经元GABA诱发抑制电流略有增强，这与GABA_A受体的表面表达数量相对应。PRIP与GABA_A受体β亚基的直接相互作用对GABA_A受体内化具有重要意义，表明PRIP对GABA_A受体的内吞作用至关重要，并控制GABA_A受体的表面表达。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