NMDA Receptor GluN2B (GluRε2/NR2B) Subunit Is Crucial for Channel Function, Postsynaptic Macromolecular Organization, and Actin Cytoskeleton at Hippocampal CA3 Synapses
NMDA Receptor GluN2B (GluRε2/NR2B) Subunit Is Crucial for Channel Function, Postsynaptic Macromolecular Organization, and Actin Cytoskeleton at Hippocampal CA3 Synapses
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DOI：
10.1523/jneurosci.5531-08.2009
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发表时间：
2009-09-02
影响因子：
5.3
通讯作者：
Sakimura, Kenji
中科院分区：
文献类型：
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作者：
Akashi, Kaori;Kakizaki, Toshikazu;Sakimura, Kenji
GluN 2B（GluR β 2/NR 2B）亚基参与突触发育、突触可塑性和认知功能。然而，它在突触表达和NMDA受体（NMDARs）在大脑中的功能仍然是未知的，因为新生儿死亡的整体敲除小鼠的角色。为了解决这个问题，我们产生了条件性基因敲除小鼠，其中GluN 2B仅在海马CA 3锥体细胞中被消融。通过免疫组织化学，GluN 2B消失，GluN 1（GluR zeta 1/NR 1）适度减少，而GluN 2A（GluR zeta 1/NR 2A）和突触后密度-95（PSD-95）在突变体CA 3中未发生变化。这与CA 3粗级分中的蛋白质含量一致：GluN 2B为对照水平的9.6%，GluN 1为对照水平的47.7%，GluN 2A为对照水平的90.6%，PSD-95为对照水平的98.0%。尽管剩余的NMDAR，NMDAR介导的电流和长时程增强几乎失去了在各种CA 3突触。然后，我们比较了突触NMDAR植入后免疫金电子显微镜和免疫印迹使用PSD馏分。在突变体CA 3中，GluN 1在免疫金（20.6-23.6%）和免疫印迹（24.6%）中均严重减少，而GluN 2A和PSD-95在免疫金中无变化，但在PSD组分中显著减少（分别为51.4%和36.5%），表明PSD分子的去污剂溶解度增加。在GluN 2A基因敲除小鼠的CA 3中，没有观察到GluN 2B的溶解度增加。此外，发现丝状肌动蛋白与球状肌动蛋白的比率（49.5%）和树突棘的密度（76.2%）显着下降。这些研究结果表明，GluN 2B是关键参与NMDAR通道功能，组织突触后大分子复合物，树突棘的形成或维持，和调节肌动蛋白细胞骨架。
GluN2B (GluR epsilon 2/NR2B) subunit is involved in synapse development, synaptic plasticity, and cognitive function. However, its roles in synaptic expression and function of NMDA receptors (NMDARs) in the brain remain mostly unknown because of the neonatal lethality of global knock-out mice. To address this, we generated conditional knock-out mice, in which GluN2B was ablated exclusively in hippocampal CA3 pyramidal cells. By immunohistochemistry, GluN2B disappeared and GluN1 (GluR zeta 1/NR1) was moderately reduced, whereas GluN2A (GluR epsilon 1/NR2A) and postsynaptic density-95 (PSD-95) were unaltered in the mutant CA3. This was consistent with protein contents in the CA3 crude fraction: 9.6% of control level for GluN2B, 47.7% for GluN1, 90.6% for GluN2A, and 98.0% for PSD-95. Despite the remaining NMDARs, NMDAR-mediated currents and long-term potentiation were virtually lost at various CA3 synapses. Then, we compared synaptic NMDARs by postembedding immunogold electron microscopy and immunoblot using the PSD fraction. In the mutant CA3, GluN1 was severely reduced in both immunogold (20.6-23.6%) and immunoblot (24.6%), whereas GluN2A and PSD-95 were unchanged in immunogold but markedly reduced in the PSD fraction (51.4 and 36.5%, respectively), indicating increased detergent solubility of PSD molecules. No such increased solubility was observed for GluN2B in the CA3 of GluN2A-knock-out mice. Furthermore, significant decreases were found in the ratio of filamentous to globular actin (49.5%) and in the density of dendritic spines (76.2%). These findings suggest that GluN2B is critically involved in NMDAR channel function, organization of postsynaptic macromolecular complexes, formation or maintenance of dendritic spines, and regulation of the actin cytoskeleton.