Calcium/calmodulin-dependent protein kinase II (CaMKII), an abundant protein in neurons, is involved in synaptic plasticity and learning. CaMKII associates with multiple proteins located at or near the postsynaptic density (PSD), and CaMKII is known to translocate from cytoplasm to PSD under excitatory conditions. The present study examined the laminar distribution of CaMKII at the PSD by immunogold labeling in dissociated hippocampal cultures under low calcium (EGTA or APV), control, and stimulated (depolarization with high K+ or NMDA) conditions. The patterns of CaMKII distribution are classified with particular reference to the two layers of the PSD: (1) the PSD core, a layer within ~ 30-40 nm to the postsynaptic membrane, and (2) the PSD pallium, a deeper layer beyond the PSD core, ~ 100-120 nm from the postsynaptic membrane. Under low calcium conditions, a subpopulation (40%) of synapses stood out with no CaMKII labeling at the PSD, indicating that localization of CaMKII at the PSD is sensitive to calcium levels. Under control conditions, the majority (~ 60-70%) of synapses had label for CaMKII dispersed evenly in the spine, including the PSD and the nearby cytoplasm. Upon stimulation, the majority (60-75%) of synapses had label for CaMKII concentrated at the PSD, delineating the PSD pallium from the cytoplasm. Median distance of label for CaMKII to postsynaptic membrane was higher in low calcium samples (68-77 nm), than in control (59-63 nm) and stimulated samples (49-53 nm). Thus, upon stimulation, not only more CaMKII translocated to the PSD, but they also were closer to the postsynaptic membrane. Additionally, there were two relatively infrequent labeling patterns that may represent intermediate stages of CaMKII distribution between basal and stimulated conditions: (1) one type showed label preferentially localized near the PSD core where CaMKII may be binding to NR2B, an NMDA receptor concentrated at the PSD core, and (2) the second type showed label preferentially in the PSD pallium, where CaMKII may be binding to Shank, a PSD scaffold protein located in the PSD pallium. Both of these distribution patterns may portray the initial stages of CaMKII translocation upon synaptic activation. In addition to binding to PSD proteins, the concentrated CaMKII labeling at the PSD under heightened excitatory conditions could also be formed by self-clustering of CaMKII molecules recruited to the PSD. Most importantly, these accumulated CaMKII molecules do not extend beyond the border of the PSD pallium, and are likely held in the pallium by binding to Shank under these conditions.

中文翻译：

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CaMKII在海马神经元突触后区室中的活动依赖性重新分布。

钙/钙调蛋白依赖性蛋白激酶II（CaMKII）是神经元中丰富的蛋白，参与突触可塑性和学习。CaMKII与位于突触后密度（PSD）或附近的多种蛋白质缔合，并且已知CaMKII在兴奋条件下从细胞质转移到PSD。本研究通过免疫金标记在低钙（EGTA或APV），对照和刺激（高K +或NMDA去极化）条件下在离体海马培养物中检测了PSD上CaMKII的层状分布。CaMKII分布的模式是根据PSD的两层进行分类的：（1）PSD核心，即距突触后膜约30-40 nm内的一层，以及（2）PSD皮层，超出了PSD核心，距突触后膜约100-120 nm。在低钙条件下，突触的亚群（40％）突显出来，在PSD处没有CaMKII标记，表明CaMKII在PSD处的定位对钙水平敏感。在对照条件下，大多数突触（〜60-70％）的CaMKII标记均匀分布在脊柱中，包括PSD和附近的细胞质。刺激后，大多数突触（60-75％）的CaMKII标记集中在PSD，从细胞质描绘了PSD皮层。在低钙样品（68-77 nm）中，CaMKII标记到突触后膜的中位距离高于对照（59-63 nm）和受刺激样品（49-53 nm）。因此，在刺激时，不仅更多的CaMKII易位至PSD，而且它们也更靠近突触后膜。另外，有两种相对少见的标记模式，它们可能代表CaMKII在基础和受激条件之间分布的中间阶段：（1）一种类型显示标记优先位于PSD核心附近，其中CaMKII可能与NR2B结合，NMDA受体集中在PSD核心（2）第二种类型优先在PSD皮层中显示标签，其中CaMKII可能与Shank（位于PSD皮层中的PSD支架蛋白）结合。这两种分布模式都可以描述突触激活后CaMKII易位的初始阶段。除了与PSD蛋白结合外，在高兴奋性条件下PSD上的浓缩CaMKII标记也可以通过募集到PSD的CaMKII分子的自聚形成。最重要的是，