Visualization of synaptic Ca2+/calmodulin-dependent protein kinase II activity in living neurons
Visualization of synaptic Ca2+/calmodulin-dependent protein kinase II activity in living neurons
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DOI：
10.1523/jneurosci.0085-05.2005
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发表时间：
2005-03-23
影响因子：
5.3
通讯作者：
Hayashi, Y
中科院分区：
文献类型：
--
作者：
Takao, K;Okamoto, KI;Hayashi, Y
钙/钙调蛋白依赖的蛋白激酶II(CaMKII)在中枢神经系统的兴奋性突触中高度丰富，在突触的可塑性、学习和记忆中起重要作用。然而，由于缺乏具体的方法，对活细胞中CaMKII活性的精确时空调控还没有得到很好的描述。我们试图通过使用荧光共振能量转移(FRET)光学检测CaMKII在激活过程中的构象变化来解决这一问题。经过改造的FRET探针Camui Alpha检测到苏氨酸286处的钙调蛋白结合和自动磷酸化，使酶具有结构性活性。结合双光子显微镜，我们证明了CAMUIα可以用来观察活神经元CaMKII活性的时空调节。
Ca2+/calmodulin-dependent protein kinase II ( CaMKII) is highly enriched in excitatory synapses in the CNS and critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, because of a lack of specific methods. We tried to address this by optically detecting the conformational change in CaMKII during activation using fluorescence resonance energy transfer (FRET). The engineered FRET probe Camui alpha detects calmodulin binding and autophosphorylation at threonine 286 that renders the enzyme constitutively active. In combination with two-photon microscopy, we demonstrate that Camui alpha can be used to observe temporal and spatial regulation of CaMKII activity in living neurons.