Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation

Because CaMKII is the critical Ca2+ sensor that triggers long-term potentiation (LTP), understanding its activation and deactivation is important. A major advance has been the development of a FRET indicator of the conformational state of CaMKII called Camui. Experiments using Camui have demonstrated that the open (active) conformation increases during LTP induction and then decays in tens of seconds, with the major fast component decaying with a time-constant of ~ 6 sec (tau1). Because this decay is faster if autophosphorylation of T286 is prevented (the autophosphorylation prolongs activity by making the enzyme active even after Ca2+ falls), it seemed likely that the fast decay is due to the T286 dephosphorylation. To test this interpretation, we studied the effect of phosphatase inhibitors on the single-spine Camui signal evoked by two-photon glutamate uncaging. We applied inhibitors of PP1 and PP2A, two phosphatases that are present at synapses and that have been shown to dephosphorylate CaMKII in vitro. The inhibitors increased the basal Camui activation state, indicating their effectiveness in cells. However, in no case did we find that tau1 was prolonged, contrary to what would be expected if the decay was phosphatase-dependent. This could either mean that decay was due to some unknown phosphatase or that the decay was not due to dephosphorylation. To distinguish between these possibilities, we expressed pseudo-phosphorylated Camui (T286D) (plus additional mutations [T/A] that prevented inhibitory 305/306 phosphorylation). This form had an elevated basal activation state, but was further activated during glutamate uncaging; importantly the activation state decayed with tau1 nearly the same as that of WT Camui. Therefore, the data strongly indicate that tau1 is not due to T286 dephosphorylation. We conclude that, although Camui is an excellent tool for observing CaMKII signaling, further experimentation is needed to determine how CaMKII is turned off by its dephosphorylation.

钙/钙调蛋白依赖性蛋白激酶II（CaMKII）是介导长时程增强（Long-term potentiation, LTP）的核心Ca²⁺传感器，阐明其激活与失活机制具有重要研究价值。领域内一项重要进展是开发出一种可检测CaMKII构象状态的荧光共振能量转移（Fluorescence Resonance Energy Transfer, FRET）指示剂——Camui。基于Camui的实验结果显示，CaMKII的开放（激活）构象在LTP诱导过程中显著升高，随后于数十秒内衰减，其中主要的快速衰减组分的时间常数约为6秒（tau1）。若抑制T286位点的自磷酸化（该自磷酸化可通过使酶在Ca²⁺浓度下降后仍保持活性，从而延长其激活时长），则衰减速率会加快，这提示快速衰减过程可能由T286位点的去磷酸化介导。为验证这一假说，我们探究了蛋白磷酸酶抑制剂对双光子谷氨酸光解笼技术所诱发的单棘突Camui信号的影响。我们选用了蛋白磷酸酶1（PP1）与蛋白磷酸酶2A（PP2A）的抑制剂——这两种磷酸酶均富集于突触，且体外实验已证实其可介导CaMKII的去磷酸化。抑制剂处理后，Camui的基础激活水平显著升高，证实其在细胞内发挥了预期的抑制效果。但无论使用何种抑制剂，均未观察到tau1出现延长——这与“衰减过程依赖蛋白磷酸酶”的预期相悖。这一结果提示两种可能性：要么快速衰减由未被靶向的未知磷酸酶介导，要么衰减过程并非依赖T286位点的去磷酸化。为区分这两种可能性，我们构建并表达了模拟磷酸化状态的Camui突变体（T286D），同时额外引入了可抑制305/306位点抑制性磷酸化的T/A突变。该突变体的基础激活水平本就升高，但在谷氨酸光解笼刺激下仍可被进一步激活；值得注意的是，其激活状态的衰减tau1与野生型（Wild Type, WT）Camui几乎一致。综上，实验数据强有力地表明，tau1对应的快速衰减并非由T286位点的去磷酸化所介导。我们得出结论：尽管Camui是观测CaMKII信号通路的优质工具，但要阐明CaMKII通过去磷酸化途径失活的具体机制，仍需开展进一步实验研究。