Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation.
Fast Decay of CaMKII FRET Sensor Signal in Spines after LTP Induction Is Not Due to Its Dephosphorylation.
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DOI：
10.1371/journal.pone.0130457
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Lisman JE
中科院分区：
文献类型：
--
作者：
Otmakhov N;Regmi S;Lisman JE
由于CaMKII是触发长时程增强（LTP）的关键Ca 2+传感器，因此了解其激活和失活非常重要。一个主要的进展是开发了一种FRET指标的构象状态的CaMKII称为Camui。使用Camui的实验表明，开放（活性）构象在LTP诱导期间增加，然后在数十秒内衰减，主要快速组分以约6秒（tau 1）的时间常数衰减。因为如果T286的自磷酸化被阻止，这种衰变会更快（自磷酸化通过使酶即使在Ca 2+福尔斯下降后仍具有活性而抑制活性），所以快速衰变似乎可能是由于T286去磷酸化。为了测试这种解释，我们研究了磷酸酶抑制剂对双光子谷氨酸释放诱发的单棘Camui信号的影响。我们应用了PP 1和PP 2A的抑制剂，这两种磷酸酶存在于突触中，并已被证明在体外使CaMKII去磷酸化。抑制剂增加了基础Camui激活状态，表明它们在细胞中的有效性。然而，在任何情况下，我们都没有发现tau 1被延长，相反，如果衰变是磷酸酶依赖性的预期。这可能意味着衰变是由于一些未知的磷酸酶或衰变不是由于去磷酸化。为了区分这些可能性，我们表达了假磷酸化的Camui（T286 D）（加上阻止抑制性305/306磷酸化的额外突变[T/A]）。这种形式具有升高的基础激活状态，但在谷氨酸释放期间被进一步激活;重要的是，激活状态与几乎与WT Camui相同的tau 1衰减。因此，数据强烈表明，tau 1不是由于T286去磷酸化。我们的结论是，虽然Camui是一个很好的工具，观察CaMKII信号，需要进一步的实验，以确定CaMKII是如何关闭其去磷酸化。
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.
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