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

Abstract

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.

CaMKII Autophosphorylation Is Necessary for Optimal Integration of Ca 2+ Signals during LTP Induction, but Not Maintenance

“…This result suggests that Thr286 phosphorylation is required for optimal integration of Ca 2+ signals (Figure 2). The fast decay of CaMKIIα T286D activity, perhaps due to the dissociation of Ca 2+ / CaM, suggests that dephosphorylation of Thr286 plays an important role in determining the optimum decay time (6 – 8 s) of CaMKII (but see also (Otmakhov et al, 2015)).…”

Memory Erasure Experiments Indicate a Critical Role of CaMKII in Memory Storage

“…During this period, autophosphorylation of CaMKII integrates the Ca 2+ signal (Lee et al, 2009) (but see (Otmakhov et al, 2015)). A key finding was that a photo-activatable CaMKII inhibitor, AIP2, could block LTP induction when activated during the first minute after induction but had no effect if activated during subsequent periods (Murakoshi et al, 2017).…”

Counteracting Protein Kinase Activity in the Heart: The Multiple Roles of Protein Phosphatases

“…Indeed several examples (see also Table 1 ) show direct PP-mediated dephosphorylation of PKs in cardiac cells. However, PP-mediated dephosphorylation of two of the most abundant and essential PKs in cardiac myocytes, namely PKA and CaMKII has only be studied in non-cardiac tissues until today ( Blitzer et al, 1998 ; Bradshaw et al, 2003 ; Humphries et al, 2005 ; Otmakhov et al, 2015 ).…”