Widely used CaMKII regulatory segment mutations cause tight actinin binding and dendritic spine enlargement in unstimulated neurons

Abstract Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is essential for long-term potentiation (LTP) of excitatory synapses that underlies learning. CaMKII responds to Ca 2+ influx into postsynaptic spines by phosphorylating proteins and forming new protein interactions. The relative importance of these enzymatic and structural functions is debated. LTP induction triggers CaMKII docking to NMDA receptors, and recent evidence suggests that LTP can proceed without kinase activity after this event. Furthermore, CaMKII interaction with α-actinin-2 is required for dendritic spine enlargement following LTP induction. CaMKII can auto-phosphorylate at T286, which enables autonomous activity after Ca 2+ /CaM dissociation. CaMKII also bears threonine at positions 305 and 306 in its regulatory segment. Experiments with CaMKII variants including a T305A/T306A (‘AA’) double substitution have led to a model whereby T305/T306 phosphorylation by autonomously active CaMKII prevents further Ca 2+ /CaM activation. However, this mechanism is not compatible with some existing data including CaMKII phospho-proteomics and measurements with reporters of CaMKII conformation. Furthermore, autonomous CaMKII activity is now thought to only endure for seconds after LTP induction. In this study, we show that the AA substitution has an unintended gain-of-function property – it enables tight binding to α-actinin-2 in unstimulated neurons. In situ labelling shows that the AA substitution elevates CaMKII-actinin interactions in neurons to a level only normally observed after induction of LTP. The AA CaMKII variant also elevates the proportion of enlarged spines in unstimulated neurons without altering synaptic currents. Calorimetric measurements with purified protein confirm that α-actinin-2 binds tightly to the AA variant of CaMKIIα with no requirement for kinase activation. Using x-ray crystallography, we show that the AA substitution enables α-actinin-2 to adopt a different tighter binding mode. Our findings reinforce the notion that CaMKII primarily fulfils a structural role in LTP.