Curtis, Ashton J; Zhu, Jian; Studniarczyk, Dorota; Church, Timothy W; Farrant, Mark; Gold, Matthew G; (2025) Widely used CaMKII regulatory segment mutations cause tight actinin binding and dendritic spine enlargement in unstimulated neurons. Journal of Neuroscience , Article e0795252025. 10.1523/JNEUROSCI.0795-25.2025. (In press).

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Abstract

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is essential for long-term potentiation (LTP) of excitatory synapses, a process fundamental to learning. CaMKII responds to Ca²⁺ 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 indicates that LTP can proceed without kinase activity after this event. Furthermore, interactions between CaMKII and α-actinin-2 that form following LTP induction are required for dendritic spine enlargement. CaMKII can auto-phosphorylate at T286, which enables autonomous activity after Ca²⁺/CaM dissociation. 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 Ca2+/CaM activation. However, this mechanism is not fully compatible with previous studies including a phospho-proteomic analysis of CaMKII and imaging using CaMKII activity reporters in live neurons. In this study, we show using rat hippocampal cultures that the AA substitution has an unintended gain-of-function property: elevated binding to α-actinin-2 in unstimulated neurons to a level only normally observed after induction of LTP. CaMKIIα AA also increases 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 CaMKIIα AA 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. / / Significance statement: New memories are encoded in the brain by long-lasting changes in the strength of neuronal synapses. The most common form of synaptic strengthening is driven by the abundant calcium-sensitive enzyme CaMKII. Most research has focused on CaMKII phosphorylation of proteins including itself with the assumption that such enzymatic activity is key to its ability to strengthen synapses. However, it is becoming apparent that formation of protein-protein interactions involving CaMKII is more critical. Here, we show that two mutations commonly used to prevent auto-phosphorylation in a regulatory region of CaMKII have an unexpected gain-of-function property: they trigger tight binding to an actin crosslinking protein leading to synaptic enlargement. Our study supports the idea that CaMKII acts primarily as a structural protein.

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