Abstract

CaMKII is a multifunctional kinase essential for synaptic plasticity and memory formation. While its canonical role involves enzymatic phosphorylation, recent evidence suggests CaMKII also functions through liquid-liquid phase separation (LLPS) with substrate proteins, including GluN2B-containing NMDA receptors. However, the physiological significance remains unclear. Here, we generated CaMKII subtype knock-in (KI) mice carrying a point mutation (I205K) in the hydrophobic pocket, a key interface required for LLPS. These mice exhibited a complete loss of structural long-term potentiation (sLTP) despite normal spine morphology, marked hyperactivity and profound deficits in aversive memory formation. Atomoxetine, an approved attention-deficit/hyperactivity disorder (ADHD) treatment, ameliorated the hyperactive phenotype. Notably, we identified a patient carrying the I205N variant presenting with ADHD and mild intellectual disability, mirroring the behavioral features observed in I205K KI mice. Additional neurodevelopmental disorder-associated variants within the same hydrophobic pocket similarly disrupted LLPS in vitro. Molecular dynamics simulations revealed these variants destabilize the CaMKII-GluN2B interaction through distinct mechanisms that perturb the dynamic stability of the binding interface. These findings establish CaMKII-mediated phase separation as critical for linking synaptic molecular assembly to cognitive function and provide a unifying molecular basis for synaptic disorganization, hyperactivity, and memory deficits associated with neurodevelopmental disorders.