Disruption of CaMKII biomolecular condensation induces attention-deficit/hyperactivity disorder-like phenotypes

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 α 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 CaMKIIα–GluN2B interaction through distinct mechanisms that perturb the dynamic stability of the binding interface. These findings establish CaMKIIα-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.