Förster resonance energy transfer-based kinase mutation phenotyping reveals an aberrant facilitation of Ca/calmodulin-dependent CaMKIIα activity in mutations related to intellectual disability.

CaMKIIα plays a fundamental role in learning and memory and is a key determinant of synaptic plasticity. Its kinase activity is regulated by the binding of Ca/CaM and by autophosphorylation that operates in an activity-dependent manner. Though many mutations in CAMK2A were linked to a variety of neurological disorders, the multiplicity of its functional substrates renders the systematic molecular phenotyping challenging. In this study, we report a new case of CAMK2A P212L, a recurrent mutation, in a patient with an intellectual disability. To quantify the effect of this mutation, we developed a FRET-based kinase phenotyping strategy and measured aberrance in Ca/CaM-dependent activation dynamics and in synaptically connected neurons. CaMKIIα P212L revealed a significantly facilitated Ca/CaM-dependent activation . Consistently, this mutant showed faster activation and more delayed inactivation in neurons. More prolonged kinase activation was also accompanied by a leftward shift in the CaMKIIα input frequency tuning curve. In keeping with this, molecular phenotyping of other reported CAMK2A mutations linked to intellectual disability revealed aberrant facilitation of Ca/CaM-dependent activation of CaMKIIα in most cases. Finally, the pharmacological reversal of CAMK2A P212L phenotype in neurons was demonstrated using an FDA-approved NMDA receptor antagonist memantine, providing a basis for targeted therapeutics in CAMK2A-linked intellectual disability. Taken together, FRET-based kinase mutation phenotyping sheds light on the biological impact of CAMK2A mutations and provides a selective, sensitive, quantitative, and scalable strategy for gaining novel insights into the molecular etiology of intellectual disability.