ATP1A3 E815K Disrupts Sodium Homeostasis and Excitability in Human Alternating Hemiplegia of Childhood Neurons
Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disorder primarily caused by heterozygous de novo mutations in the ATP1A3 gene, which encodes the neuron-specific catalytic 3 subunit of the P-type Na/K-ATPase. Among these, the E815K variant is the second most frequent ATP1A3 mutation in AHC and is associated with the most severe clinical phenotypes, characterized by frequent epileptic and dystonic episodes and profound developmental impairment, making it a key target for mechanistic study. Although E815K was shown to disrupt pump function in heterologous systems and animal models, its impact on human neurons remain poorly understood. To investigate its disease-relevant pathophysiology, we generated induced pluripotent stem cells (iPSCs) from an AHC patient carrying the E815K mutation and differentiated them into mature neurons. Patient-derived neurons displayed normal differentiation and synaptogenesis, with preserved evoked synaptic transmission. However, 3-specific Na/K pump currents were nearly abolished, whereas 1 activity remained intact. This selective dysfunction was associated with a depolarized resting membrane potential, reduced input resistance, and diminished intrinsic excitability. Patient neurons generated fewer action potentials in response to depolarizing current steps, displayed altered firing patterns, and showed significantly decreased voltage-gated sodium current densities, while potassium conductance remained unaffected. Taken together, these results indicate that the ATP1A3 E815K mutation disrupts intracellular sodium homeostasis and consequently modifies the excitability of human neurons. Overall, these findings provide mechanistic insight into the molecular pathophysiology underlying this AHC variant and thereby establish a patient-derived neuronal model which serves as a platform for future drug screening approaches and therapeutic investigation.