Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy

K_v7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability. Mutations in K_v7.2 and K_v7.3 subunits cause epileptic encephalopathy (EE), yet the underlying pathogenetic mechanism is unclear. Here, we used novel statistical algorithms and structural modeling to identify EE mutation hotspots in key functional domains of K_v7.2 including voltage sensing S4, the pore loop and S6 in the pore domain, and intracellular calmodulin-binding helix B and helix B-C linker. Characterization of selected EE mutations from these hotspots revealed that L203P at S4 induces a large depolarizing shift in voltage dependence of K_v7.2 channels and L268F at the pore decreases their current densities. While L268F severely reduces expression of heteromeric channels in hippocampal neurons without affecting internalization, K552T and R553L mutations at distal helix B decrease calmodulin-binding and axonal enrichment. Importantly, L268F, K552T, and R553L mutations disrupt current potentiation by increasing phosphatidylinositol 4,5-bisphosphate (PIP₂), and our molecular dynamics simulation suggests PIP₂ interaction with these residues. Together, these findings demonstrate that each EE variant causes a unique combination of defects in K_v7 channel function and neuronal expression, and suggest a critical need for both prediction algorithms and experimental interrogations to understand pathophysiology of K_v7-associated EE.