Reduced axonal surface expression and phosphoinositide sensitivity in K_v7 channels disrupts their function to inhibit...

Neuronal K_v7/KCNQ channels are voltage-gated potassium channels composed of K_v7.2/KCNQ2 and K_v7.3/KCNQ3 subunits. Enriched at the axonal membrane, they potently suppress neuronal excitability. De novo and inherited dominant mutations in K_v7.2 cause early onset epileptic encephalopathy characterized by drug resistant seizures and profound psychomotor delay. However, their precise pathogenic mechanisms remain elusive. Here, we investigated selected epileptic encephalopathy causing mutations in calmodulin (CaM)-binding helices A and B of K_v7.2. We discovered that R333W, K526N, and R532W mutations located peripheral to CaM contact sites decreased axonal surface expression of heteromeric channels although only R333W mutation reduced CaM binding to K_v7.2. These mutations also altered gating modulation by phosphatidylinositol 4,5-bisphosphate (PIP₂), revealing novel PIP₂ binding residues. While these mutations disrupted K_v7 function to suppress excitability, hyperexcitability was observed in neurons expressing K_v7.2-R532W that displayed severe impairment in voltage-dependent activation. The M518 V mutation at the CaM contact site in helix B caused most defects in K_v7 channels by severely reducing their CaM binding, K⁺ currents, and axonal surface expression. Interestingly, the M518 V mutation induced ubiquitination and accelerated proteasome-dependent degradation of K_v7.2, whereas the presence of K_v7.3 blocked this degradation. Furthermore, expression of K_v7.2-M518V increased neuronal death. Together, our results demonstrate that epileptic encephalopathy mutations in helices A and B of K_v7.2 cause abnormal K_v7 expression and function by disrupting K_v7.2 binding to CaM and/or modulation by PIP₂. We propose that such multiple K_v7 channel defects could exert more severe impacts on neuronal excitability and health, and thus serve as pathogenic mechanisms underlying Kcnq2 epileptic encephalopathy.