Reduced axonal surface expression and phosphoinositide sensitivity in Kv7 channels disrupts their function to inhibit neuronal excitability in Kcnq2 epileptic encephalopathy

• Published in: Neurobiology of disease Vol. 118; pp. 76 - 93
• Main Authors: Kim, Eung Chang, Zhang, Jiaren, Pang, Weilun, Wang, Shuwei, Lee, Kwan Young, Cavaretta, John P., Walters, Jennifer, Procko, Erik, Tsai, Nien-Pei, Chung, Hee Jung
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.10.2018; Elsevier
• Subjects: Calmodulin; Current; Epileptic encephalopathy; Excitability; Kcnq2; Kv7 channels; Mutation; Phosphoinositide; Surface expression; Epileptic encephalopathy; Phosphoinositide; YFP; BFNE; EEG; Surface expression; AKAP; Excitability; Kv7 channels; K; AIS; TLE; ER; CaM; AP; Kcnq2; Mutation; Current; Calmodulin; PIP2
• ISSN: 0969-9961; 1095-953X
• DOI: 10.1016/j.nbd.2018.07.004

Neuronal Kv7/KCNQ channels are voltage-gated potassium channels composed of Kv7.2/KCNQ2 and Kv7.3/KCNQ3 subunits. Enriched at the axonal membrane, they potently suppress neuronal excitability. De novo and inherited dominant mutations in Kv7.2 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 Kv7.2. 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 Kv7.2. These mutations also altered gating modulation by phosphatidylinositol 4,5-bisphosphate (PIP2), revealing novel PIP2 binding residues. While these mutations disrupted Kv7 function to suppress excitability, hyperexcitability was observed in neurons expressing Kv7.2-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 Kv7 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 Kv7.2, whereas the presence of Kv7.3 blocked this degradation. Furthermore, expression of Kv7.2-M518V increased neuronal death. Together, our results demonstrate that epileptic encephalopathy mutations in helices A and B of Kv7.2 cause abnormal Kv7 expression and function by disrupting Kv7.2 binding to CaM and/or modulation by PIP2. We propose that such multiple Kv7 channel defects could exert more severe impacts on neuronal excitability and health, and thus serve as pathogenic mechanisms underlying Kcnq2 epileptic encephalopathy. [Display omitted] •Epileptic encephalopathy mutations enriched in calmodulin binding sites of Kv7.2•Impairment of calmodulin binding by M518V mutation at the calmodulin contact site•Loss of current and axonal surface expression of Kv7 channels by M518V mutation•Abnormal PIP2 sensitivity by mutations peripheral to the calmodulin contact site•Expression of mutant Kv7.2 fails to reduce excitability or causes cell death.