Crosstalk of KCNH1 and KCNH5 gain-of-function mutations leading to epilepsy and neurodevelopmental disorders

Voltage-gated K⁺ (Kv) channels are tetrameric complexes of proteins encoded by KCN genes. Gain-of-function (GoF) mutations in KCNH1 (Kv10.1, hEAG1) and KCNH5 (Kv10.2, hEAG2) give rise to developmental disorders, intellectual disability, and epilepsy. Currently, clinical symptoms are not straightforwardly associated with functional properties of mutated channels. Here we investigated how members of the KCNH subfamily are affected by heteromerization with mutant Kv10.1 or Kv10.2 protein subunits. The de novo variant Kv10.1-G496E, which leads to impaired neurodevelopment and epilepsy, was expressed alone or with other wild-type subunits in HEK293T cells and characterized using whole-cell patch clamp. While Kv10.1-G496E alone did not yield functional K⁺ channels, coexpression with Kv10.1 or Kv10.2 shifted the half-maximum voltage of activation in the hyperpolarizing direction. Likewise, the homologous mutation Kv10.2-G465E did not yield functional channels but also induced GoF upon coexpression with wild-type Kv10.1 or Kv10.2. By contrast, the mutants did not affect the function of Kv11.1 (KCNH2, hERG1) channels. To infer the relevance of Kv10 GoF mutations under physiological conditions, we used the fluorescent genetically encoded voltage indicator mK2-rEstus and found that both, Kv10.1 and Kv10.2, hyperpolarized HEK293T cells, and that coexpression of the GoF mutants augmented this hyperpolarization. Our findings imply that interpretation of clinical symptoms related to Kv10 GoF mutations requires considering the functional crosstalk with Kv10.1 and Kv10.2 subunits, which are both expressed in glutamatergic neurons in cortical Layers III and IV.