Epileptic encephalopathy-related Kv2.1 mutants impair clustering but not neuronal excitability

The voltage-gated potassium channel Kv2.1, encoded by the epileptic encephalopathy-associated gene KCNB1, is a primary driver of delayed-rectifier K⁺ currents in neurons. These currents contribute to high-frequency firing by preventing depolarization block due to Na⁺ channel inactivation. Wild-type (WT) channels are localized at the soma, proximal dendrites, and the initial segment of the axon, forming aggregates (clusters) via their C-terminal proximal restriction and clustering domain (PRC). This study investigated the biophysical and functional consequences of two C-terminal truncation mutations (Y529^∗ and R579^∗), identified in patients with epileptic encephalopathy, which disrupt this critical clustering domain. The mutations induced a change of Kv2.1 subcellular distribution towards distal dendrites and the axon. Cluster formation was impaired, though not abolished, in neurons expressing mutated subunits together with endogenous WT subunits. Consistent with this clustering deficit, single-molecule imaging revealed altered lateral diffusion of mutated channels. While WT channels remained largely immobile (stabilized), mutated forms exhibited intermittent diffusion punctuated by transient immobilization events. The percentage of stabilized trajectories was lower for mutated channels in the soma, dendrites and distal axons but not in the proximal portion of the axons. The differential diffusive behavior of WT and mutated channels was reproduced by diffusion-capture simulations considering channels with different numbers of PRC domains (i.e. heterotetramers with mutant subunits) and labile scaffolding interactions. Patch-clamp recordings revealed no significant difference in excitability between WT- and mutant-expressing neurons. However, we observed increased firing in both conditions compared to non-transfected neurons. Altogether, our results suggest that 1) mutated subunits form heterotetramers with endogenous WT subunits; 2) several PRC per channel are needed to efficiently immobilize Kv2.1 in large clusters; 3) increasing Kv2.1 channel expression level, independently of clustering, could drive neuronal hyperexcitability.