Forward genetics reveals a conserved CNBHD-CNBHD interface that restrains EAG channel activation to regulate sleep.

KCNH voltage-gated potassium channels regulate neuronal excitability, yet how their hallmark cyclic nucleotide-binding homology domains (CNBHDs) influence channel gating to regulate behavior remains unclear. Using an unbiased forward genetic screen in Caenorhabditis elegans, we identified a gain-of-function mutation in the EAG-subfamily KCNH channel EGL-2 that suppresses sleep. The mutation (G574E) alters a highly conserved glycine residue at the interface between CNBHDs of adjacent channel subunits. EGL-2 functions cell-autonomously in the central sleep-promoting neuron ALA. In vivo calcium imaging showed that the gain-of-function mutation suppresses sleep by inhibiting ALA neuronal activity, consistent with enhanced potassium channel activity. Genetic analysis demonstrated that the sleep-suppressing phenotype of G574E requires both an intact potassium selectivity filter and the intrinsic ligand-occupied CNBHD. A second substitution at the same glycine (G574R), which introduces a positively charged side chain in contrast to the negatively charged side chain in G574E, similarly suppresses sleep, indicating that perturbation of the CNBHD-CNBHD interface, rather than the specific amino acid change, underlies the phenotype. Moreover, mutations at the corresponding residue in the ERG-subfamily KCNH channels C. elegans UNC-103 and human hERG also produce gain-of-function effects, and electrophysiological analysis showed that hERG(G749R) shifts voltage-dependent channel activation to more negative potentials and accelerates activation kinetics. These findings identify EGL-2 as an important sleep regulator and uncover a conserved molecular mechanism by which the CNBHD-CNBHD interface restrains KCNH channel activation to regulate neuronal excitability and behavior.