Functional validation of human SK channels variants causing NEDMAB and Zimmermann–Laband syndrome-3 in <i>C. elegans</i>

Small conductance Ca²⁺-activated K⁺ channels (SK channels) are widely expressed in the central nervous system, where they play a crucial role in modulating neuronal excitability. Recent studies have identified missense variants in the genes encoding SK2 and SK3 channels as the cause of two rare neurodevelopmental disorders: NEDMAB and ZLS3, respectively. Here, we used <i>Caenorhabditis elegans</i> as an <i>in vivo</i> model to investigate the functional consequences of these patient variants. The <i>C. elegans</i> orthologue KCNL-1 regulates neuronal and muscle excitability in the egg-laying system, a well-characterized model circuit. To visualize KCNL-1 expression and localization, we generated a fluorescent translational reporter at the endogenous <i>kcnl-1</i> locus. We then introduced eight point mutations corresponding to pathogenic variants reported in NEDMAB or ZLS3 patients. Our study confirmed the molecular pathogenicity of the ZLS3-associated mutations, revealing a gain-of-function effect that led to increased <i>in utero</i> egg retention, likely due to electrical silencing of the egg-laying circuitry. NEDMAB mutations exhibited more complex phenotypic effects. Most caused a loss-of-function phenotype, indistinguishable from null mutants, while one displayed a clear gain-of-function effect. Additionally, a subset of NEDMAB variants altered KCNL-1 localization, suggesting an impairment in channel biosynthesis, trafficking or stability. These findings provide new insights into the molecular mechanisms underlying NEDMAB and ZLS3 physiopathology, enhancing our understanding of SK channel dysfunction in human disease. Moreover, they establish <i>C. elegans</i> as a robust and cost-effective <i>in vivo</i> model for rapid functional validation of new SK channel mutations, paving the way for future investigations.