Abstracts

Rationale: Mutations in the KCNQ2 gene encoding for K+ channel subunits are responsible for early-onset neonatal seizures with wide clinical outcome, ranging from Benign Familial Neonatal Seizures (BFNS) (Bellini et al., 2010, GeneReviews®) to severe Early-Onset Epileptic Encephalopathy (EOEE) (Weckhuysen et al., 2012, Ann Neurol 71:15-25). The molecular basis for such phenotypic heterogeneity is unknown, but the mutation-induced functional changes in the ionic currents seem to play a major role (Miceli et al., 2013, Proc Natl Acad Sci USA 110:4386-4291).Methods: Mutations found in EOEE-affected patients (A265T, R325G, and S195P) (Weckhuysen et al., 2013, Neurology 81:1697-1703) were engineered in a KCNQ2-encoding plasmid for mammalian expression. Wild-type (WT) and mutant plasmids were then transiently transfected in Chinese Hamster Ovary (CHO) cells to investigate the mutation-induced biochemical and functional consequences and the ability of KCNQ modulators to counteract such changes.Results: Patch-clamp recordings performed 24 h after transfection revealed that homomeric KCNQ2 A265T or R325G mutant channels were non-functional. When mutant channels were expressed in heteromeric configuration with WT KCNQ2 and KCNQ3 subunits, a significant reduction in maximal current density was measured, suggesting a loss-of-function as a pathogenetic mechanism. Biotinylation assays revealed that mutations-induced KCNQ2 currents reduction was not due to changes in plasma membrane levels of mutant subunits. Exposure to the KCNQ opener retigabine (10 μM) (Barrese et al., 2010, Clin Pharmacol 2:225-236) significantly increased KCNQ2/KCNQ2 A265T/KCNQ3 or KCNQ2/KCNQ2 R325G/KCNQ3 current density, rescuing defective channels to wild-type levels. By contrast, KCNQ2 S195P mutant subunits, both when expressed in homomeric or heteromeric configuration with KCNQ2/3 subunits, showed a significant increase in the maximal current density and a robust leftward shift in the voltage-dependence of activation, revealing that a gain-of-function mechanism is instead associated to this mutation, as it occurs with other mutations affecting the proximal S4 region (Miceli et al., 2015, J Neuroscience 35:3782-3793). Exposure to the potent and selective KCNQ2 blocker ML252 (100 nM) (Cheung et al., 2012, J Med Chem 55:6975-6979) similarly reduced by about 50% the currents carried by WT or KCNQ2 S195P homomeric channels.Conclusions: Altogether, these results suggest that EOEE-associated mutations can lead to divergent biophysical consequences on KCNQ2 channels, ranging from gain- to loss-of function effects. These mutation-specific effects should be taken into account when planning patient-tailored pharmacological treatments. Sources of funding. This work has been supported by Telethon Italy and the University of Molise.