Abstracts

Rationale: Many epilepsy-related phenotypes in mice, including induced, spontaneous convulsive and non-convulsive seizures, are significantly modified by genetic background. The identification of the underlying genes is valuable as modifier effects are likely to contribute to genetic complexities of human epilepsy. Loss of function mutations in Gria4 (encoding GluRD) were previously shown to cause absence seizures in mice (Beyer et al. 2008 Hum Molec Genet. 17:1738-49). Recently a genetic modifier of this phenotype was mapped to a region of Chr 15 that harbors Kcnq3 (Frankel et al. 2014 PLOS Genetics, 10: e1004454), encoding the Kv7.3 potassium channel. KCNQ3 variants are known to be associated with diverse epilepsies, ranging from benign neonatal seizures to severe epileptic encephalopathy. Public sequence data revealed that several mouse strains encode an in-frame 3 amino acid deletion at the distal N-terminus of Kv7.3, including the C3H strain which contributed the enhancer allele – tripling Gria4 spike-wave discharge (SWD) incidence and doubling duration with great variation, including some mice that had SWD lasting over 1 minute. Interestingly, two other strains, CBA and NOD with this in-frame variant also have spontaneous SWD (Letts et al. 2014 Genes Brain Behav. 13:519-26).Methods: To test whether this deletion variant confers altered channel function, we cloned the N-terminal region of wild-type (wt) and deleted mouse Kcnq3 (from mouse strains C57BL/6J and C3HeB/FeJ, respectively) into the pIRES expression vector containing rat Kcnq3 and mouse Kcnq2, whose co-expression is required for the classical M-current.Results: Heterologous expression in HEK293T cells revealed that mutant Kcnq3 conferred a significant increase in current amplitude at resting membrane potentials. This effect is likely due to increase PIP2 affinity in the mutant as suggested by the faster recovery of the KCNQ2/3 currents following PIP2 depletion using a voltage-activated phosphatase. Currently we are extending these studies, and also to confirm its causality as the modifier we are targeting the deletion de novo in the C57BL/6J strain. Once confirmed we will determine whether this deletion variant affects other seizure types.Conclusions: KCNQ or Kv7 channels respond to action potentials by raising their threshold. As such they are attractive as modifier candidates and as targets for anti-seizure treatment, and thus there is much attention on K+ channel openers (such as retigabine) that act on Kv7 channels. The mechanism of this gain-of-function effect is not known, although we suspect that PIP2 regulation may be involved because of its role in controlling the KCNQ2/3 channel probability of opening and thus current amplitude.