Abstracts

Rationale: Inherited KCNQ2 mutations are the most frequent cause of benign familial neonatal seizures (BFNS). Recent clinical case series have identified novel KCNQ2 missense mutations, typically de novo, in patients with severe early-onset epileptic encephalopathy (Q2EE) [1, 2]. Previously, we analyzed the locations of Q2EE mutations, and hypothesized that these mutations might often act as strong dominant-negatives, capable of producing up to 8-fold lower current with heterozygous expression than null alleles. Experimental confirmation of that hypothesis would have implications for therapy. Methods: We introduced missense mutations from Q2EE patients into KCNQ2 cDNAs in the mammalian expression vector, pcDNA3. We used KCNQ2 G279S, a variant with known dominant-negative properties in vitro and in vivo, as a control. We obtained whole cell voltage-clamp recordings from CHO cells transfected with wild-type (wt) and mutant constructs. Results: Expression of KCNQ2 alone or KCNQ2 and KCNQ3 together gave large currents. Replacement of KCNQ2 with a 1:1 mix of KCNQ2 and empty vector reduced current 50% as expected. Co-expression of wt KCNQ2 with mutants G279S, T274M, and G290D 1:1 yielded currents of between ~6 and 15% of wild type (+40 mV/1 sec). Heteromeric KCNQ2/KCNQ3 currents were also strongly suppressed by coexpression of mutant KCNQ constructs. Conclusions: Although partial dominant-negative effects have been seen in KCNQ2 mutations affecting the voltage sensor, previous studies of pore loop mutations causing BFNS have shown only partial loss-of-function. The Q2EE mutations we studied, by contrast, exhibit dominant negative suppression, to levels near the maximum predicted by a model assuming equal subunit expression and random association. Further experiments in vitro and in vivo are needed to fully elucidate the basis for this suppression and its relationship to clinical KCNQ2 encephalopathy. Nonetheless, suppression is subtotal. This indicates by the raudom associated model that a small proportion of wt channels remain, lacking any mutant subunits. These residual functional channels are potential targets for KCNQ2 opener therapy.