Abstracts

Rationale: KCNQ2 and KCNQ3 encode voltage-gated potassium channel subunits that are broadly expressed in both the central and peripheral nervous system, where they control the excitability of neurons. Variants in KCNQ2 (or, more rarely, KCNQ3) cause benign familial neonatal epilepsy (BFNE). More recently, de novo KCNQ2 mutations have emerged as an important cause of early infantile epileptic encephalopathy (EIEE). To better understand the basis for the severe KCNQ2 encephalopathy phenotype resulting from these variants and the potential for therapy based on reversing the pathogenic effects pharmacologically, we functionally analyzed three different human de novo KCNQ2 missense variants at Ala265 (A265), and tested the effects of ICA-069673 and SF0034, two KCNQ2 openers with distinct binding mechanisms. Methods: Combinations of wild type (WT) KCNQ2, WT KCNQ3, and/or A265P, A265T or A265V were heterologously expressed at different ratios in Chinese hamster ovary (CHO) cells. At near physiological temperature (35?), currents were recorded under whole cell voltage clamp before and after application of 1 M SF0034 or 10 M ICA-069673. Results: The KCNQ2 variants studied, A265P, A265T and A265V were found in 1, 4 and 6 unrelated EIEE patients, respectively. Expressed in mammalian CHO cells, each of the variants alone yielded minimal currents just above the background. Expressed with WT KCNQ2 in a 1:1 ratio to mimic the heterozygous state, such co-expression yielded currents that did not reach half of control levels, indicative of dominant-negative effects. The G-V curve showed no significant effect on the voltage-dependence of activation. In second series of experiments, Co-expression of WT KCNQ2, the A265 variant, and wild type KCNQ3 in a ratio of 1:1:2 to mimic heterozygous heteromeric channels produced significant reductions in current density again without a shift in voltage-dependence. Activation and deactivation rates were not significantly changed except for A265T, which showed slower activation as well as faster deactivation at some subthreshold voltages. These findings suggested that these three variants all strongly reduce current densities, an in vitro signature found in many variants found in EIEE patient. Furthermore, we tested the effects of KCNQ channel openers, ICA-069673 and SF0034 and the combination of these two drugs on the WT and mutation channels. Both drugs increased current densities in cells expressing the variants, particularly in the subthreshold range (1M SF0034: 35033% of control, 10 M ICA-069673: 40029% of control at -40 mV, n=11~15). Conclusions: In CHO cells, these three de novo KCNQ2 encephalopathy mutations reduced current density strongly, with only minor effects on voltage-dependence of activation. KCNQ2/3 openers reverse some of the variant effects and merit consideration for development as treatments for this patient group. Funding: NINDS R01 NS49119, CURE Pediatric Epilepsy Award, SciFluor Life Sciences