Fig 1.
A. Molecular structure of the KCNQ2 subunit. The KCNQ2 subunit has six transmembrane domains (S1–S6), of which the 4th transmembrane domain (S6) functions as a voltage sensor, whereas a loop between the 5th and 6th transmembrane domains (S5 and S6, respectively) forms an ion pore along with three other counterpart subunits. In general, the KCNQ2 and KCNQ3 subunits form a heterotetrametric potassium channel, i.e., Kv7.2/Kv7.3. The Kv7.2/Kv7.3 channel generates M-currents that control the subthreshold excitability of the membrane. p.Tyr284 and p.Ala306, where the mutations examined in this study (Y284C and A306T) are located, occur at the outer mouth and the inner lining (S6) of the channel pore, respectively. B. Three dimensional images of the Kv7.2/Kv7.3 channels bearing Y284C and A306T. The images indicate the outer mouth of the channel seen from an extracellular position. Left: The heterotetrametric Kv7.2/Kv7.3 channel consists of two KCNQ3 subunits (Black), one normal KCNQ2 subunit (White), and the mutant KCNQ2 subunit (Red) bearing the Y284C mutation. The cysteine residue introduced by the Y284C mutation, which is indicated with balls, is located at the outer mouth of the channel. Right: The heterotetrametric Kv7.2/Kv7.3 channel consists of two KCNQ3 subunits, one normal KCNQ2 subunit, and one mutant KCNQ2 subunit bearing the A306T mutation. The tyrosine residue introduced by the A306T mutation, which is indicated with balls, is located at the inner lining of the channel pore. C. Spike burst. Top: A spike burst recorded during a kainic acid challenge test on an electroencephalogram (EEG) is indicated with a double sided arrow Bottom: A simultaneous electromyogram (EMG) recording. A spike burst is a cluster of high-amplitude and high frequency spikes, each of which lasts a few seconds to several minutes.
Table 1.
Modified Racine’s scale.
Fig 2.
Representative electroencephalograms at each score of Modified Racine’s scale.
Electroencephalogram (EEG) and electromyogram (EMG) were obtained as described in Method. Each panel shows a representative EEG recording at Score 0 to 5 of a Modified Racine’s scale [19]. Spikes and sharp waves were considered part of seizure activities only when they were associated with abnormal behaviors which were confirmed on video monitoring and electromyogram. A spike burst is defined as a cluster of high-amplitude and high frequency spikes, each of which lasts a few seconds to several minutes on EEG during kainic acid challenge tests [17].
Fig 3.
Relationship between heterozygous Kcnq2 mutations and seizures.
A. The distribution of seizure scores in WT mice (n = 13) (upper panel, in white color), Kcnq2Y284C/+ mice (n = 6) (middle panel, in gray color), and Kcnq2 A306T /+ mice (n = 7) (lower panel, in light gray color). Differences in the distribution of seizure scores among and between groups were examined by the logistic regression analysis, as described in the Methods. B and C. Box-and-whisker plots showing the mean (●), median (middle bar in the rectangle), and 10th (bottom bar), 25th (bottom of rectangle), 75th (top of rectangle), and 90th (top bar) percentiles of the number (B) and duration (C) of spike bursts in WT (n = 13) (in white color), Kcnq2Y284C/+ (n = 6) (in gray color), and Kcnq2 A306T /+ (n = 7) mice (in light gray color). The number and duration of spike bursts were compared among and between groups by using Negative binomial model, as described in the Methods. Raw data for bar and box plots are included in S1 File.
Fig 4.
Effects of phenobarbital (PB) and retigabine (RTG) at low and high doses on seizures in Kcnq2Y284C/+ mice.
A. Distribution of seizure scores in Kcnq2Y284C/+ mice administered vehicle (n = 6) (upper panel, in gray color), PB at doses of 5 mg/kg (n = 7) and 15 mg/kg (n = 7) (middle panel, in light gray and gray color, respectively), or RTG at doses of 5 mg/kg (n = 10) and 15 mg/kg (n = 10) (lower panel, in light gray and gray color, respectively). Between and within drug differences in the distribution of seizure scores were examined by the Jonckheere−Terpstra test, as described in the Methods. B and C. Box-and-whisker plots showing the mean (●), median (middle bar in the rectangle), and 10th (bottom bar), 25th (bottom of rectangle), 75th (top of rectangle), and 90th (top bar) percentiles of the number (B) and duration (C) of spike bursts in Kcnq2Y284C/+ mice administered vehicle (n = 6), PB at doses of 5 mg/kg (n = 7) and 15 mg/kg (n = 7), or RTG at doses of 5 mg/kg (n = 10) and 15 mg/kg (n = 10). PB and RTG at doses of 5 mg/kg are shown in light gray color. Between and within drug differences in terms of the number and duration of spike bursts were examined using Negative binomial model, as described in the Methods. Raw data for bar and box plots are included in S1 File.
Fig 5.
The effects of phenobarbital (PB) and retigabine (RTG) at low and high doses on seizure in Kcnq2A306T/+ mice.
A. Distribution of seizure scores in Kcnq2A306T/+ mice administered vehicle (n = 7) (upper panel, in gray color), PB at doses of 5 mg/kg (n = 5) and 15 mg/kg (n = 7) (middle panel, in light gray and gray color, respectively), or RTG at doses of 5 mg/kg (n = 6) and 15 mg/kg (n = 8) (lower panel, in light gray and gray color, respectively). Between and within drug differences in the distribution of seizure scores were examined by the Jonckheere−Terpstra test, as described in the Methods. B and C. Box-and-whisker plots showing the mean (●), median (middle bar in the rectangle), and 10th (bottom bar), 25th (bottom of rectangle), 75th (top of rectangle), and 90th (top bar) percentiles of the number (B) and duration (C) of spike bursts in Kcnq2A306T/+ mice administered vehicle (n = 7), PB at doses of 5 mg/kg (n = 5) and 15 mg/kg (n = 6), or RTG at doses of 5 mg/kg (n = 6) and 15 mg/kg (n = 8). PB and RTG at doses of 5 mg/kg are shown in light gray color. Between and within drug differences in terms of the number and duration of spike bursts were examined using Negative binomial model, as described in the Methods. Raw data for bar and box plots are included in S1 File.