EEG of a bilateral onset seizure in Gly256Trp/+ individual 1, age 16 days.

The recording is continuous from A to B. A. Seizure onset with diffuse, bilateral amplitude EEG attenuation (red arrow), which is obscured in several electrodes by high frequency muscle artifact (muscle artifact is better seen in Figure 1—figure supplement 1, movie). B. Seizure electrographic evolution to post-ictal voltage attenuation (red arrow). Settings: LFF 3 Hz, HFF 70 Hz, sensitivity 7uV/mm, 35 sec/panel.

EEG recording including pre-ictal, post-ictal attenuation and recovery of background of seizure excerpted in Figure 1.

As labeled, onset was preceded by eyeblink and muscle artifact. The interval of uninterrupted voltage attenuation between the end of the high voltage fast activity to the first epileptiform burst was 61 sec. Interburst length progressively shortened in length, over about 3 min. Settings as in Figure 1. Link to movie F1-S1.

Examples of awake and sleep EEG background. There is evidence of state change, with more discontinuity during sleep. The awake excerpt shows variable frequency composition, and excess multifocal sharps. Excess discontinuity and sharps indicate dysmaturity, but burst-suppression is not seen. Settings as in Figure 1, except LFF changed to 1 Hz.

Gly256 is linked to the selectivity filter bridge segment via a hydrogen bond network among residues distinct to KCNQ2.

A, B. Cartoons showing KCNQ2 membrane topology, including transmembrane segments S1-S6 and the P-loop (turret segment, purple; H5 or P-helix, cyan; and selectivity filter segment, yellow). Positions of the K+ selective pore, and the G256W substitution within the turret are indicated. C. Alignment of human KCNQ4 and KCNQ5 sequences with KCNQ2 sequences of major vertebrate groups. Background colors match panels A-B, and the five selectivity filter lining residues are boxed in red. At four aligned positions within the turret and one in the SFB, KCNQ2 substitutions have evolved in amphibians and tetrapods (residues highlighted in red). D. Rendering of the WT KCNQ2-calmodulin tetrameric structure obtained by cryoEM (PDB 7cr3), highlighting one subunit and the position of the G256W substitution near the channel’s extracellular domain apex. The Trp256 sidechain is at scale but its rotamer is chosen arbitrarily. The subunit closest to the viewer is partially deleted to reveal the highlighted subunit more clearly. E. Ribbon rendering of the extracellular part of the PGD. For clarity, only two opposing side subunits are shown (as schematically in A). A Trp side chain is added at one Gly256 α-carbon. The distance between the G256 α-carbon and Y280 carbonyl oxygen at the selectivity filter mouth is labeled. F. Top down view of the KCNQ2 regions as in panel E, but showing 4 subunits. The Trp rotamer is different from panels D-E. The S5, S6 and P-helices are labeled. G. Hydrogen bonding network of the KCNQ2 turret. All predicted bonds are shown as dashed orange lines. The network extends from the S5 helix (Y251) via the labelled turret residue atoms to bonds involving residues of the SFB. As in C, five residues that diverge invertebrates are colored red. H, I. The turret peptide region near G256, which is boxed with a grey dashed line in G. The main chain is shown as ball-and-stick; side chains as stick. A tight turn occurs at K255 to N258, stabilized by hydrogen bonding between the G256 carbonyl oxygen and N258 amide. I. The G256-E257 peptide deviates from planarity (ω = +/-180°) by 11.6° (∼2.6 sd). In and out arrows indicate N and C termini, respectively. Abbreviations: mya, million years ago; VSD, voltage-sensor domain; HA-HB, the cytoplasmic helices A and B; CaM, calmodulin.

Movie illustrating position of the G256W substitution within the KCNQ2 channel pore turret and its distance to the selectivity filter. Link to movie F2-S1

The G256W variant affects a divergent neuronal KCNQ turret structure enabling forming a bonding network linked to the ion selective pore.

A-C. Aligned structural models of the extracellular portions of PGDs of KCNQ1, KCNQ4 and KcsA with that of KCNQ2. Single subunits are shown. D. Cartoon of structural model of turret region of KCNQ4 highlighting the predicted hydrogen bonding network. Several bonds are conserved between KCNQ2 and KCNQ4, but the KCNQ4 network has fewer bonds (compare with Figure 2G). E. Cladogram summarizing evolutionary relationships among several voltage-gated potassium channel genes. Gene duplication(s) are indicated by red circles, and are labeled by a common ancestor (or their extant descendant) possessing both duplicate genes. F. Sequence alignments of KCNQ1-5 P loops and flanking S5 and S6 regions reveal relative conservation of KCNQ5, KCNQ4, and KCNQ2 (as in Figure 2C), and divergence of KCNQ1 and KCNQ3.

Movie illustrating locations of residues contributing to a non-covalent bonding network extending from S5 to the selectivity filter. Link to movie F2-S3

KCNQ2 G256W co-expression suppresses current in KCNQ2/KCNQ3 heteromeric channels.

A. Cartoon showing the expected combinations of WT and G256W subunits under heterozygosity based on a simple random association model and preferred 2:2 stoichiometry for KCNQ2 and KCNQ3. B-G. In vitro dissection of effects of G256W heterozygosity on currents. B-C. Mean current families are shown for the indicated combinations of expression of KCNQ2 and KCNQ3 prior to and after addition of 10 μM ezogabine (n = 60, 50; 40, 31; 28, 24 for the upper, middle, and lower conditions). ScaleD-E. Current/voltage and conductance/voltage relationships for the indicated WT only and G256W/WT electroporations into KCNQ3 stable expressing cells. F. Current/voltage relationship for G256W (‘homozygous”) heteromeric channels, compared with the subset of WT control cells studied in parallel by automated patch recording. G. Replot of data from panel D. At each voltage, mean current is normalized to mean current at +40 mV in absence of ezogabine.

KCNQ2 G256W co-expression suppresses current in KCNQ2/KCNQ3 heteromeric channels recorded by manual patch-clamp.

A. Representative current families for the indicated ratios of subunits. Note currents are larger than in Figure 3. B-C. Current/voltage and conductance/voltage relationships for the indicated WT only and G256W/WT cells.

KCNQ2 G256W co-expression suppresses current in KCNQ2 homomeric channels recorded by manual patch-clamp.

A. Representative current families for the indicated ratios of subunits. Homomeric currents are smaller than in Figure 3—Figure supplement 1. B-C. Current/voltage and conductance/voltage relationships for the indicated WT only and G256W/WT cells. D. Cartoon showing the expected combinations of WT and G256W subunits under heterozygosity based on a simple random association model. Mutant subunits are included in 15/16 of channel tetramers.

Immature heterozygous G256W mice exhibit normal development and have infrequent epileptic seizures.

A. Upper, map of the Kcnq2 constructs. Lower, sequence alignments for the region between the middle of exon 5 and the beginning of exon 6. Although the human G256W variant is a single base substitution, Crispr/Cas9 editing introduced two substitutions, since the WT G256 codons differ between mouse (GGT) and human (GGG). Also aligned is the DNA and protein sequences of the frameshift mutation. B. WT and G256W/+ mice showed no difference in weight gain during development. C. WT and G256W/+ mice performed similarly in the developmental milestone assays for negative geotaxis, surface righting, and cliff aversion. D-H. Screenshots of stages of a generalized seizure in a P10 G256W/+ mouse (see also: Figure 4—Figure supplement 3-Movie). D. Onset with immobility and myoclonic tail and forelimb shaking. E. Abrupt fall to side with flexion posturing. F. Evolution to hindlimb and tail extension posture. G. Immobility with flaccid appearance, interrupted by brief episodes of tail, individual limb myoclonus or clonus. H. Arouses, quickly regains upright posture, then normal mobility. Labels: time in 15 min source video.

DNA, RNA, and predicted protein consequences of the G256W and E254fs*16 mutations.

Upper, DNA and predicted protein alignment of the frameshift mutation. Lower, Sanger sequence for cDNA from hippocampal mRNA of an E254fs/+ mouse. Splicing occurs at the WT junction, resulting in the predicted in-frame stop codon.

Western blotting reveals no evidence of the predicted E254fs truncated protein product.

A. Western blot of WT and E254fs/+ cortical homogenates (3 biological replicates per genotype, all males), probed with KCNQ2 N-terminal antibody. Black arrow indicates the monomer (Mr ∼85kDa), red arrow indicates the estimated relative mobility (∼29.7 kDa), of the truncated protein product made from the E254fs allele. Red asterisk indicates a ∼28 kDa band equally detected in both WT and E254fs/+. B. Same blot as in A but windowed to show higher molecular weight bands. Bands at ∼180 kDa and ∼360 kDa consistent with predicted mobility of KCNQ2 dimers and tetramers, respectively. A band at ∼250 kDa appears in all immunoblots of whole brain homogenates using our KCNQ2 N-terminal antibody. Nano-LC tandem mass spectrometry of peptides from an in-gel tryptic digest of this band showed high peptide counts for multiple abundant proteins and few KCNQ2 peptides (Supplementary Data). C. Quantification of ∼28 kDa band from A.

Generalized seizure in a P10 heterozygous G256W mouse.

This movie includes from 9:01 to 11:05 of a 15:00 min period of open field observation. Animal recovers upright posture at 1:45 in the clip. Link to movie F4-S3

Heterozygous G256W mice have increased CA1 pyramidal cell excitability.

A. Representative voltage responses to increasing current injection steps (step duration 1 sec) in CA1 pyramidal neurons from WT and G256W/+ mice. The resting membrane potential was held at -65 mV. B. Representative voltage responses to decreasing current injections steps (1s) in CA1 pyramidal neurons from WT and G256W/+ mice. C. Time to first action potential following step stimulus is not significantly different between groups (3 animals per group; WT and G256W/+, n=16 cells each).D. Summary graph showing the effect of one copy of G256W on the action potential count (3 animals per group; WT and G256W/+, n=16 cells each, F(12,180)=5.8, **** is P<0.0001).. Data are presented as mean and s.e.m.

Several neuronal biophysical properties are unchanged.

A. Resting membrane potential. B. Action potential amplitude. C. Action potential width. D. Action potential rise slope. E. Action potential decay. F. Input resistance. For all panels, 3 animals per genotype, n=16 cells/genotype).

Convulsive seizures in adult heterozygous G256W mice show stereotyped electrographic features and reduce survival.

A-B. EEGs of non-fatal and subsequent fatal seizure captured in a P54 male G256W/+ mouse (animal 2, panel C). Electrographic seizures were characterized by fast spiking, high amplitude activity lasting 15-20 s (highlighted in gray). C. Summary showing the sex, ages, duration of recordings and timing of seizures in 8 animals undergoing EEG. Turqoise hashmarks denote a survived seizure, red hashmark denote a fatal seizure. Black bars are periods on EEG; some recording were performed on a 6 hr/day schedule. D. Survival curve of WT vs G256W/+ mice, hashmarks indicate censored mice. G256W/+ mice had signifcant mortality, P = 0.0348 Cox propotional hazards model.

Video of a fatal convulsive seizure in a 4 month old heterozygous G256W mouse.

Seizure onset (not shown) occurred 5-10 sec prior to the start of recording with wild running and jumping, followed by arrest, then resumed (start of video). This was again followed by loss of postural control, followed by sustained forelimb flexor/hindlimb extensor posturing. Attempts to resuscitate the animal were begun immediately and were unsuccessful. Link to movie F6-S1

No significant mortality in heterozygous E254fs mice.

Survival curve of WT vs E254fs/+ mice, hashmarks indicate censored mice. E254fs/+ mice showed no significant mortality, P = 0.452 Cox propotional hazards model.

RT-qPCR shows incompletely efficient nonsense mediated decay of Kcnq2 E254fs/+ mRNA and increased Kcnq3 mRNA in E254fs/+ and G256W/+ mice.

A. Upper, Kcnq2 mRNA levels in P21 hippocampus and neocortex. Levels were analyzed using TaqMan probes binding to both WT and variant Kcnq2 alleles (allele non-selective, NS), or binding only to WT only (WT selective, WS). Total Kcnq2 mRNA in E254fs/+ samples were significantly higher than 50% of WT (hippocampus: P = 1.28x10-5, neocortex: P = 3.5x10-7, one sample t-test). Lower, Kcnq2 mRNA levels in P100 hippocampus and neocortex, using the probes as above. Total Kcnq2 mRNA levels in E254fs/+ samples were significantly higher than the expected 50% of WT in hippocampus (p = 0.0003) and neocortex: (p = 0.0007). B. Total and WT Kcnq2 mRNA, tested in parallel, by individual. Age, sex (male, open symbols), brain region, genotype, and probe are indicated. In all four tissues tested, E254fs/+ mice have greater total Kcnq2 mRNA than WT Kcnq2 mRNA (P21 hippocampus, P = 0.0001; P100 hippocampus, P = 0.005; P21 neocortex, P = 0.0007; P100 neocortex, P = 0.0053; pairwise t-test). C. In P21 G256W/+ mice, Kcnq3 mRNA was significantly increased: 1.15-fold (+/- 0.10, P = 0.0043) in the hippocampus and 1.12-fold (+/- 0.09, P = 0.00213) in the neocortex. In P100 E254fs/+ mice, Kcnq3 mRNA significantly increased (1.11 +/- 0.02-fold, P = 0.0245) in the hippocampus only. One way ANOVA, * = p0.05, ** = p0.005, *** = p0.0005. (See Supplemental Data for statistical test calculations).

A. Probes used for allele non-selective and WT allele-selective RT-qPCR. B. Upper, WT sequence (bases 758-771). Lower, Sanger trace of amplified Kcnq2 cDNA from WT hippocampus. C. Upper, WT sequence; the red line indicates bases deleted in the E254fs allele. Lower, Sanger trace of cDNA from an E254fs/+ mouse. Blue shading highlights the shift following the deletion at positions 761-767. Peaks corresponding to E254fs transcripts are labeled and are smaller than WT peaks. D. Upper, alignment of WT and missense variant DNA sequences, red lines highlight the two base substitutions at codon 256. Below, Sanger trace of amplified cDNA from G256W/+ hippocampus. Double peaks are visible at the bases mutated by Crispr.

Heterozygous G256W mice show reduced KCNQ2 and KCNQ3 labeling of CA1 pyramidal cell AISs and increased labeling of neuronal somata.

Identically processed age P21 tissue sections of WT (upper) and G256W/+ (lower) mice; area CA1B was imaged under identical settings. Confocal image stacks are shown as maximal intensity projections. In the animation, channels for the indicated markers are allowed to fade into the next, enabling evaluation of colabeling. DAPI marks cell nuclei. AnkG strongly marks AISs and lightly labels somata and proximal apical dendrites. An arrow highlights one stratum oriens interneuron somatically labeled for KCNQ2 only. Labels: DAPI, 4’,6-diamidine-2’-phenylindole; so, Stratum oriens; sp, Stratum pyramidale, sr, Stratum radiatum. Scale: 50 μm. Link to movie F8

In CA1, the KCNQ2 and KCNQ3 cellular and subcellular immunolabeling patterns appear similar for WT and heterozygous E254fs mice.

Ankyrin-G marks position of AISs. KCNQ2 and KCNQ3 strongly label CA1 AISs in E254fs/+ mice, and do not show increased somatic labeling compared to WT. Highlighted by an arrow is one interneuron in stratum pyramidale that was somatically labeled for KCNQ2 only. Scale: 50 μm. Link to movie F8-S1

Heterozygous G256W mice show increased CA3 pyramidal cell somatic labeling and reduced mossy fiber labeling for KCNQ2 and KCNQ3.

Yellow lines demarcate the borders of sp; the sp-sl border is cut obliquely through the tissue section in the G256W/+ sample. PanNav strongly labels the unmylenated axons of the mossy fibers in stratum lucidum of both samples. PanNav also labels the obliquely cut AISs of pyramidal cell neurons, which are mostly located within sp. Scale: 50 μm. Link to movie F8-S2

G256W/+ mouse Interneurons in CA1 show somatic KCNQ2 labeling.

A, D. Wider views of CA, including more of S. radiatum and s. oriens. (D, same as shown in Figure 8-Movie). In WT, positions of three S. radiatum interneurons are boxed, but higher magnification (e.g., B, C) shows lack somatic labeling for KCNQ2 or KCNQ3 In G256W/+ image (D), four interneurons somatically labelled for KCNQ2 but not KCNQ3 are enclosed by red boxes. The interneuron indicated with an arrow in Figure 8-Movie (KCNQ2 labeled, KCNQ3 unlabeled), is again highlighted. The yellow box encloses an interneuron somatically co-labeled for KCNQ2 and KCNQ3. E. Individual laser channels for the interneuron enclosed by yellow box in D. The soma is labeled for both KCNQ2 and KCNQ3. The nearby AIS showing AnkG, KCNQ2, and KCNQ3 may arise from this or a different cell, as its origin was not verified by higher resolution re-imaging. F. Interneuron somatically labeled for KCNQ2, not KCNQ3. Its AIS appears to arise from a KCNQ2 labeled (white arrowheads). In C-F, yellow arrowheads show distal AISs strongly labeled for AnkG, and weakly for KCNQ2 and KCNQ3. Scales: A, 50 µm; B, 10 µm.

Single marker grey-scale images and selected merged images of CA1, related to Figure 8 movie.

In lower merge images, yellow arrows indicate KCNQ2/KCNQ3 overlap, white and green arrows indicate AnkG-only labeling of proximal AIS. Scales: 50 µm, upper; 10 µm, middle and lower.

Single marker grey-scale images and selected merged images of CA1, related to Figure 8 —figure supplement 1 movie.

In lower merge images, yellow arrows indicate KCNQ2/KCNQ3 overlap, white and green arrows indicate AnkG-only labeling of proximal AIS. Scales: 50 µm, upper; 10 µm, middle and lower.

Single marker grey-scale images and selected merged images of CA3, related to Figure 8 —figure supplement 2 movie. Scales: 50 µm.

The ratios of axonal to somatic KCNQ2 and KCNQ3 labeling are reduced in CA1 and CA3 in heterozygous G256W mice.

A-B. The ratios of AIS to somatic immunofluorescence intensity is significantly reduced for KCNQ2 and KCNQ3 in CA1 (A) and CA3 (B) for G256W/+ but not E254fs/+ mice. C. The ratio of mossy fiber to somatic KCNQ2 and KCNQ3 immunofluorescence intensity is reduced in the CA3 for G256W/+ but not E254fs/+ mice. D. In the dentate gyrus, the ratio between GCL and PML intensity is significantly reduced for KCNQ3 but not KCNQ2 in G256W/+ but not E254fs/+ mice. n=3 per genotype. One way ANOVA, * = P0.05, ** = P0.005, *** = P0.0005.

KCNQ2 protein is reduced in neocortex of P21 heterozygous E254fs and G256W mice.

A-B. Representative western blots for all three genotypes probed for KCNQ2 and KCNQ3. C. Quantified KCNQ2 signal relative to WT, normalized to protein loaded as assayed by BCA. D. Quantified KCNQ3 signal relative to WT, n=3 per genotype, all males. One way ANOVA, * = P0.05, ** = P0.005.

KCNQ2 antibodies, unlike KCNQ3, show complex electrophoretic banding pattern, and reduced levels in E254fs and G256W mice.

PVDF filter with electrotransferred brain proteins was cut between the 70 and 50 kDa markers. Each lane contains homogenate from an individual animal. Upper portion of the filter (blue bar at L) was initially probed for KCNQ2; lower portion (green bar at L) was initially probed for tubulin, then stripped and reprobed for KCNQ2. Next, the entire filter was stripped and probed again for KCNQ3. A. KCNQ2 blots. Arrowheads point to KCNQ2 monomer band (Mr ∼80 kDa), and candidate dimeric and oligomeric bands of Mr ∼180 to ∼400 kDa. B. Sequential probe of same filter from A using KCNQ3 and tubulin antibodies. Arrowhead points to predicted KCNQ3 monomer with a Mr ∼100 kDa. C. The indicated bands from the KCNQ2 blots, and the total Individual lane intensities, means of genotypes, and SEMs are shown. One way ANOVA, * = P0.05.