Plural molecular and cellular mechanisms of pore domain KCNQ2 encephalopathy

KCNQ2 variants in children with neurodevelopmental impairment are difficult to assess due to their heterogeneity and unclear pathogenic mechanisms. We describe a child with neonatal-onset epilepsy, developmental impairment of intermediate severity, and KCNQ2 G256W heterozygosity. Analyzing prior KCNQ2 channel cryoelectron microscopy models revealed G256 as keystone of an arch-shaped non-covalent bond network linking S5, the pore turret, and the ion path. Co-expression with G256W dominantly suppressed conduction by wild-type subunits in heterologous cells. Ezogabine partly reversed this suppression. G256W/+ mice have epilepsy leading to premature deaths. Hippocampal CA1 pyramidal cells from G256W/+ brain slices showed hyperexcitability. G256W/+ pyramidal cell KCNQ2 and KCNQ3 immunolabeling was significantly shifted from axon initial segments to neuronal somata. Despite normal mRNA levels, G256W/+ mouse KCNQ2 protein levels were reduced by about 50%. Our findings indicate that G256W pathogenicity results from multiplicative effects, including reductions in intrinsic conduction, subcellular targeting, and protein stability. These studies reveal pore “turret arch” bonding as a KCNQ structural novelty and introduce a valid animal model of KCNQ2 encephalopathy. Our results, spanning structure to behavior, may be broadly applicable because the majority of KCNQ2 encephalopathy patients share variants near the selectivity filter.

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.

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Figure 1 -supplement 2. Awake and sleep EEG background.Settings as in Figure 1, except LFF changed to 1 Hz.

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Figure 2 -supplement 1. 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 Figure 2 -supplement 2. 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

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Figure 2 -supplement 3. Movie illustrating locations of residues contributing to a noncovalent bonding network extending from S5 to the selectivity filter.Link to movie F2-S3

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Figure 3 -supplement 2. 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 -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.

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Figure 4 -supplement 1. 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.

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Figure 4 -supplement 2. Western blotting reveals a lower molecular weight band (Mr ~ 28 kDa) that is equally detected in WT and heterozygous E254fs, but no evidence of the 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 ~90kDa), red arrow indicates the estimated relative mobility (~29.73 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 digest of this band showed multiple abundant proteins and few KCNQ2 peptides.C. Quantification of ~28 kDa band from A.

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Figure 7 -supplement 1. E254fs and G256W variant transcripts are detectable in cDNA from heterozygous animals.A. Upper, WT sequence (bases 758-771).Lower, Sanger trace of amplified Kcnq2 cDNA from WT hippocampus.B. 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.C. 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 at the mutated bases positions are visible.

Figure 8 -Figure 8 -Figure 8 -
Figure 8 -supplement 1.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 Figure 8 -supplement 2. 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

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Figure 9 -supplement 2. Images of entire filter used for western blot of lysates, probed for KCNQ2 and KCNQ3. A. PVDF filter with electrotransferred brain proteins was cut below the 75 kDa marker.Upper portion was probed for KCNQ2; lower portion was probed for tubulin.Black arrow points to KCNQ2 monomer band (Mr ~90 kDa).Red arrow points to a band at ~250 kDa.Mass spectrometry of in gel digest of this band showed multiple abundant proteins and few KCNQ2 peptides.Also visible are bands likely representing KCNQ2 dimer formation (Mr ~180 kDa and higher molecular weight).B. KCNQ3 filter was cut below 75 kDa in order to probe for Tubulin.Black arrow points to predicted KCNQ3 monomer with a Mr ~100 kDa.The red asterisk indicates a bad sample that was not included in the analysis seen in Figure9-supplement 1. C. KCNQ2 band intensity normalized to tubulin.D. KCNQ3 band intensity signal normalized to Tubulin.

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Figure 2-figure supplement 2 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made