Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Domain structures of short and long Ank2 protein variants (220 and 440 kDa, respectively). Note that exon 4 deletion (red star) affects both short and long variants. MBD membrane-binding domain, SBD spectrin-binding domain; linker, a linker for the MBD and SBD domains; giant exon, a domain uniquely present in the long Ank2 variant; DD death domain, CT C-terminal region. b Immunoblot analysis of WT and Ank2-cKO cortical and hippocampal lysates (P21). ( n = 4 mice [WT, cKO], one sample t -test). c Hyperactivity of Ank2-cKO mice (P19–21) in the open-field test (100 lux), as shown by distance moved. The reduced time spent in the center region suggests anxiety-like behavior. ( n = 25 [WT; males and females mixed here and in all other behavioral experiments], 19 [cKO], Mann-Whitney test [total distance moved, center time], two-way repeated-measures/RM-ANOVA with Sidak’s test [distance moved]). d Anxiolytic-like behavior in Ank2-cKO mice (P19–21) in the light-dark test (600 lux), as shown by light-chamber time/entry. ( n = 20 [WT], 17 [cKO], Student’s t -test). e Anxiolytic-like behavior in Ank2-cKO mice (P24–25) in the elevated plus-maze test, as shown by open-arm time/entry. ( n = 18 [WT], 10 [cKO], Student’s t -test). f Normal social interaction and impaired social novelty recognition in Ank2-cKO mice (P19–20) in the three-chamber test, as shown by sniffing time. S1/S2, novel/familiar stranger; O, object. ( n = 18 [WT], 14 [cKO], Mann–Whitney test [WT-S1/O, WT-S1/S2, and cKO-S1/O], Student’s t-test [cKO-S1/S2]). g Abnormally increased social interaction in Ank2-cKO mice (P23–25) in a direct social-interaction test. ( n = 9 [WT], 7 [cKO], Student’s t -test). h Decreased self-grooming and digging, without a change in jumping, in Ank2-cKO mice (P25–27). Note that hyperactivity is observed here, similar to the open-field hyperactivity. ( n = 24 [WT], 17 [cKO], Mann-Whitney test [digging/self-grooming]). Source data for uncropped immunoblot images are provided as a Source Data file. The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/- SEM. P values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Membrane, Binding Assay, Variant Assay, Western Blot, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Examples of epileptiform spike discharges in Ank2-cKO mice (P35; parietal right lobe) monitored until death, as shown by electroencephalography (EEG). b Epileptiform discharges in EEG observed in an Ank2-cKO mouse until death. c Juvenile seizure-related death occurring in Ank2-cKO mice during ~P20–50. ( n = 23 [WT], 23 [cKO], Log-rank test). d – h Increased pentylenetetrazole (PTZ)-induced seizures in Ank2-cKO mice (P25–27), as shown by final seizure stage (1–4) reached, latency to stage 2 seizure, animals reached stage 2, and frequency and total duration of stage 2. The stage 2 was mainly compared because of its predominance in the mutant mice. ( n = 17 [WT], 10 [cKO], chi-square test [final seizure stage], Mann-Whitney test [stage 2 latency/duration]). The statistical tests involved two-sided analyses. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Mutagenesis, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Examples of neuronal firing in the somatosensory cortex (SSC) in brain slices from Ank2-cKO mice, as measured by multielectrode array (MEA; 64 electrodes in 8 × 8 arrays; cutoff for significant firing > 4.5 x standard deviation). b , c Increased mean firing rate and mean burst rate (inter-spike-interval <10 ms) in the Ank2-cKO SSC (P19–22), as measured by MEA. ( n = 9 slices/3 mice [WT], 10, 3 [cKO], Student’s t -test [total], Welch’s test [layer 2/3], Mann–Whitney test [layer 5]). d Unaltered number of spikes in a single burst in the Ank2-cKO SSC (P19–22) measured by MEA. ( n = 9, 3 [WT], 10, 3 [cKO], Student’s t-test [total], Mann–Whitney test [layer 2/3 and 5]). e Unaltered percentage of spikes in bursts in the Ank2-cKO SSC (P19–22) measured by MEA. ( n = 9, 3 [WT], 10, 3 [cKO], Welch’s test [total], Mann-Whitney test [layer 2/3 and 5]). f Diagram showing examples of local field potentials (LFPs) and depicting parameters of LFP (length and amplitude). g – i Increased duration but normal frequency and amplitude of LFPs in layer 2/3 but not layer 5 in the Ank2-cKO SSC (P19–22) measured by MEA. ( n = 9, 3 [WT], 10, 3 [cKO], Student’s t -test [LFP frequency-layer 2/3, amplitude-total and layer 2/3, duration-total and layer 5], Mann–Whitney test [frequency/amplitude-layer 5], Welch’s test [duration-layer 2/3]). The statistical tests involved two-sided analyses. Data are presented as mean values +/- SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Standard Deviation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Normal resting membrane potential in Ank2-cKO SSC layer 2/3 pyramidal neurons (P19–22). ( n = 12 neurons/8 mice [WT], 22,8 [cKO], Mann–Whitney test). b , c Increased input resistance in Ank2-cKO SSC layer 2/3 neurons (P19–22). ( n = 10, 3 [WT], 12, 3 [cKO], two-way RM-ANOVA with Sidak’s test and Student’s t -test). d Increased current-firing curve slope in Ank2-cKO SSC layer 2/3 neurons (P19–22). ( n = 7, 3 [WT], 9, 3 [cKO], two-way RM-ANOVA with Sidak’s test). e Diagram explaining mAHP. f Decreased mAHP amplitude in Ank2-cKO SSC layer 2/3 neurons (P19–22). ( n = 16, 5 [WT], 21, 5 [cKO], Welch’s test). g Diagram explaining AP threshold and AP shape-related parameters. h – l Normal AP threshold and AP shape-related parameters in Ank2-cKO SSC layer 2/3 neurons (P19–22), as indicated by AP-threshold voltage and the height, width, and time-dependent voltage changes (dV/dt; maximal/depolarizing and minimal/repolarizing) of APs. ( n = 13, 9 [WT], 18, 11 [cKO], Student’s t -test [AP threshold and Max dV/dt and min dV/dt], Mann-Whitney test [FWHM and amplitude]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Membrane, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Diagram depicting procedures of PTM (posttranslational modification) analyses for samples from WT and Ank2-cKO cortex and hippocampus; (P19–22) ( n = 3 groups from 9 mice [3 mice/group] [WT, cKO]). b , c DAVID gene ontology (GO) analyses of biological functions for the proteins with up- and downregulated PTM levels (PTM proteins). d – f Volcano plots highlighting the PTM proteins with significant fold changes (>1.5) and p -values (<0.05, Welch’s t-test) and the presence of potassium channels in the down-PTM proteins (fold change > 1.5 and p < 0.05 [Welch’s t -test]). g Diagram depicting procedures of proteomic analysis of total (whole-lysate) proteins in Ank2-cKO mice (cortex + hippocampus; P19–22). h , i Volcano plots showing the differentially expressed proteins (DEPs) derived from the analysis of total (whole-lysate) proteins in Ank2-cKO mice (cortex and hippocampus; P19–22) with significant fold changes (>1.5) and p -values (<0.05) and the presence of Kv7.2 and Kv7.3 in the downregulated total DEPs ( p < 0.05 [Welch’s t -test] but not fold change > 1.5). ( n = 3 mice [WT, cKO]). j Decreased total and crude synaptosomal levels of Kv7.2 and Kv7.3 in Ank2-cKO brains (cortex + hippocampus; P19–23). Note that the levels of Kv2.1, Kv3.1, and Kv1.2 (unrelated control) were not changed. ( n = 4 mice [WT, cKO] except for Kv7.3 in total lysates ( n = 5 mice [WT, cKO]), one sample t -test). k Decreased surface levels of Kv7.2 and Kv7.3 in cultured cortical neurons (DIV 17). ( n = 15 dishes [5 + 5 + 5] from 3 biologically independent experiments [Kv7.2] and 14 dishes [5 + 5 + 4] from 3 biologically independent experiments [Kv7.3], one sample t-test). Source data for uncropped immunoblot images are provided as a Source Data file. The statistical tests involved two-sided analyses. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Modification, Derivative Assay, Control, Cell Culture, Western Blot

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a – c Increased AIS area (marked by Ank3; arrows) in cultured hippocampal Ank2 fl/fl neurons infected with AAV-Cre (DIV 4 ~ 10–14 ~ 28), and decreased mean intensities of Kv7.3 and Ank3 in the AIS area; these changes together yield normal total intensities of Kv7.3 and Ank3. Note that the width of Ank3 was not changed. ( n = 36 neurons from three independent experiments [WT], 36 [cKO], Welch’s test [Ank3 mean intensity], Mann–Whitney test [Ank3 area/total intensity; Kv7.3 mean/total intensity in AIS]). Scale bar, 10 µm. d – g Increased AIS length and area (marked by Ank3) in the Ank2-cKO somatosensory cortex (P19–23), as determined by three-dimensional imaging and quantification of expanded brain tissues. ( n = 60 neurons from 4 mice [WT], 60, 4 [cKO], Student’s t-test [Ank3 length], Welch’s t -test [Ank3 area]). Scale bar, 4 µm. h – j The Kv7.2/3 antagonist, XE991, increases the slope of the current-spike curve in WT, but not Ank2-cKO, SSC layer 2/3 pyramidal neurons (P19–23). ( n = 7 neurons from 3 mice [WT], 14,4 [WT + XE991], 9,3 [cKO], 13,5 [cKO+XE991], two-way RM-ANOVA with Sidak’s test [i] and with Tukey’s test [j]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Cell Culture, Infection, MANN-WHITNEY, Imaging

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a , b Retigabine treatment (from P16–17) rescues the increased neuronal excitability and firing in Ank2-cKO SSC layer 2/3 pyramidal neurons (P19–23) without affecting WT neurons, as shown by current-firing curves. ( n = 8 neurons from 4 mice [WT-Veh], 8,4 [WT-RTG], 8,4 [cKO-Veh], 8,4 [cKO-RTG], two-way RM-ANOVA with Tukey’s test [a], two-way RM-ANOVA with Sidak’s test [b]). c and d Retigabine treatment (from P16–17) rescues the decreased mAHP (medium afterhyperpolarization) amplitude in Ank2-cKO SSC layer 2/3 neurons (P19–23) without affecting that of WT neurons. ( n = 8, 4 [WT], 8,4 [WT-RTG], 8,4 [cKO], 8,4 [cKO-RTG], two-way RM-ANOVA with Tukey’s test [c], two-way RM-ANOVA with Sidak’s test [d]). e , f Retigabine treatment (from P16–17) does not affect input resistance in Ank2-cKO or WT SSC layer 2/3 neurons (P19–23). ( n = 7,4 [WT], 7,4 [WT-RTG], 7,4 [cKO], 7,4 [cKO-RTG], two-way RM-ANOVA with Tukey’s test [e], two-way RM ANOVA with Sidak’s test [f]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/- SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques:

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a , b Chronic retigabine treatment (from P16–17) improves juvenile seizure-related death of Ank2-cKO mice without affecting WT mice. ( n = 11 mice [WT-Veh/vehicle], 12 [WT-RTG/retigabine], 16 [cKO-Veh], 10 [cKO-RTG], Log-rank test). c , d Cessation of chronic retigabine treatment at around ~P42 (~26 days after the initiation of retigabine treatment) eliminates the treatment effect of retigabine on juvenile seizure-related death in Ank2-cKO mice. ( n = 14 mice [cKO-Veh], 13 [cKO-RTG], Log-rank test). e – g Chronic retigabine treatment (from P16–17) does not improve hyperactivity or anxiety-like behavior (center time) of Ank2-cKO mice in the open-field test. ( n = 11 mice [WT-Veh], 12 [WT-RTG], 11 [cKO-Veh], 10 [cKO-RTG], two-way ANOVA with Tukey test). h – k Acute retigabine treatment (5 mg/kg) at P21/22 improves hyperactivity but not anxiety-like behavior (center time) of Ank2-cKO mice in the open-field test. Drug treatment groups were divided into two (retigabine/saline first) to minimize cross-treatment effects. ( n = 13 mice [WT-Veh], 15 [WT-RTG], 16 [cKO-Veh], 17 [cKO-RTG], two-way ANOVA with Tukey’s test [distance moved_4min], Sidak’s test [distance moved_total]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Saline

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet:

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Virus, Recombinant, Software

Journal: bioRxiv

Article Title: Isolation and characterization of multi-protein complexes enriched in the K-Cl co-transporter 2 from brain plasma membranes

doi: 10.1101/2020.04.30.071076

Figure Lengend Snippet: A. Primary cultured neurons from P1 pups were infected with CamKII AAV-GFP at DIV 3 (to visualize cell morphology and to identify excitatory neurons) and fixed at DIV 21. The cells were immunostained for Kcc2 and components of the spectrin/ankyrin subnetwork; Sptan1, Sptbn1, Ank2, Itpr1. Colocalization was measured using line scans (white lines), and represent the normalised fluorescent intensity of single pixels (approximately 1 μm) on dendrites (n=3).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (Icc): Ank2 (mouse, Icc, Invitrogen 33-3700), Ank2 (rabbit, IB, Bioss BS-6967R-TR), Cntn1 (rabbit, IB), Abcam Ab66265), Itpr1 (rabbit, IB/Icc, Alomone Acc-019), Kcc2 (mouse, IP/Icc, Neuromab 75-013), Kcc2 (rabbit, IB/Icc, Millipore 07-432), Scn2a (mouse, Neuromab 75-024), Scn2a (rabbit, IB, Alomone ASC-002), Sptan1 (mouse, Icc, Abcam Ab11755), Sptan1 (rabbit, IB, Cell Signaling 21225), Sptbn1 (rabbit, IB/Icc, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Cell Culture, Infection

Journal: bioRxiv

Article Title: Isolation and characterization of multi-protein complexes enriched in the K-Cl co-transporter 2 from brain plasma membranes

doi: 10.1101/2020.04.30.071076

Figure Lengend Snippet: A. Primary cultured neurons from P1 pups were infected with CamKII AAV-GFP at DIV 3 (to visualize cell morphology and to identify excitatory neurons) and fixed at DIV 21. The cells were immunostained for Kcc2 and components of the spectrin/ankyrin subnetwork; Sptan1, Sptbn1, Ank2, Itpr1. Colocalization was measured using line scans (white lines), and represent the normalised fluorescent intensity of single pixels (approximately 1 μm) on dendrites (n=3).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (Icc): Ank2 (mouse, Icc, Invitrogen 33-3700), Ank2 (rabbit, IB, Bioss BS-6967R-TR), Cntn1 (rabbit, IB), Abcam Ab66265), Itpr1 (rabbit, IB/Icc, Alomone Acc-019), Kcc2 (mouse, IP/Icc, Neuromab 75-013), Kcc2 (rabbit, IB/Icc, Millipore 07-432), Scn2a (mouse, Neuromab 75-024), Scn2a (rabbit, IB, Alomone ASC-002), Sptan1 (mouse, Icc, Abcam Ab11755), Sptan1 (rabbit, IB, Cell Signaling 21225), Sptbn1 (rabbit, IB/Icc, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Cell Culture, Infection

Journal: International Journal of Molecular Medicine

Article Title: Regulation of drug resistance and metastasis of gastric cancer cells via the microRNA647-ANK2 axis

doi: 10.3892/ijmm.2018.3381

Figure Lengend Snippet: Primer sequences for reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Rabbit anti-human antibodies specific to focal adhesion kinase (FAK; ab40794), matrix metalloproteinase (MMP)2 (ab92536), MMP12 (ab52897), cluster of differentiation (CD)44 (ab51037), snail family transcriptional repressor 1 (SNAIL1; ab82846) and GAPDH (ab9485); and mouse anti-human antibodies specific to ankyrin-B (ANK2; ab131419) were obtained from Abcam (Cambridge, UK).

Techniques: