Journal: bioRxiv

Article Title: A pain-reducing Kv6.4 variant spares other Kv6 channels, offering a target for uterine pain

doi: 10.64898/2025.12.18.692915

Figure Lengend Snippet: SH-SY5Y cells were transfected with either Kv6.4-HA wild-type, Kv6.4-Met418-HA and immnuostained with anti-HA antibody (red) and NaLJ/KLJ-ATPase (green). (A) Immunostaining of Kv6.4-HA wild-type (top panel) or Kv6.4Met418-HA (second panel) and NaLJ/KLJ-ATPase. (B) Pearson colocalization coefficients between HA-tagged Kv6.4 and NaLJ/KLJ-ATPase were analyzed to assess membrane localization. Kv6.4Met418 analysis shows a Pearson colocalization coefficient with NaLJ/KLJ-ATPase comparable to that of Kv6.4 WT, indicating that Kv6.4Met418 successfully reaches the plasma membrane. The box-and-whisker plot displays Pearson correlation values for each condition, with each dot representing an individual stained cell. Experiments were independently repeated four times ( N = 4 ). Statistical comparisons between wild-type and mutant co-expression conditions were performed using Student’s t-test. ( C) Co-immunoprecipitation of Kv6.4-HA or Kv6.4-HAMet418 using an anti-HA antibody. Following pulldown, Kv2.1 (95 kDa) was detected using an anti-Kv2.1 antibody. The results show that Kv6.4Met418 interacts with Kv2.1 similarly to wild-type Kv6.4, although interaction strength varied between experiments. The blot has been cropped; full-length blots are provided in Supplementary Fig S7D. *Input = lysate from transfected cells; IP = co-immunoprecipitated sample.

Article Snippet: Following blocking, membranes were incubated overnight at 4°C with primary antibodies: anti-Flag (BioLegend, 1:1000) and anti-Kv2.1 rabbit polyclonal (Alomone, 1:500).

Techniques: Transfection, Immunostaining, Membrane, Analysis, Clinical Proteomics, Whisker Assay, Staining, Mutagenesis, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: A pain-reducing Kv6.4 variant spares other Kv6 channels, offering a target for uterine pain

doi: 10.64898/2025.12.18.692915

Figure Lengend Snippet: SH-SY5Y cells were co-transfected with either wild-type Kv6.1-HA wild type or the Kv6.4Met425-HA mutant (both shown in white), along with wild-type Kv6.1-Flag wild type, Kv6.2-Flag wild type, or Kv6.3-Flag wild type (shown in red). Cells were co-stained with anti-HA and anti-Flag antibodies, as well as NaLJ/KLJ-ATPase (green) to mark the plasma membrane. ( A) Co-immunostaining of Kv6.1-HA wild-type (top panel) or Kv6.1Met425-HA (second panel) with Kv6.1-Flag wild type and NaLJ/KLJ-ATPase. (B) Co-immunostaining of Kv6.1-HA wild-type (top panel) or Kv6.1Met425-HA (second panel) with Kv6.2-Flag wild type and NaLJ/KLJ-ATPase. (C) Co-immunostaining of Kv6.1-HA wild-type (top panel) or Kv6.1Met425-HA (second panel) with Kv6.3-Flag wild type and NaLJ/KLJ-ATPase. (D) Pearson colocalization coefficients between Flag-tagged Kv6 subunits and NaLJ/KLJ-ATPase were quantified to assess membrane localization. Kv6.1, Kv6.2, and Kv6.3 maintained effective membrane localization when co-expressed with Kv6.1Met425. The box-and-whisker plot displays Pearson correlation values for each condition, with each dot representing an individual stained cell. Experiments were independently repeated four times ( N = 4 ). Statistical comparisons between wild-type and mutant co-expression conditions were performed using Student’s t-test. (E) SH-SY5Y cells were co-transfected with Kv6.1-HA (WT) and either Kv6.2-Flag (WT) or Kv6.3-Flag (WT). Kv6.1 was immunoprecipitated using an anti-HA antibody, and the presence of co-immunoprecipitated Kv6.2 or Kv6.3 was assessed using an anti-Flag antibody. Co-immunoprecipitation results show that Kv6.1 does not interact with either Kv6.2 or Kv6.3. A weak interaction between Kv6.1 and Kv6.2 was observed in a single replicate (data not shown). (F) SH-SY5Y cells expressing Kv6.1Met425-HA were subjected to co-immunoprecipitation using an anti-HA antibody, and the presence of Kv2.1 was assessed using an anti-Kv2.1 antibody. The results show that the Met425 variant disrupts the interaction between Kv6.1 and Kv2.1. Cropped blots shown; the full-length blots are available in Supplementary Fig S8 *Input = lysate from transfected cells; IP = co-immunoprecipitated sample.

Article Snippet: Following blocking, membranes were incubated overnight at 4°C with primary antibodies: anti-Flag (BioLegend, 1:1000) and anti-Kv2.1 rabbit polyclonal (Alomone, 1:500).

Techniques: Transfection, Mutagenesis, Staining, Clinical Proteomics, Membrane, Immunostaining, Whisker Assay, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Variant Assay

Journal: bioRxiv

Article Title: Epileptic encephalopathy-related Kv2.1 mutants impair channel clustering and membrane distribution but not neuronal excitability

doi: 10.1101/2025.10.13.681825

Figure Lengend Snippet: (A) Schematic representation of Kv2.1 subunit chimeras (WT and mutant) indicating the locations of the fluorescent tag (GFP or Dendra2) and the PRC domain. (B) Top panels: confocal maximum projection images of WT and mutant Kv2.1-GFP expressed in cultured hippocampal neurons, labeled on the surface with AlexaFluor647-coupled anti-GFP nanobodies. Bottom panels: higher magnification of the regions indicated by white squares. Scale bars: 30 µm. (C) Average fluorescence intensity of Kv2.1-GFP staining as in A in proximal (prox., 0-50 µm from soma), intermediate (interm, 50-100 µm) and distal (dist, 100-150 µm) dendrites, normalized to the average intensity on the soma (horizontal discontinuous line). Median and 10%-90% IQR. ANOVA with Friedman tests. ns: not significant; ** p <0.01; *** p <0.001; **** p <0.0001. n = 10 to 11 pyramidal neurons from 4 independent cultures. (D) Single-molecule localization microscopy analysis of clustering. D1-2 : Pointillistic (D1) and rendered (D2) images of STORM detections of surface Kv2.1 GFP labeled with AlexaFluor647-coupled anti-GFP nanobodies. The focal plane corresponds to the top of the soma of cultured hippocampal neurons expressing WT and mutant channels. D3 : Detection of clusters. The detected clusters appear in different colors. The area indicated by the black squares is shown at higher magnification in the bottom panels. Scale bars: 1 µm. (E) Quantification of Kv2.1 cluster’s area (E1) and percentage of detections in clusters (E2). Median and 10%-90% IQR. Kruskal-Wallis with Dunn tests. ns: not significant; ** p <0.01; **** p <0.0001. n = 7 to 10 pyramidal neurons from 2 independent cultures.

Article Snippet: Subsequently, neurons were incubated for 1h with rabbit anti-Kv2.1 antibody (1:100; Alomone Labs, Israel) diluted in blocking solution and 45 min at RT with the corresponding secondary antibody (AlexaFluor647-or Cy3-tagged donkey anti-rabbit; Jackson Immunoresearch, USA).

Techniques: Mutagenesis, Confocal, Cell Culture, Labeling, Fluorescence, Staining, Microscopy, Analysis, Expressing

Journal: bioRxiv

Article Title: Epileptic encephalopathy-related Kv2.1 mutants impair channel clustering and membrane distribution but not neuronal excitability

doi: 10.1101/2025.10.13.681825

Figure Lengend Snippet: (A) Epifluorescence images of hippocampal cultured neurons co-transfected with WT or mutant Kv2.1-Dendra2 (top panels) and Homer-DsRed (middle panels), immunolabeled with an anti-Kv2.1 antibody targeting the C-ter absent in mutant subunits (bottom panels). Scale bar: 20 µm. (B) Top panels: epifluorescence images of dendrites labelled with anti-Kv2.1 antibody (top panels) in non-transfected and transfected cells as in A. Bottom panels: merge images of anti-Kv2.1 immunolabelling (magenta) and Homer-DsRed fluorescence (cyan). Scale bar: 5 µm. (C) Quantification of endogenous Kv2.1 (anti-Kv2.1 staining) in non-transfected or mutant-expressing neurons: average fluorescence along dendrites (mean with SEM). (D) Total fluorescence of clusters (D1) and density of clusters (D2, number of clusters per unit length along dendrites). Each symbol represents a cell (horizontal line: median value). Kruskal-Wallis with Dunn tests; ns: not significant; * p <0.05; *** p <0.001. (E-F) Quantification of anti-Kv2.1 staining (endogenous and transfected) in non-transfected or Kv2.1WT expressing neurons: average fluorescence along dendrites (E, mean with SEM), total fluorescence of clusters (F1) and density of clusters (F2, number of clusters per distance unit along dendrites). In F1 and F2 each symbol represents a cell (horizontal line: median value). (D-F) n = 27 to 30 pyramidal neurons from 3 independent cultures. Mann-Whitney (E) or Kruskal-Wallis with Dunn tests (F1,2). ns: not significant; **** p <0.0001.

Article Snippet: Subsequently, neurons were incubated for 1h with rabbit anti-Kv2.1 antibody (1:100; Alomone Labs, Israel) diluted in blocking solution and 45 min at RT with the corresponding secondary antibody (AlexaFluor647-or Cy3-tagged donkey anti-rabbit; Jackson Immunoresearch, USA).

Techniques: Cell Culture, Transfection, Mutagenesis, Immunolabeling, Fluorescence, Staining, Expressing, MANN-WHITNEY, Immunofluorescence

Journal: bioRxiv

Article Title: Epileptic encephalopathy-related Kv2.1 mutants impair channel clustering and membrane distribution but not neuronal excitability

doi: 10.1101/2025.10.13.681825

Figure Lengend Snippet: (A1) Schematic representation of quantum dots labelling of Kv2.1. (A2) Epifluorescence image of Kv2.1WT-GFP expressing neuron (cyan) labelled with QD-coupled anti-GFP antibodies (magenta). Scale bar: 10 µm. (B) Cumulative frequency distributions of the diffusion coefficient ( D) of Kv2WT-GFP (black), Kv2Y529*-GFP (red), Kv2R579*-GFP (blue), and a1m (green) trajectories. Kruskal-Wallis with Dunn tests. p <0.0001, n = 974-1658 trajectories, 3 independent experiments. (C) Examples of trajectory reconstruction with detection of stabilization events by Pc analysis. Representative trajectories of QD-bound Kv2.1WT (C1), Kv2.1Y529* (C2) Kv2.1R579* (C3) or a1m (C4). In color: segments recognized as stabilization events (one color per event) following Pc analysis. Left: position of the stabilization events on the trajectories. Right: Pc metric in time for the trajectories on the left, showing the detection of stabilization events based on the thresholding of Pc . Note the different scale of Pc values between the examples. (D) Percentage of trajectories with at least one stabilization event (independently of the duration of the event). Each symbol represents one recording. Horizontal lines: median value. Kruskal-Wallis with Dunn tests. ns: not significant; *p<0.05; ***p<0.001; ****p<0.0001, n = 20-30 recordings. (E) Percentage of time spent in stabilization events (median and 10%-90% IQR). (F) Number of stabilization events per minute (violin plots). Kruskal-Wallis with Dunn tests. ns: not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Subsequently, neurons were incubated for 1h with rabbit anti-Kv2.1 antibody (1:100; Alomone Labs, Israel) diluted in blocking solution and 45 min at RT with the corresponding secondary antibody (AlexaFluor647-or Cy3-tagged donkey anti-rabbit; Jackson Immunoresearch, USA).

Techniques: Expressing, Diffusion-based Assay, Analysis, Immunofluorescence