Journal: Experimental & Molecular Medicine

Article Title: Rare KCNQ4 variants found in public databases underlie impaired channel activity that may contribute to hearing impairment

doi: 10.1038/s12276-019-0300-9

Figure Lengend Snippet: a Exon structure of human KCNQ4 cDNA and domain structure of KCNQ4 are shown. Only variants identified in individuals with DFNA2 are shown. KCNQ4 contains 14 exons; the positions of the start (ATG) and stop (TGA) codons are indicated. Loss-of-function mutations, such as nonsense and frameshift mutations, are indicated. b Missense and in-frame deletion mutations are indicated. Note that mutations are clustered in exons 5 and 6, which encode part of the transmembrane (TM) 5, pore region, and TM6. The pathogenic variants identified by our group are shown in blue

Article Snippet: Complementary DNAs (cDNAs) of human KCNQ4 were purchased from OriGene Technologies (Rockville, MD, USA) and subcloned into the pENTR-D-TOPO vector (Invitrogen, Carlsbad, CA, USA).

Techniques:

Journal: Experimental & Molecular Medicine

Article Title: Rare KCNQ4 variants found in public databases underlie impaired channel activity that may contribute to hearing impairment

doi: 10.1038/s12276-019-0300-9

Figure Lengend Snippet: Loss-of-function KCNQ4 variants from the genome Aggregation Database

Article Snippet: Complementary DNAs (cDNAs) of human KCNQ4 were purchased from OriGene Technologies (Rockville, MD, USA) and subcloned into the pENTR-D-TOPO vector (Invitrogen, Carlsbad, CA, USA).

Techniques:

Journal: Experimental & Molecular Medicine

Article Title: Rare KCNQ4 variants found in public databases underlie impaired channel activity that may contribute to hearing impairment

doi: 10.1038/s12276-019-0300-9

Figure Lengend Snippet: Missense variants corresponding to the pore region of KCNQ4 from the genome Aggregation Database

Article Snippet: Complementary DNAs (cDNAs) of human KCNQ4 were purchased from OriGene Technologies (Rockville, MD, USA) and subcloned into the pENTR-D-TOPO vector (Invitrogen, Carlsbad, CA, USA).

Techniques: Mutagenesis

Journal: Experimental & Molecular Medicine

Article Title: Rare KCNQ4 variants found in public databases underlie impaired channel activity that may contribute to hearing impairment

doi: 10.1038/s12276-019-0300-9

Figure Lengend Snippet: a Immunofluorescence of wild-type (WT) and variant KCNQ4 proteins in HEK 293 cells. Cells were immunostained with anti-Myc, anti-BiP, and anti-GOLGB1 antibodies. Nuclei were stained with DAPI. BiP and GOLGB1 indicate the endoplasmic reticulum and Golgi apparatus, respectively. All KCNQ4 variant proteins were observed on the plasma membrane. b Cell surface biotinylation. Proteins on the plasma membrane were labeled with biotin, isolated with avidin beads, and assessed by western blotting. Surface expression of KCNQ4 variant proteins was similar to that of the WT protein. c Coimmunoprecipitation. Cells were cotransfected with FLAG-tagged WT KCNQ4 and Myc-tagged WT or mutant KCNQ4 clones. After transfection (36 h), whole-cell lysates were subjected to immunoprecipitation using anti-FLAG beads and immunoblotted. All KCNQ4 variant proteins interacted with the WT protein

Article Snippet: Complementary DNAs (cDNAs) of human KCNQ4 were purchased from OriGene Technologies (Rockville, MD, USA) and subcloned into the pENTR-D-TOPO vector (Invitrogen, Carlsbad, CA, USA).

Techniques: Immunofluorescence, Variant Assay, Staining, Labeling, Isolation, Avidin-Biotin Assay, Western Blot, Expressing, Mutagenesis, Clone Assay, Transfection, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: Rare KCNQ4 variants found in public databases underlie impaired channel activity that may contribute to hearing impairment

doi: 10.1038/s12276-019-0300-9

Figure Lengend Snippet: a Representative whole-cell current traces recorded in CHO cells transfected with wild-type (WT) and mutant KCNQ4 with step pulses from −80 mV to 40 mV in 20 mV steps. b Mean current-voltage (I–V) relation curves obtained from steady-state currents elicited at each step pulse voltage. c Summary bar graph of steady-state currents elicited at 40 mV normalized to membrane area (capacitance), obtained from whole-cell current recordings as presented in A. Averaged whole-cell current densities were 151.9 ± 11.6 pA/pF ( n = 30), 61.5 ± 8.8 pA/pF ( n = 19), 68.6 ± 7.8 pA/pF ( n = 17), 81.8 ± 16.4 pA/pF ( n = 12), 61.6 ± 4.3 pA/pF ( n = 15), 82.3 ± 9.5 pA/pF ( n = 19), 38.5 ± 5 pA/pF ( n = 16), 30.2 ± 3.8 pA/pF ( n = 12), 30.4 ± 5.9 pA/pF ( n = 8), 142.5 ± 20.5 pA/pF ( n = 4), and 32.4 ± 4.2 pA/pF ( n = 9) for WT, p.T278A, p.S273A, p.L281M, p.L295P, p.R433W, p.N264S, p.S269F, p.W276S, p.H455Q and green fluorescent protein only, respectively. Data represent the mean ± SEM. *** P < 0.001 compared to WT. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison. d Dominant-negative effects of mutants on KCNQ4-mediated currents were analyzed with a 1:1 ratio of WT and mutants KCNQ4 and step pulses from −80 mV to 40 mV in 20 mV steps. The mean I–V relation curve obtained from the steady-state current values elicited at each step pulse voltage is shown. e Summary bar graph of the steady-state currents elicited at 40 mV normalized to capacitance. The averaged whole-cell current densities were 143.6 ± 24.5 pA/pF ( n = 7), 56.6 ± 4.5 pA/pF ( n = 11), 69.4 ± 1.1 pA/pF ( n = 9), 72 ± 16.4 pA/pF ( n = 12), 66.1 ± 6.7 pA/pF ( n = 10), 72.9 ± 15.9 pA/pF ( n = 10), 46.7 ± 3.4 pA/pF ( n = 9), 40.6 ± 6.04 pA/pF ( n = 9), 58.8 ± 4.2 pA/pF ( n = 10), and 129.2 ± 32.5 pA/pF ( n = 4) for WT, p.T278A, p.S273A, p.R433W p.L281M, p.L295P, p.N264S, p.S269F, p.W276S, and p.H455Q, respectively. The red line is the predicted current density if there is no dominant-negative effect. Data represent the mean ± SEM. ** P < 0.01, *** P < 0.001 compared to the WT. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison

Article Snippet: Complementary DNAs (cDNAs) of human KCNQ4 were purchased from OriGene Technologies (Rockville, MD, USA) and subcloned into the pENTR-D-TOPO vector (Invitrogen, Carlsbad, CA, USA).

Techniques: Transfection, Mutagenesis, Dominant Negative Mutation

Journal: Experimental & Molecular Medicine

Article Title: Rare KCNQ4 variants found in public databases underlie impaired channel activity that may contribute to hearing impairment

doi: 10.1038/s12276-019-0300-9

Figure Lengend Snippet: a Enhancement of voltage-gated potassium channel activity of wild-type (WT) KCNQ4 by retigabine. b Summary bar graph of current produced by KCNQ4 mutants and rescued by retigabine versus that of WT, obtained from steady-state currents elicited at 40 mV shown in Supplementary Fig. . Current values were 131.4 ± 37.6 pA/pF ( n = 11), 194 ± 28.3 pA/pF ( n = 9), 229.2 ± 62.2 pA/pF ( n = 6), 129.7 ± 23.2 pA/pF ( n = 8), 126.4 ± 17.8 pA/pF ( n = 10), 58.8 ± 21.5 pA/pF ( n = 6), 24.4 ± 3.3 pA/pF ( n = 4), 37.4 ± 8.6 pA/pF ( n = 4), and 30.4 ± 5.8 pA/pF ( n = 5) for p.T278A, p.S273A, p.L281M, p.L295P, p.R433W, p.N264S, p.S269F, p.W276S, and green fluorescent protein, respectively. c Effect of retigabine on thallium influx in cells expressing WT and mutant KCNQ4. Data represent the mean ± SEM. ** P < 0.01, *** P < 0.001 compared to WT ( a , b ) or mock ( c ). Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison

Article Snippet: Complementary DNAs (cDNAs) of human KCNQ4 were purchased from OriGene Technologies (Rockville, MD, USA) and subcloned into the pENTR-D-TOPO vector (Invitrogen, Carlsbad, CA, USA).

Techniques: Activity Assay, Produced, Expressing, Mutagenesis