Journal: International Journal of Molecular Sciences

Article Title: H1153Y- KCNH2 Mutation Identified in a Sudden Arrhythmic Death Syndrome Case Alters Channel Gating

doi: 10.3390/ijms22179235

Figure Lengend Snippet: Molecular position of the KCNH2 H1153Y variant (NM_0002383; rs 199473035) detected using the Ion torrent TM PGM platform and confirmed with Sanger sequencing. ( A ) The reads of NGS sequencing and the DNA sequence chromatogram illustrate the heterozygous c:3457C > T nucleotide substitution that results in the substitution of a Histidine-H (positively charged amino acid) for a Tyrosine-Y (polar uncharged amino acid) at amino acid residue 1153. ( B ) Cartoon of one Kv11.1 channel subunit. The circle denotes the approximate location of the p.H1153Y variant in the C-term region.

Article Snippet: The membranes were incubated over night at 4 °C with primary rabbit anti-potassium channel Kv11.1 intracellular antibody (1:100; Sc-20130; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Variant Assay, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: H1153Y- KCNH2 Mutation Identified in a Sudden Arrhythmic Death Syndrome Case Alters Channel Gating

doi: 10.3390/ijms22179235

Figure Lengend Snippet: Representative microscopy images of HEK293 cells expressing WT-Kv11.1 and H1153Y-Kv11.1. GFP fluorescence is shown in green. Anti-Kv11.1 extracellular immunostaining (Alexa 647 fluorescence) is shown in red. Overlapping immunostaining of red and green signals of similar intensities is shown as yellow, and the cell nuclei are labeled blue. The scale bar represents 100 µm at 20× magnification and 50 µM at 63× magnification.

Article Snippet: The membranes were incubated over night at 4 °C with primary rabbit anti-potassium channel Kv11.1 intracellular antibody (1:100; Sc-20130; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Microscopy, Expressing, Fluorescence, Immunostaining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: H1153Y- KCNH2 Mutation Identified in a Sudden Arrhythmic Death Syndrome Case Alters Channel Gating

doi: 10.3390/ijms22179235

Figure Lengend Snippet: Flow cytometry qualitative ( A ) and quantitative ( B , C ) assessment of WT-Kv11.1 and H1153-Kv11.1 channel expression. ( A ) Representative histograms for unstained cells (corresponding to non-transfected cells) and cells transiently transfected with WT-Kv11.1 and H153Y-Kv11.1 stained with antiKv11.1 conjugated antibody. ( B ) Quantification of surface Kv11.1 channel (Alexia 647 fluorescence) for WT and H1153 variants in non-permeabilized cells. ( C ) Quantification of total Kv11.1 channel (GFP fused to the N-terminus of Kv11.1) for WT and H1153-Kv11.1 in non-permeabilized cells. The Median Kv11.1 Intensity of Fluorescence (MIF) represents the median of fluorescence intensity of 10,000 cells counted for each experiment ( p > 0.05). Each value represents the median+ S.E.M. of four independent experiments.

Article Snippet: The membranes were incubated over night at 4 °C with primary rabbit anti-potassium channel Kv11.1 intracellular antibody (1:100; Sc-20130; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Flow Cytometry, Expressing, Transfection, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: H1153Y- KCNH2 Mutation Identified in a Sudden Arrhythmic Death Syndrome Case Alters Channel Gating

doi: 10.3390/ijms22179235

Figure Lengend Snippet: Trafficking phenotype of WT-Kv11.1 and H1153-Kv11.1 channel protein. ( A ) Representative Western blot analyses of HEK293 cells transiently transfected with pcDNA3.1-N-eGFP (control cells), WT-Kv11.1, or H1153-Kv11.1, with or without pK treatment (200 μg/mL, 30 min); Kv11.1 channel protein was detected with an anti-Kv11.1 antibody targeting intracellular domain. Kv11.1 WT and H1153 showed two bands corresponding to a mature complex-glycosylated 182 kDA Kv11.1 band (155 kD + 27 kDA of GFP) completely digested by pK and one band at slightly lower molecular weight (162 kD) resistant to pK cleavage corresponding to immature core-glycosylated Kv11.1 protein band. ( B – D ) Semi-quantification of mature ( B ) and immature proteins ( C ) in WT and H1153Y cells with and without pK. Total protein measured with stain-free method was used to normalize, n = 5 independent experiments (** ( p < 0.01) = for difference between WT and pK-treated WT, and for difference between mutant and pK-treated mutant; ns = no significant difference ( p > 0.05) between mutant and WT. ( D ) The relative amount of mature Kv11.1 protein (mature/total Kv11.1) based on immunoblot analyses for each set of experiments is plotted ( p > 0.05). The error bars represent SEM.

Article Snippet: The membranes were incubated over night at 4 °C with primary rabbit anti-potassium channel Kv11.1 intracellular antibody (1:100; Sc-20130; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Western Blot, Transfection, Molecular Weight, Staining, Mutagenesis