Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: A & B, illustration of a HCN4 -subunit with the cytoplasmic NH2- and COOH-terminus, six transmembrane segments (S1–S6), including the S4 voltage sensor (‘+’ sign denotes amino acid residues with positive charge), the pore loop between S5 and S6, and the C-linker (CL) with the cyclic-nucleotide binding domain (CNBD). The red (seven) and blue (three) circles denote the location of the novel variants identified in the early-onset AF cases and referents, respectively.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Binding Assay

Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: A, current recordings of wild type HCN4, p.Lys189Arg and p.Gly1077Ser channels (see methods). B, plots of activation curves for wild type HCN4, p.Lys189Arg and p.Gly1077Ser. C & D, plots of V1/2 and k for wild type HCN4 and the seven variants. The dashed black line through p.Pro257Ser denotes the lack of measureable current (see figure 3). The V1/2 and k for the six variants expressing current were not significantly different from wild type (t-test, p> 0.05). The numbers in the parentheses represent the number of cells.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Activation Assay, Expressing

Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: Electrophysiology properties of wild type and variant HCN4 channels.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Variant Assay, Expressing

Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: A, Currents were elicited from a holding current of -35 mV to a test pulse of -150 mV (fully-activated voltage) for 4 seconds and returned back to the holding current. The wild type HCN4 channel produced a current and the p.Pro257Ser mutant did not. B, confocal micrographs of wild type HCN4 and p.Pro257Ser channels expressed in CHO cells. The cells were stained with rabbit anti-HCN4 antibody (green) and DAPI (blue). The wild type HCN4 channel is expressed on the cell membrane and in the cytoplasm, whereas the p.Pro257Ser mutant channel is restricted to the cytoplasm. The scale bar denotes 50μm.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Produced, Mutagenesis, Staining

Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: A, current recordings of wild type HCN4, p.Asn688Ser and p.Arg1068His channels. B, plots of activation curves for wild type HCN4, p.Asn688Ser and p.Arg1068His channels. C & D, plots of V1/2 and k for wild type HCN4 and the three mutants. The V1/2 and k for the three mutants were not significantly different from wild type (t-test, p> 0.05). The numbers in parentheses represent the number of analyzed cells.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Activation Assay

Journal: Heart rhythm : the official journal of the Heart Rhythm Society

Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

doi: 10.1016/j.hrthm.2014.03.002

Figure Lengend Snippet: A, current recordings of wild type HCN4 (2 μg) and wild type HCN4 (1 μg)+p.Pro257Ser (1 μg). B, plot of current density (pA/pF) measured at -150 mV for wild type HCN4 and wild type HCN4+p.Pro257Ser. C, plots of activation curves for wild type HCN4 and wild type HCN4+p.Pro257Ser. The number in parentheses represents the number of cells. D, confocal micrographs of co-expressed wild type HCN4 and p.Pro257Ser constructs tagged with unique C-terminal epitopes in CHO cells (see methods); i) wild type HCN4-myc (green)+ wild type HCN4-V5 (red) and ii) wild type HCN4-myc (green)+p.Pro257Ser-V5(red). Co-expressed wild type HCN4-myc and wild type HCN4-V5 channels both traffick and are distributed together on cell membrane. ii) wild type HCN4-myc +p.Pro257Ser-V5 images show the wild type HCN4-myc channel expressed on the cell membrane and the p.Pro257Ser-V5 channel distributed in the cytoplasm and not on the cell membrane. Cells were also stained with DAPI (blue) to visualize the nucleus which is shown in the merged images. The scale bar denotes 50μm.

Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

Techniques: Activation Assay, Construct, Staining