Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Mutagenesis, Binding Assay, Sequencing

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Western Blot, Transfection, Mutagenesis, Produced, Molecular Weight, Expressing, Cotransfection, Immunoprecipitation, Construct, Binding Assay

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Dominant Negative Mutation, Construct, Transfection, Activation Assay

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Staining, Transfection, Immunocytochemistry, Confocal Microscopy, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Expressing, Transfection, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Incubation, Western Blot, Cotransfection

Journal: PLoS ONE

Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

doi: 10.1371/journal.pone.0018273

Figure Lengend Snippet: A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.

Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

Techniques: Incubation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels *

doi: 10.1074/jbc.M114.558379

Figure Lengend Snippet: The N-Cap/PAS domain of Kv11. 1 is not interchangeable with that of Kv10.1. A, cartoon representation of a single subunit of either WT Kv11.1 channels (panel (i)), N-truncated (Δ2–137) Kv11.1 channels (panel (ii)), Kv11.1 channels with the N-Cap and PAS domains replaced by those of Kv10.1 (panel (iii)), or Kv11.1 channels with only the PAS domain of Kv10.1 (panel (iv)). Corresponding deactivation current traces are shown in the right hand panels for voltages between −50 and −150 mV in 20-mV increments. B, exemplary fits (red lines) of two exponential components to raw current traces measured at −120 mV (black lines) for WT and Δ2–137 channels. The fast (τfast) and slow (τslow) time constants are indicated. C, mean (± S.E.) rates of deactivation for WT Kv11.1 (black circles, n = 16), N-truncated Kv11.1 (gray triangles, n = 5), Kv11.1 with the N-Cap and PAS domain of Kv10.1 (green diamonds, n = 5), or Kv11.1 with only the PAS domain of Kv10.1 (open squares, n = 5). Note that error bars are included but are often within the symbols. Mean ± S.E. values for τfast, τslow, and the relative amplitudes of τfast to τslow for WT and all mutants are given in supplemental Table S1.

Article Snippet: This fragment was then inserted into the Kv11.1 backbone using Bpu10I and BstEII restriction enzymes (New England Biolabs, Ipswich, MA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels *

doi: 10.1074/jbc.M114.558379

Figure Lengend Snippet: Positive charges in the N-Cap/PAS are important for function. A, surface representation of the N-Cap/PAS domains of Kv11.1 channels, showing the location of the positively charged arginine residues Arg4, Arg5, Arg20, and Arg56 (shown in dark blue). B, side chains of Arg4 (panel (i)), Arg5 (panel (ii)), Arg20 (panel (iii)), and Arg56 (panel (iv)) were mutated to either a positively charged lysine (blue squares), negatively charged aspartate (red triangles), or glutamate (orange inverted triangles) side chains, and mean (± S.E.) rates of deactivation were compared with the WT arginine (black circles). Note that error bars are included but are often within the symbols. Mean ± S.E. values for τfast, τslow, and the relative amplitudes of τfast to τslow for WT and all mutants are given in supplemental Table S1. For each mutated residue, the inset shows representative current traces at −120 mV for WT arginine (black), lysine (blue) or aspartate (red) side chains. C, mean (± S.E.) changes in natural log of the fast time constant of deactivation measured at −120 mV (ln·(τf,−120 mV) for mutant channels compared with WT (see also supplemental Table S1).

Article Snippet: This fragment was then inserted into the Kv11.1 backbone using Bpu10I and BstEII restriction enzymes (New England Biolabs, Ipswich, MA).

Techniques: Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels *

doi: 10.1074/jbc.M114.558379

Figure Lengend Snippet: Several charged cNBH domain residues are critical for the slow deactivation kinetics of Kv11. 1 channels. A, surface representation homology model of the C-linker/cNBH domain of Kv11.1 channels showing the location of 18 negatively charged residues (red spheres). B, mean (± S.E.) rates of deactivation for the 18 negatively charged cNBH domain residues individually mutated to arginine. Three mutations exhibited substantially faster deactivation kinetics: D774R (panel (i), red triangles, n = 6), E788R (panel (ii), red diamonds, n = 21), and D803R (panel (iii), red squares, n = 8), whereas the remainder had deactivation kinetics similar to, or slower than, WT (panel (iv), gray symbols, n = 4–21). Note that error bars are included but are within the symbols. Mean ± S.E. values for τfast, τslow and the relative amplitudes of τfast to τslow for WT and all mutants are given in supplemental Table S1. Representative current traces at −120 mV for D774R, E788R, and D803R are shown in the inset. C, summary of all individual mutant effects on deactivation kinetics measured at −120 mV. Mutant perturbations were considered significant only when the mean was outside ± two standard deviations of the WT Kv11.1 (indicated by gray band). D774R, E788R, D803R, D821R, and E857R exhibited fast deactivation kinetics, whereas D727R and D829R were significantly slower than WT. * denotes that E807R did not express functional channels.

Article Snippet: This fragment was then inserted into the Kv11.1 backbone using Bpu10I and BstEII restriction enzymes (New England Biolabs, Ipswich, MA).

Techniques: Mutagenesis, Functional Assay

Journal: The Journal of Biological Chemistry

Article Title: Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels *

doi: 10.1074/jbc.M114.558379

Figure Lengend Snippet: PAS domain residue Arg56 forms a charge-charge interaction with the cNBH domain residue Asp803. A, mean (± S.E.) rates of deactivation for N-Cap mutants R4D (panel (i)), R5D (panel (ii)), R20D (panel (iii)), or the PAS domain mutant R56D (panel (iv)), either alone (blue triangles, n = 5–14) or in the presence of the cNBH domain mutation D803R (purple circles, n = 7–16). WT Kv11.1 (black circles, n = 16) and D803R (red squares, n = 8) are shown for comparison. Note that error bars are included but are within the symbols. Mean ± S.E. values for τfast, τslow, and the relative amplitudes of τfast to τslow for WT and all mutants are given in supplemental Table S1. Representative current traces at −120 mV are shown within insets, with mutants color-coded as above. B, summary (means ± S.E.) showing complete restoration of slow deactivation kinetics, measured as Δln(τfast,−120 mV) compared with WT Kv11.1, only when R56D was combined with D803R, indicating a direct functional interaction between the native residues. C and D, summary (mean ± S.E.) ΔΔG0 values calculated from Boltzmann energy fits of steady-state g-V curves (see “Experimental Procedures” for details) for charge reversal (C) or alanine (D) mutant channels compared with the WT. Gray bars represent the theoretical additive values of the two individual mutant perturbations combined. Note that the double mutant R56D/D803R exhibits a reduced ΔΔG0 compared with the two single mutants alone. Mean ± S.E. values are given in supplemental Table S2. E, surface representation Kv11.1 homology model of the N-Cap/PAS domains (blue) interacting with the C-linker/cNBH domains (red), with the boxed region, expanded in the right panel, showing a charge-charge interaction between the side chains of the PAS domain residue Arg56 and Asp803 in the cNBH domain.

Article Snippet: This fragment was then inserted into the Kv11.1 backbone using Bpu10I and BstEII restriction enzymes (New England Biolabs, Ipswich, MA).

Techniques: Mutagenesis, Functional Assay

Journal: The Journal of Biological Chemistry

Article Title: Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels *

doi: 10.1074/jbc.M114.558379

Figure Lengend Snippet: cNBH domain residue Glu788 does not appear to interact with the N-Cap/PAS domain arginine residues. A, mean (± S.E.) rates of deactivation for N-Cap mutants R4E (panel (i)), R5E (panel (ii)), R20E (panel (iii)), or the PAS domain mutant R56E (panel (iv)), either alone (blue triangles, n = 11–14) or in the presence of the cNBH domain mutation E788R (purple circles, n = 12–17). WT Kv11.1 (black circles, n = 16) and E788R (red squares, n = 21) are shown for comparison. Note that error bars are included but are within the symbols. Mean ± S.E. values for τfast, τslow, and the relative amplitudes of τfast to τslow for WT and all mutants are given in supplemental Table S1. Representative current traces at −120 mV are shown within the insets, with mutants color-coded as above. B and C, summary (mean ± S.E.) showing no restoration of slow deactivation kinetics (Δln(τfast, −120mV), B) or of chemical potential energy (ΔΔG0, C), compared with WT Kv11.1, with any of the combined N-Cap/PAS arginine mutants. Gray bars in C represent the theoretical additive values of the two individual mutant perturbations combined. Means ± S.E. for chemical potential energy parameters are given in supplemental Table S2.

Article Snippet: This fragment was then inserted into the Kv11.1 backbone using Bpu10I and BstEII restriction enzymes (New England Biolabs, Ipswich, MA).

Techniques: Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels *

doi: 10.1074/jbc.M114.558379

Figure Lengend Snippet: cNBH domain residue Glu788 interacts with the N-Cap residue Asn12. A, surface representation homology model of the N-Cap/PAS domain (blue) interacting with the C-linker/cNBH domain (red). The N-Cap residue Asn12 (blue spheres) interacts with Glu788 (red spheres) of the cNBH domain. B, mean (± S.E.) rates of deactivation for WT Kv11.1 (black circles, n = 16), N-Cap mutant N12E (blue triangles, n = 5), cNBH domain mutant E788R (red squares, n = 21), and combined mutant N12E/E788R (purple circles, n = 8). Note that error bars are included but are within the symbols. Mean ± S.E. values for τfast, τslow, and the relative amplitudes of τfast to τslow for WT and all mutants are given in supplemental Table S1. Inset, representative current traces at −120 mV for each mutant are color-coded as above. C, summary (mean ± S.E.) showing that the combined mutant N12E/E788R has slower deactivation kinetics, measured as smaller Δln(τ−120 mV) compared with WT Kv11.1, than either of the two single mutants alone. D and E, summary (mean ± S.E.) ΔΔG0 for charge reversal (D) or alanine (E) mutant channels compared with the WT. Note that the effects of the two single mutants are not additive when combined in the double mutant N12E/E788R. Gray bars represent the theoretical additive values of the two individual mutant perturbations combined. Means ± S.E. are given in supplemental Table S2.

Article Snippet: This fragment was then inserted into the Kv11.1 backbone using Bpu10I and BstEII restriction enzymes (New England Biolabs, Ipswich, MA).

Techniques: Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels *

doi: 10.1074/jbc.M114.558379

Figure Lengend Snippet: N-Cap residues Arg4/Arg5 may interact with the C-linker residues Glu698/Glu699. A, surface representation homology model showing that residues Arg4 and Arg5 (shown in “blue” spheres) of the N-Cap/PAS domains (blue) are in close proximity to residues Glu698 and Glu699 (shown in red spheres) of the C-linker/cNBH domains (red) in Kv11.1 channels. B, mean (± S.E.) rates of deactivation for WT Kv11.1 (black circles, n = 16), N-Cap double mutant R4E/R5E (blue triangles, n = 5), C-linker double mutant E698R/E699R (red squares, n = 5), and combined charge reversal mutant R4E/R5E+E698R/E699R (EERR, purple circles, n = 5). Note that error bars are included but are within the symbols. Mean ± S.E. values for τfast, τslow, and the relative amplitudes of τfast to τslow for WT and all mutants are given in supplemental Table S1. Inset, representative current traces at −120 mV for each mutant are color-coded as above. C, summary (means ± S.E.) showing that the charge reversal mutant R4E/R5E+E698R/E699R (EERR) exhibits altered deactivation kinetics, measured as Δln(τ−120 mV) compared with WT Kv11.1, that are similar to the C-linker double mutant E698R/E699R, but much slower (i.e. smaller shift) than the N-Cap double mutant R4E/R5E. D and E, summary (mean ± S.E.) ΔΔG0 for charge reversal (D) or alanine (E) mutant channels compared with the WT. Note that the effects of the N-Cap double mutant (R4E/R5E) and the C-linker/cNBH domain double mutant (E698R/E699R) are not additive when combined in the quadruple mutant. Gray bars represent the theoretical additive values of the two individual mutant perturbations combined. Means ± S.E. are given in supplemental Table S2.

Article Snippet: This fragment was then inserted into the Kv11.1 backbone using Bpu10I and BstEII restriction enzymes (New England Biolabs, Ipswich, MA).

Techniques: Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels *

doi: 10.1074/jbc.M114.558379

Figure Lengend Snippet: N-Cap and PAS domains share a common mechanism for the regulation of slow deactivation gating kinetics. A, mean (±S.E.) rates of deactivation for WT Kv11.1 (black circles, n = 16), the N-Cap domain double mutant R4D/R5D (orange inverted triangles, n = 5), the PAS domain mutant R56D (red triangles, n = 5), and the N-Cap/PAS triple mutant R4D/R5D+R56D (purple diamonds, n = 6). Note that error bars are included but are within the symbols. Mean ± S.E. values for τfast, τslow, and the relative amplitudes of τfast to τslow for WT and all mutants are given in supplemental Table S1. Inset, representative current traces at −120 mV for each mutant are color-coded as above. B and C, all mutants show similar accelerated deactivation kinetics (Δln(τ−120 mV), B) and similar changes in chemical potential energy (ΔΔG0, C) compared with WT Kv11.1, indicating a shared mechanism for slowing deactivation kinetics. Gray bars in C represent the theoretical additive values of the two individual mutant perturbations combined. Means ± S.E. for chemical potential energy parameters are given in supplemental Table S2.

Article Snippet: This fragment was then inserted into the Kv11.1 backbone using Bpu10I and BstEII restriction enzymes (New England Biolabs, Ipswich, MA).

Techniques: Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels *

doi: 10.1074/jbc.M114.558379

Figure Lengend Snippet: Proposed molecular mechanism for slow deactivation in Kv11. 1 channels. Cartoon of two opposing subunits of a tetrameric Kv11.1 channel shown in the closed (left) and open (right) conformations. In both conformations, Arg56 in the PAS domain (blue triangles) and Asp803 in the cNBH domain (red squares) form a highly stable interaction (denoted by black star). In the open conformation, Arg4 and Arg5 in the N-Cap domain (shown in purple) and Glu698 and Glu699 in the C-linker (shown in maroon) form an additional, but more transient, interaction (denoted by gray star) that stabilizes the open state, presumably by stabilizing the S6 activation gate (represented by dark gray bar) in the open conformation. The stable PAS-cNBH domain interaction is critical because it positions the N-Cap domain in the correct location to interact with the C-linker. These interactions underlie the slow deactivation gating observed in Kv11.1 channels.

Article Snippet: This fragment was then inserted into the Kv11.1 backbone using Bpu10I and BstEII restriction enzymes (New England Biolabs, Ipswich, MA).

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: A Comprehensive Evaluation of Sdox, a Promising H 2 S-Releasing Doxorubicin for the Treatment of Chemoresistant Tumors

doi: 10.3389/fphar.2022.831791

Figure Lengend Snippet: Effect of Dox and Sdox on K V 11.1 (hERG) current recorded in hERG-HEK293 cells. Concentration-dependent effect of Dox and Sdox of hERG tail currents. On the ordinate scale, current amplitude is reported as a percentage of the value recorded just before the addition of the first concentration of the drug. Data points are the mean ± SD ( n = 6).

Article Snippet: hERG-HEK293 cells (BPS Bioscience, San Diego, United States), expressing hERG K + channel (Kv11.1), were cultured in Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% Na pyruvate, 1% penicillin/streptomycin, and 400 μg/ml geneticin ( ).

Techniques: Concentration Assay