KCNQ1 p.D446E Variant as a Risk Allele for Arrhythmogenic Phenotypes: Electrophysiological Characterization Reveals a Complex Phenotype Affecting the Slow Delayed Rectifier Potassium Current (IKs) Voltage Dependence by Causing a Hyperpolarizing Shift and a Lack of Response to Protein Kinase A Activation

Figures

• 

  Figure 1. Resting ECG of index case 1, KCNQ1 p.D446E heterozygous. Early repolarization manifested as terminal QRS slurring and J point elevation in V2, V3, V4 and V5.

• 

  Figure 2. Family pedigrees. Pedigrees of probands heterozygous for KCNQ1-D446E variant where at least one first-degree relative was available for screening; (A) index case 1, (B) index case 2. Index cases are indicated with an arrow, circles indicate females, squares males and symbols with diagonal lines deceased individuals. Symbols half-filled in black are KCNQ1-D446E heterozygous, and symbols half-filled with dark gray have LQTS.

• 

  Figure 3. Activation curves. (A) Representative IKs currents of all genotypes without and with 8Br-cAMP and voltage-clamp protocols. Holding potential was −85.5 mV for all cells; however, different voltage step ranges are indicated, as activation curves were obtained at different voltage ranges in the presence or absence of 8Br-cAMP. (B) Activation curves (G-V) illustrating the variance in voltage dependence (V1/2, midpoint of Boltzmann fit) and voltage sensitivity (slope factor) of experimental series without (line-fit) and with (dash line-fit) 8Br-cAMP. Data points are mean values ± SE. (C) Box plots showing the differences and distribution of Boltzmann fit parameters; each point represents one cell. Data without and with 8Br-cAMP are represented as filled and empty circles, respectively. Differences were assessed using a two-way ANOVA followed by a post hoc Tukey test of significance. Actual p values for comparisons are indicated above the box plots. The number of cells was as follows: WT, n = 24; 446 D/E, n = 12; 446 E/E, n = 15; 446 D/D-cAMP, n = 8; 446 D/E-cAMP, n = 9; 446 E/E-cAMP, n = 7. wo: without 8Br-cAMP; cAMP: with 8Br-cAMP.

• 

  Figure 4. Mean rise time estimations of all IKs series according to voltage without and with 8Br-cAMP stimulation in different genotypes. (A) Comparison among genotypes without 8Br-cAMP. (B) With 8Br-cAMP. (C–E) Effect of 8Br-cAMP on rise time for all tested potentials in each genotype. Data without and with 8Br-cAMP are represented as filled and empty circles, respectively. Asterisks indicate significant differences without and with 8Br-cAMP as assessed by a two-way ANOVA followed by a post hoc Tukey test of significance. Number of cells was as follows: WT, n = 24; 446 D/E, n = 12; 446 E/E, n = 15; 446 D/D-cAMP, n = 8; 446 D/E-cAMP, n = 9; 446 E/E-cAMP, n = 7.

• 

  Figure 5. Deactivation kinetics. (A) Representative traces of each experimental series showing the IKs deactivation rate from 34.5 mV to −125.5 mV. The prepulse to activate IKs was 5 s at 34.5 mV and the test pulse to deactivate was 1 s at −125.5 mV. (B) Box plots showing statistically significant differences between experimental series without and with 8Br-cAMP. Filled circles represent data without 8Br-cAMP and empty circles with the 8Br-cAMP effect. Wo: without 8Br-cAMP; cAMP: with 8Br-cAMP. Differences were assessed using a two-way ANOVA followed by a post hoc Tukey test of significance. WT n = 10; WT-cAMP n = 8; Homo n = 7; Homo-cAMP n = 6; Hetero n = 5; Hetero-cAMP n = 6.

• 

  Figure 6. Structural model of the Kv7.1 channel. The vertical dotted line shows the channel axis. The attached conformation is represented in green, while the detached conformation is shown in red. The amino and carboxyl domains, whose experimental structures have not been determined, are denoted by N and C, respectively. (A) Superposition of Kv7.1 monomers highlighting the dynamic movement of our modelled domain 5 upon detachment of the VSD domain. Domain 5 is delineated by green or red outlines, while calmodulin is depicted in orange or yellow. Notably, domain 5 changes both its position and orientation, as evident by the positioning of the variant site and its adjacent nitrosylation site (magenta) in the different conformations. (B) Kv7.1 tetramers in attached (left) and detached (right) conformations, with the same color codes as in panel A except for calmodulin, which is yellow in both cases. The sizes of the amino and carboxyl domains are comparable to that of domain 5. N and C represent the locations of the chains of each domain within the cytosol. (C) Comparison of upper and lateral views showcasing the conformational differences between aspartic and glutamic acid side chains.…