Disruption of a Conservative Motif in the C-Terminal Loop of the KCNQ1 Channel Causes LQT Syndrome

Figures

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  Figure 1. Schematic diagram of Kv7.1 ion channel organization. One alpha (KCNQ1) and one beta (KCNE1) subunit are shown. VSD—voltage-sensing domain; PD—pore domain; CaM—calmodulin.

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  Figure 2. (A) Pedigree of family with genetic variant c.1264A > G (K422E) in the KCNQ1 gene. Family members with QTc prolongation—closed symbols; healthy individuals—open symbols. Arrow—proband. (B) Confirmation of the heterozygous c.1264A > G variant in the KCNQ1 gene by Sanger sequencing. Arrow—a.a. substitution. (C) Fragment of the proband’s ECG (5-year-old male, standing). Sinus rhythm, HR, 102 bpm, PQ, 142 ms, QTc, 570–587 ms.

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  Figure 3. (A) Immunoblot analysis of Kv7.1 expression in whole-cell lysates of CHO-K1 cells. WT—cells transfected with control Kv7.1 protein; K422E—cells, transfected with Kv7.1 protein with K422E mutation; VEC—cells transfected with an empty vector; NT—non-transfected cells. Primary antibodies against V5-tag and alpha-tubulin. (n = 3 blots from three transfections). (B) Immunofluorescent staining of CHO-K1 cells expressing the recombinant Kv7.1 protein: wildtype (top left) and mutant (top right). Primary antibody against V5-tag, secondary antibody conjugated to alexa−488 dye (green channel). Nuclei are stained with DAPI (blue channel). Bottom—intensity of green signal across the cell (along the white lines in the images in the top row) with the two peaks corresponding to cell plasma membrane (M1 and M2). (C) Statistical analysis of the surface plots of Kv7.1 fluorescence intensity signal, mean ± SE fluorescence of Kv7.1 (WT, 18 cells, left; and K422E mutant, 28 cells, right) normalized to the average cell fluorescence intensity signal.

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  Figure 4. Electrophysiological properties of IKs current in transfected CHO-K1 cells. (A–C) Original recordings of the current from 3 different representative cells transfected with plasmid containing KCNE1 cDNA and either the WT Kv7.1 cDNA (A), or the same cDNA with introduced K422E mutation (B) or 1:1 combination of these two plasmids (C). The current was elicited by the 2-step square-pulse depolarization (shown in the inset), from the holding potential of −80 mV. The second step to −40 mV was used to analyze the outward tail current. (D–G) Comparison of the main parameters of IKs recorded from cells transfected with plasmid containing the WT Kv7.1 cDNA (n = 15), the same cDNA with introduced K422E mutation, homozygous K422E/K422E (n = 17), or 1:1 combination of these two plasmids (heterozygous WT/K422E, n = 16). The data is mean ± SEM. (D) I-V curves of peak tail IKs current in 3 groups of cells. * Significant difference from WT group, p < 0.05, two-way ANOVA with Tukey’s posthoc test. (E) Activation curves of IKs in 3 groups of cells. G/Gmax is the ratio between actual and maximal conductance. (F) Comparison of tail current deactivation kinetics (τD) after +40 mV depolarization in 3…

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  Figure 5. Simulated action potential (left panel) and IKs current (right panel) in control (WT) and heterozygote (WT/K422E) mutation models.

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  Figure 6. (A) Multiple sequence alignment for the 412–431 fragments of the human Kv7.1 and corresponding fragments of their relative sequences. Asterisks mark fully conserved positions. (B) Sequence logo for the fragment of an alignment of several Kv7.1 sequences. The height of each residue is proportional to its frequency, while the height of the overall stack of residues is inversely proportional to Shannon entropy. (C) Electrostatic potential maps showing positive (blue) and negative (red) potential near the Kv7.1-KCNE3-CaM-PIP2 complex. Blue and red electrostatic potential maps correspond to +400 kT/e and −400 kT/e, respectively. Protein chains are colored according to their IDs. Four PIP2 molecules are shown in space-filling representation. Eight Ca2+ ions are shown with brown spheres. Red circles correspond to the regions with the highest values of negative potential.