KCNH2 ‐ L693P Causes Long QT Syndrome Type 2 Through hERG Channel Dysfunction: Functional Validation of a Variant of Uncertain Significance

Figures

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  FIGURE 1. Clinical and genetic characteristics of the proband and family members. (A) Electrocardiogram (ECG) recorded after the proband's most recent syncopal episode at a local hospital, showing frequent multiform premafture ventricular contractions with markedly prolonged QT interval (QTc 600 ms). (B) Follow‐up ECG obtained at our center demonstrating QT prolongation (QTc 530 ms) with bifid T waves. (C) Pedigree of the proband's family. Squares represent males and circles represent females. Filled black symbols indicate carriers of the L693P mutation, open symbols indicate unaffected individuals, and the arrow denotes the proband. (D) Representative Sanger sequencing chromatograms showing the c.2078 T>C (p.Leu693Pro) variant in different family members. The proband (II‐4) and her father (I‐1) are heterozygous carriers of the mutation, indicated by overlapping peaks (red arrows), whereas the proband's sister (II‐2) displays the wild‐type sequence at the same position.

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  FIGURE 2. Electrocardiographic abnormalities in family members carrying the KCNH2‐L693P mutation. (A) ECG of the proband's father (I‐1) showing QTc prolongation (470 ms) with bifid T waves. (B) ECG of the proband's aunt (II‐3) demonstrating QTc prolongation (465 ms) with bifid or flattened T waves. (C) ECG of the proband's niece (III‐1) revealing QTc prolongation (480 ms) with bifid T waves. (D) ECG of the proband's nephew (III‐7) showing QTc prolongation (460 ms) with bifid T waves.

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  FIGURE 3. Expression and maturation of hERG protein in WT‐hERG, L693P‐hERG, and WT/L693P‐L693P cells. (A) Representative Western blot showing hERG protein expression and maturation status. The upper band at 155 kDa corresponds to the fully glycosylated mature hERG protein localized to the plasma membrane, whereas the lower band at 135 kDa represents the core‐glycosylated immature hERG protein retained in the endoplasmic reticulum (ER). Mature 155 kDa bands are clearly detected in WT‐hERG and WT/L693P‐hERG groups but are absent in the L693P‐hERG group. Tubulin (55 kDa) served as the loading control. Each lane was loaded with 30 μg of protein. (B) Quantitative analysis of the relative expression levels of the 135 kDa hERG protein in WT, L693P, and WT/L693P cells (n = 3). **p < 0.01 vs. WT; ***p < 0.001 vs. WT. (C) Quantitative analysis of the relative expression levels of the 155 kDa hERG protein in WT, L693P, and WT/L693P cells (n = 3). ***p < 0.001 vs. WT; ****p < 0.0001 vs. WT.

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  FIGURE 4. Subcellular localization of hERG protein in WT, L693P, and WT/L693P cells. Confocal microscopy images of HEK293 cells transfected with WT‐KCNH2‐GFP, L693P‐KCNH2‐GFP, or WT/L693P‐KCNH2‐GFP plasmids together with pDsRed2‐ER plasmids. The first column shows nuclear staining with DAPI (blue); the second column shows GFP‐labeled hERG protein (green); the third column shows the ER marker pDsRed2‐ER (red); and the fourth column shows merged images. Row 1: WT group; Row 2: WT/L693P group; Row 3: L693P group. In WT cells, hERG protein is predominantly localized to the plasma membrane. In WT/L693P cells, hERG is mainly retained in the endoplasmic reticulum with partial trafficking to the plasma membrane. In contrast, in L693P cells, hERG protein is almost exclusively retained in the endoplasmic reticulum with no detectable membrane localization.

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  FIGURE 5. Whole‐cell patch‐clamp analysis of membrane current and channel activation kinetics. (A) Voltage protocol to assess the effects of the L693P mutation on hERG current and activation kinetics. Membrane potential was clamped at −80 mV and subjected to stepwise pulses from −50 mV to +60 mV (increments of 10 mV) for 3 s, followed by a repolarization pulse at −40 mV for 2 s. Sampling frequency was 20.0 kHz, with a filter frequency of 2.9 kHz and a stimulus interval of 15 s. (B) Currents recorded from HEK293 cells transfected with 4 μg WT‐KCNH2, 2 μg WT‐KCNH2, 2 μg WT‐KCNH2 + 2 μg L693P‐KCNH2, and 4 μg L693P‐KCNH2 according to the described voltage protocol. (C‐D) I‐V curves of tail currents (C) and peak currents (D) recorded from the HEK293 cells transfected with 4 μg WT‐KCNH2 (circles) and 2 μg WT‐KCNH2 + 2 μg L693P‐KCNH2 (squares) (*p < 0.05 vs. WT 4 μg, **p < 0.01 vs. WT 4 μg). (E) Steady‐state activation curves obtained by fitting the normalized hERG tail currents with the Boltzmann equation. WT, wild‐type; MT, L693P mutant.

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  FIGURE 6. Effects of L693P mutation on channel inactivation and steady‐state inactivation kinetics. (A) Voltage clamp protocol for channel inactivation. Membrane potential was held at −80 mV, depolarized to +60 mV for 200 ms for activation and subsequent inactivation of potassium channels, then hyperpolarized to −80 mV for 10 ms, followed by pulse stimulation from −20 mV to +60 mV (increments of 10 mV) lasting 300 ms. (B) Currents recorded using the inactivation voltage clamp protocol from HEK293 cells transfected with 4 μg WT‐KCNH2 and 2 μg WT‐KCNH2 + 2 μg L693P‐KCNH2. (C) Voltage dependence of channel inactivation time constants from single exponential fitting in cells transfected with 4 μg WT‐KCNH2 (circles) and 2 μg WT‐KCNH2 + 2 μg L693P‐KCNH2 (squares). (D) Voltage clamp protocol for steady‐state inactivation. Membrane potential was clamped at −80 mV, initially depolarized to +20 mV for 4 s for slow activation, followed by inactivation. Cells were then subjected to test pulses from −130 mV to +20 mV (increments of 10 mV) for 20 ms, followed by a third depolarization to +20 mV at 0.1 Hz stimulation frequency. (E) Currents recorded using a steady‐state inactivation protocol. (F)…

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  FIGURE 7. Effects of L693P mutation on the deactivation and recovery kinetics. (A) Voltage clamp protocol for channel deactivation. Membrane potential was held at −80 mV, depolarized to +60 mV for 1 s to fully activate tail currents, followed by pulses from −100 mV to −20 mV (increments of 10 mV) for 5 s, and then repolarized to −80 mV. (B) Currents recorded using the deactivation protocol. (C) Voltage dependence of deactivation time constants (fast and slow components) in cells transfected with 4 μg WT‐KCNH2 (fast‐blue circles, slow‐blue squares) and 2 μg WT‐KCNH2 + 2 μg L693P‐KCNH2 (fast‐green squares, slow‐blue triangles). (D) Voltage‐clamp protocol for channel recovery. Membrane potential was clamped at −80 mV, depolarized to +60 mV for 200 ms to activate and inactivate potassium channels, then subjected to pulses from −80 mV to −20 mV (increments of 10 mV) for 300 ms. (E) Currents recorded under the recovery protocol. (F) Recovery time constants derived from single exponential fitting plotted against voltage for 4 μg WT‐KCNH2 (circles) and 2 μg WT‐KCNH2 + 2 μg L693P‐KCNH2 (squares). *p < 0.05 vs. WT 4 μg; **p < 0.01 vs. WT 4 μg; ***p < 0.001 vs. WT 4 μg; ****p < 0.0001 vs. WT 4 μg.