Rescue of expression and function of long QT syndrome-causing mutant hERG channels by enhancing channel stability in the plasma membrane

Figure 1. Reduced temperature (27 °C) culture rescues expression and function of G601S mutant hERG channels.A, expression of hERG proteins in HEK cells stably expressing WT hERG as well as G601S mutant hERG cultured at 37 °C or 27 °C for 24 h. Intensities of 155-kDa hERG bands were normalized to their respective actin loading controls then to WT and summarized as box plots with mean ± SD beneath a representative Western blot image (n = 8). ∗∗∗∗p < 0.0001 between indicated groups. A one-way ANOVA with Tukey’s post hoc test was used. B, families of hERG currents elicited by the voltage protocol shown above in HEK cells stably expressing WT hERG as well as G601S hERG cultured at 37 °C or 27 °C for 24 h. The pulse (IhERG-Pulse) and tail (IhERG-Tail) currents were summarized from cells in 4 to 6 independent experiments beneath representative current traces (n = 15 for WT hERG, n = 12 for G601S at 37 °C, and n = 13 for G601S at 27 °C). ∗∗p < 0.01; ∗∗∗∗p < 0.0001 for voltage at 10 mV for pulse currents, and for voltages ≥10 mV for tail currents between G601S at 27 °C and at 37 °C as well as between WT and G601S at 27 °C. ANOVA with Tukey’s post hoc test was used. Error bars in B represent SD.…

Figure 2. Reduced temperature (27 °C) culture slows both forward trafficking and degradation of mature hERG channels.A and B, effects of reduced temperature culture on hERG forward trafficking. WT hERG-HEK cells were treated with proteinase K (PK, 200 μg/ml, 20 min) to completely remove the 155-kDa hERG protein and thus IhERG. After the treatment, cells were cultured at 27 °C or 37 °C and collected at different time points for Western blot analysis (A) and whole-cell patch clamp recording (B). ∗p < 0.05 between 27 °C and 37 °C, ANOVA with Tukey’s post hoc test was used. C and D, effects of reduced temperature culture on mature hERG degradation. hERG-HEK cells were cultured in the presence of BFA (10 μM) under 37 °C or 27 °C and collected at different time points for Western blot analysis (C) and whole-cell patch clamp recording (D). ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 between 27 °C and 37 °C. ANOVA with Tukey’s post hoc test was used. For hERG expression in A and C, densities of the 155-kDa bands relative to control (CTL, or 0 h) were plotted against time (n = 4). For IhERG in B and D, peak tail currents from cells in three independent experiments are summarized as box plots with mean…

Figure 3. Reduced temperature (27 °C) culture slows degradation rate of WT hERG in 0 mM K+omedium and prerescued G601S hERG in normal culture conditions.A and B, reduced temperature culture or MG132 (10 μM) slows the degradation rate of WT hERG in 0 mM K+o medium. hERG-HEK cells were cultured with BFA (10 μM) in 0 mM K+ medium at 37 °C, 27 °C, or 37 °C + MG132 for various periods. After culture, cells were collected for Western blot analysis (A) and whole-cell patch clamp recordings (B). ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 between 37 °C and 27 °C or 37 °C + MG132. ANOVA with Tukey’s post hoc test was used. C and D, reduced temperature culture or MG132 (10 μM) slows the degradation rate of pre-rescued G601S hERG in normal culture medium. G601S hERG cells were cultured at 27 °C for 24 h to restore the 155-kDa expression. The cells were then cultured with BFA (10 μM) in normal medium at 37 °C, 27 °C, or 37 °C + MG132 for various periods. After culture, cells were collected for Western blot analysis (C) and whole-cell patch clamp recordings (D). ∗∗p < 0.01; ∗∗∗p < 0.001 between 37 °C and 27 °C or 37 °C + MG132. ANOVA with Tukey’s post hoc test was used. For Western blot analyses in A and…

Figure 4. G601S hERG channels can be labelled with an antibody that only targets cell-surface hERG channels. Control HEK cells and HEK cells stably expressing WT, G601S, or Y611H were cultured for 30 min in medium containing FITC-conjugated anti-hERG antibody that binds to an epitope in the extracellularly exposed S1-S2 linker of hERG channels. After washing unbound antibodies from the medium, live-cell images were taken using an inverted fluorescence microscope. The scale bar represents 10 μm. HEK, human embryonic kidney; hERG, human ether-g-go-go-related gene.

Figure 5. Intragenic addition of S624T mutation rescues the membrane expression and current of the G601S hERG channel.A, representative Western blot images of whole-cell lysate from HEK cells stably expressing WT, S624T, G601S, or G601S/S624T hERG channels (n = 7). B, families of IhERG traces elicited by the voltage protocol shown in Figure 1B from HEK cells stably expressing WT, S624T, G601S, or G601S/S624T hERG channels. C and D, summarized IhERG-Pulse- and IhERG-Tail-voltage relationships of these channels from cells in three independent experiments (n = 12 for WT, n = 9 for S624T, n = 12 for G601S, and n = 9 for G601S/S624T). Error bars in C and D represent SD. HEK, human embryonic kidney; hERG, human ether-g-go-go-related gene.

Figure 6. Intragenic addition of S624T substitution rescues the membrane expression and current of N470D and P596R, but not A561T or Y611H mutant hERG channels. A, Western blot images of hERG expression from whole-cell lysate of HEK cells expressing WT, N470D, A561T, P596R, and Y611H hERG channels (n = 5). B, Western blot images of hERG expression from whole-cell lysate of HEK cells stably expressing WT, N470D/S624T, A561T/S624T, P596R/S624T, or Y611H/S624T hERG channels (n = 9). C, families of IhERG traces elicited by the voltage protocol shown in Figure 1B from HEK cells stably expressing WT, N470D/S624T, A561T/S624T, P596R/S624T, or Y611H/S624T hERG channels. D and E, summarized IhERG-Pulse- and IhERG-Tail-voltage relationships from cells in three to four independent experiments (n = 12 for WT, n = 10 for N470D/S624T, n = 10 for P596R/S624T). Error bars in D and E represent SD. HEK, human embryonic kidney; hERG, human ether-g-go-go-related gene.

Figure 7. Intragenic addition of the “trafficking-defective” mutation G601S, N470D, or P596R makes S624T sensitive to 0 mM K+exposure. HEK cells stably expressing WT, S624T, G601S/S624T, N470D/S624T, or P596R/S624T hERG channels were cultured in either 5 mM K+ or 0 mM K+ medium for 6 h. After the treatment, cells were collected for Western blot analysis and whole-cell voltage clamp recordings. A, representative Western blot images (n = 3–5). B, peak tail current from cells in three to five independent experiments are summarized as box plots with mean ± SD beneath representative current traces. NS, not significant; ∗∗∗∗p < 0.0001 compared to 5 mM K+ medium. A two-tailed unpaired Student’s t test was used. HEK, human embryonic kidney; hERG, human ether-g-go-go-related gene.