Volume 10, Issue 1, Pages 128-136 (January 2013) RNA interference targeting E637K mutation rescues hERG channel currents and restores its kinetic properties  Xiaoli Lu, MD, Xi Yang, MD, Xiaoyan Huang, MD, Chen Huang, MD, PhD, Huan Huan Sun, MD, Lihua Jin, MD, Weifeng Xu, MD, Haiyan Mao, MD, Junming Guo, PhD, Jianqing Zhou, MD, Jiangfang Lian, MD, PhD  Heart Rhythm  Volume 10, Issue 1, Pages 128-136 (January 2013) DOI: 10.1016/j.hrthm.2012.09.124 Copyright © 2013 Heart Rhythm Society Terms and Conditions

Figure 1 Analysis of hERG protein expression in HEK293 cells. A: hERG protein expressions in WT-hERG, WT/E637K-hERG, E637K-hERG, and WT/E637K-hERG/siRNA transfected cells. hERG levels in membrane fraction are shown in the lower panel. B, C: Knocking-down efficiencies of different siRNAs against E637K-hERG (B) or WT-hERG (C). Note that none of the 5 siRNAs had any effect on WT-hERG protein levels. D, E: Cell growth (D) and viability (E) were not affected by any of the siRNAs. Heart Rhythm 2013 10, 128-136DOI: (10.1016/j.hrthm.2012.09.124) Copyright © 2013 Heart Rhythm Society Terms and Conditions

Figure 2 Confocal imaging of pmCherry-WT-hERG and pEGFP-E637K-hERG channels in U2OS cells. U2OS cells were transfected with WT-hERG and/or E637K-hERG plasmids, in the presence or absence of siRNA. Whereas WT-hERG (first column) localizes to both the plasma membrane and cytoplasm, the E637K-hERG mutant (second column) localizes exclusively to cytoplasm. Membrane localization of WT-hERG is lost when cells coexpress E637K-hERG mutant (third row), and this loss is rescued by siRNA (fourth row). Scale bar = 5 μm. Heart Rhythm 2013 10, 128-136DOI: (10.1016/j.hrthm.2012.09.124) Copyright © 2013 Heart Rhythm Society Terms and Conditions

Figure 3 Effect of siRNA on voltage-dependent activation of hERG channel. Inset shows the voltage-clamp protocol. A–F: Representative current traces in HEK293 cells transfected with different plasmids in the presence or absence of siRNA. G, H: Current–voltage relationships for peak currents and tail currents amplitudes of WT-hERG or WT/E637K-hERG transfected cells in the presence or absence of siRNA. I: Amplitudes of tail currents of WT-hERG or WT/E637K-hERG channel in the presence or absence of siRNA plotted as a function of the test potential and fitted to a Boltzmann function (n = 6). Heart Rhythm 2013 10, 128-136DOI: (10.1016/j.hrthm.2012.09.124) Copyright © 2013 Heart Rhythm Society Terms and Conditions

Figure 4 Effect of siRNA on steady-state inactivation of hERG channel. Inset shows the voltage-clamp protocol. A–D: Representative current traces in HEK293 cells transfected with WT-hERG or WT/E637K-hERG plasmids in the presence or absence of siRNA. E: Normalized steady-state inactivation curves in cells transfected with WT-hERG or WT/E637K-hERG plasmids in the presence or absence of siRNA (n = 6). Heart Rhythm 2013 10, 128-136DOI: (10.1016/j.hrthm.2012.09.124) Copyright © 2013 Heart Rhythm Society Terms and Conditions

Figure 5 Effect of siRNA on time courses of inactivation of hERG channel. Inset shows the voltage-clamp protocol. A–D: Representative current traces of time courses and inactivation time constants (tau [τ])in HEK293 cells transfected with WT-hERG or WT-hERG/E637K-hERG plasmids in the presence or absence of siRNA. E: The τ was measured by fitting inactivation currents of WT-hERG or WT-hERG/E637K-hERG channel in the presence or absence of siRNA during test pulse at each potential with a single exponential function (n = 6). Heart Rhythm 2013 10, 128-136DOI: (10.1016/j.hrthm.2012.09.124) Copyright © 2013 Heart Rhythm Society Terms and Conditions

Figure 6 Effect of siRNA on recovery from inactivation of hERG channel. Inset shows the voltage-clamp protocol. A–D: Representative recovery from inactivation in HEK293 cells transfected with WT-hERG or WT/E637K-hERG plasmids in the presence or absence of siRNA. E: Time constants (tau [τ]) for hERG channel recovery from inactivation plotted as a function of test voltages for siRNA to hERG channel (n = 6). Heart Rhythm 2013 10, 128-136DOI: (10.1016/j.hrthm.2012.09.124) Copyright © 2013 Heart Rhythm Society Terms and Conditions

Figure 7 Effect of siRNA on deactivation of hERG channel. Inset shows the voltage-clamp protocol. A–D: Representative deactivation in HEK293 cells transfected with WT-hERG or WT/E637K-hERG in the presence or absence of siRNA. Arrow marks the deactivation phase. E: Fast and slow components of deactivation time constants (tau [τ]) plotted as a function of test potentials for siRNA to hERG channel (n = 6). Heart Rhythm 2013 10, 128-136DOI: (10.1016/j.hrthm.2012.09.124) Copyright © 2013 Heart Rhythm Society Terms and Conditions