Determinants of Isoform-Specific Gating Kinetics of hERG1 Channel: Combined Experimental and Simulation Study

Figures

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  Figure 1. (A) Topology of Kv11.1 or hERG channel. (B) Comparison of a-isoform and b-isoform. Exons of each of the two splice variants, with a-isoform on the top and b-isoform on the bottom.

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  Figure 2. Proposed kinetic mechanisms using Markov models (M-models) for WT hERG (Perissinotti et al., 2015; Romero et al., 2015) showing the transition rates with the corresponding labels.

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  Figure 3. Representative current traces of hERG1a and hERG1b channels and their current-voltage relationship at room temperature. (A) Selected current traces recorded from HEK cells and elicited by the voltage protocol shown in the top. (B) Normalized tail currents against voltage. The solid lines correspond to the fitted Boltzmann functions and symbols correspond to data. All data are listed as mean ± SEM. hERG1a (n = 10), hERG1b (n = 10). (C) Currents measured at the end of each step were used to construct the current-voltage (I-V) relationship. All data are shown as mean ± SEM. hERG1a (n = 10), hERG1b (n = 10).

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  Figure 4. Activation kinetics of hERG1a and hERG1b channels expressed in HEK cells. An envelope of tails protocol was used to measure the activation properties at +40 mV. (A) Representative current traces elicited by the protocol shown at the top corresponding to 5–500 ms of activation are shown. (B)The data were normalized to the maximum amplitude of the tail current and plotted against time. hERG1a (n = 10); hERG1b (n = 10). All data are shown as mean ± SEM.

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  Figure 5. Deactivation Kinetics of hERG1a and hERG1b channels measured in HEK cells. (A) Representative current traces elicited by the voltage protocol shown in the top for both isoforms. (B) Superimposed raw deactivation time courses for voltages −40, −60, −100, and −120 mV for hERG1a (n = 10) and hERG1b (n = 10). Average time course is shown with a solid white line.

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  Figure 6. M-model 1 optimization to experimental data. (A) Activation curve of a-isoform and b-isoform. (B) Steady State Activation curve for both isoforms. (C) Simulated deactivation curves at −120, −100, −60, and −40 mV. Simulated data is shown as solid lines and symbols are used for experimental time course data (average, n = 10). Superimposed raw time course data (n = 10) is shown in the background for deactivation at each voltage.

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  Figure 7. M-model 2 fit to experimental data. (A) Activation curve of a-isoform and b-isoform. (B) Steady State Activation curve for both isoforms. (C) Simulated deactivation curves at −120, −100, −60, and −40 mV. Simulated data is shown as solid lines and symbols are used for experimental time course data (average, n = 10). Superimposed raw time course data (n = 10) is shown in the background for deactivation at each voltage.

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  Figure 8. Simulated currents M-model 1 and M-model 2. (A) Simulated currents elicited by the SSA voltage protocol (Figure 3) for both M-models and Isoforms. (B) Simulated I-V steady state current (x104 pA) relationship for M-model 1 and 2. a-Isoform is shown as black dashed line and b-isoform as red dashed line. Experimental data is shown in the background as symbols with the corresponding error bar (Figure 3).

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  Figure 9. Models of the quaternary structure of homomeric hERG tetramers. Side view (A) of closed state model in the left panel and open state hERG Cryo-EM structure, right panel. In both structures N-cap PAS can be seen interacting with VSD (black arrow). When the TM domain is aligned, cytoplasmic domains are slightly rotated respects each other. Orange arrow show the rotation direction when transitioning from closed to open states. (B) N-cap PAS interaction with VS (S1 & potentially S4-S5), CNBD, and C-linker for closed in the left panel and open states, right panel.

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  Figure 10. Structural alignment of closed (light colors) and open (dark colors) models of hERG. TM and C-linker side view shows that the C-linker/TM are closer to each other in the open state compared to the closed (A). Distances between residues Gly669 show that a closer C-linker is a consequence of a more open pore in the open state (B). Key salt bridge interactions between Glu544 and Arg681 can only be found in the open state (C) but not in the closed state (D). (E) Current traces from experiments previously published by Durdagi et al. for hERG1a, hERG1b, and hERG1a-E544L elicited by the voltage protocol at the top. E544L mutant displays a faster deactivation rates than WT but not as much as hERG1b.

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  Figure 11. Proposed mechanism based on the structures modeled. When the channel opens the cytoplasmic domains rotate toward the membrane as the S6 end twists in the same direction.

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  Figure 12. Simulated action potential duration (APD) in cells and virtual pseudo-ECGs in a transmural 1-D tissue model. (A) Single cells simulations show concentration response for different ratios of a- and b-isoforms. (B) APD for the 1,000th paced beat at 1 Hz in single endocardial cells. hERG 1a shows a longer APD compared to hERG 1b. (C) The Pseudo ECGs of hERG 1b (red) indicates shorter QT intervals than hERG 1a.