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	Figure 1. Mode shift of hERG channels. (A) Example traces recorded in response to protocols shown to measure the voltage-dependent equilibria of activation (i) and deactivation (ii) of E518C hERG channels. Insets are expansions of the highlighted regions. Peak currents were measured at the points marked with asterisks. (B) Example fluorescent traces recorded from MTSR-labeled 518C using voltage-clamp fluorometry in response to the protocols shown to measure activation/upward motion (i) and return (ii) of the VSD of E518C hERG. (C) Summary of voltage dependence of equilibria of activation and deactivation. Solid lines are fits of the Boltzmann equation to current data, whereas broken lines are fits of the Boltzmann equation to the fluorescent data. V0.5 values were −4 ± 0.9 mV and −44 ± 2 mV for current activation and deactivation, respectively (SEM; n = 10) and 1.4 ± 2 mV and −50.9 ± 1 mV for upward motion and return of the VSD, respectively (SEM; n = 12).
	Figure 2. Effect of N-terminal truncation on mode shift of hERG channels. (A) Example traces recorded in response to protocols shown to measure the voltage-dependent equilibria of activation (i) and deactivation (ii) of Δ2–25 hERG channels. Insets are expansions of the highlighted regions. Peak currents were measured at the points marked with asterisks. (B) Example fluorescent traces recorded using voltage-clamp fluorometry in response to the protocols shown to measure activation/upward motion (i) and return (ii) of the VSD of Δ2–25 hERG. (C) Summary of voltage dependence of equilibria of activation and deactivation. Solid lines are fits of the Boltzmann equation to current data, whereas broken lines are fits of the Boltzmann equation to the fluorescent data. V0.5 values were 2 ± 0.3 mV and −10 ± 0.4 mV for current activation and deactivation, respectively (SEM; n = 7). V0.5 values were measured as 0 ± 1 mV (SEM; n = 8) and −43 ± 7 mV (SEM; n = 11) for activation and return of the VSD, respectively.
	Figure 3. Effect of N-terminal truncation on kinetics of hERG activation. (A) Typical examples of currents recorded in response to the envelope of tails protocols shown for E518C (i) and Δ2–25 (ii) hERG channels. In these examples, the activating potential was 100 mV. (B) Peak tail currents plotted against the duration of the activating pulse for the examples shown in A. The black line is a fit of a single exponential to the activation time course. (C) Summary of the voltage dependence of rates of activation of ionic current. Error bars are SEM; n ≥ 8. (D) Summary of the voltage dependence of the rates of VSD activation/upward motion derived from single exponential fits to the fluorescent time course using the same protocol illustrated in Fig. 1 B (i). Error bars are SEM; n ≥ 7. The inset shows example fits to fluorescent traces from MTSR-labeled E518C recorded at 100 mV.
	Figure 4. Effect of N-terminal truncation on kinetics of hERG deactivation. (A) Typical examples of ionic current (i) and fluorescence (ii) recorded after repolarization to −120 mV from a depolarized holding potential for E518C and Δ2–25 hERG. The fast time constant (τfast) for deactivation of ionic current was derived from a fit of a double exponential function to the decaying phase of the current after repolarization. Time constants for VSD return were derived from fits of a single exponential to the decaying phase of the fluorescence record. (B) Summary of the voltage dependence of rates of deactivation and VSD return for E518C and Δ2–25 hERG channels. Error bars are SEM; n ≥ 7.
	Figure 5. Alanine scan of N-terminal tail. (A) Example traces recorded in response to protocols shown to measure the voltage-dependent equilibria of activation (i) and deactivation (ii) ionic current in R4A hERG channels. Insets are expansions of the highlighted regions. (B) Summary of voltage-dependent equilibria of activation (i) and deactivation (ii) for R4A, R5A, and G6A mutants. Solid lines are fits of the Boltzmann equation to data. V0.5 values for activation were measured as 3.7 ± 1.1 mV, 2.8 ± 1.3 mV, and −3.4 ± 1.5 mV for R4A, R5A, and G6A, respectively (SEM; n > 4). For deactivation of ionic current, V0.5 values were measured as −19.3 ± 2.5 mV, −10 ± 0.9 mV, and −16.1 ± 1.2 mV for R4A, R5A, and G6A, respectively (SEM; n > 5). (C) Mutation-dependent changes (termed ΔΔΔG0) in the difference (ΔΔG0) between the free energy terms associated with the voltage-dependent equilibrium of deactivation (ΔG0, deact) and activation (ΔG0, act) relative to wild-type hERG (SEM; n ≥ 21). Three amino acid residues in this region, R4A, R5A, and G6A, contribute strongly to coupling with ΔΔΔG0 of ∼3 kcal/mol.
	Figure 6. Voltage-clamp fluorometry of residues contributing to coupling. (A) Summary of the voltage-dependent equilibria of activation (i) and deactivation (ii) of ionic current for R4A, R5A, and G6A mutants in the E518C background. Solid lines are fits of the Boltzmann equation to data. (B) Example fluorescent traces recorded from MTSR-labeled R4A E518C hERG using voltage-clamp fluorometry in response to the protocols shown to measure upward motion (i) and return (ii) of the VSD. (C) Summary of the voltage-dependent equilibria of VSD upward motion and return. V0.5 values for VSD upward motion were −1.4 ± 2 mV, 20.9 ± 2.6 mV, 10 ± 3.5 mV, and 6.2 ± 3.8 mV for E518C, R4A, R5A, and G6A, respectively (SEM; n > 4). V0.5 values for VSD return were −46.4 ± 1 mV, −34.5 ± 7.2 mV, −29.6 ± 3.8 mV, and −29.0 ± 4.4 mV for E518C, R4A, R5A, and G6A, respectively (SEM; n > 4). (D) Difference between free energy terms (ΔΔG0) associated with the voltage-dependent equilibrium of current deactivation relative to current activation (open) and VSD return relative to VSD upward motion (gray; SEM; n ≥ 6).
	Figure 7. Mode shift of ionic current in S631A hERG. (A) Example traces recorded in response to the protocols shown to measure the voltage-dependent equilibria of activation (i) and deactivation (ii) of ionic current in inactivation-deficient S631A hERG channels. Insets are expansions of the highlighted regions. (B) Summary of the voltage-dependent equilibria of activation and deactivation. Solid lines are fits of the Boltzmann equation to activation data, whereas broken lines are fits of the Boltzmann equation to the deactivation data. Neither the V0.5 values for activation (−20.32 ± 0.5 mV and −21.7 ± 0.7 mV for wild type and S631A, respectively, SEM; n ≥ 5) nor deactivation (−54.9 ± 1.6 mV and −50.72 ± 0.9 mV for wild type and S631A, respectively, SEM; n = 4) were significantly different from wild type (Student’s t test, P < 0.05).
	Figure 8. A simple domain level model of coupling of VSD mode shifting to the gate during hERG activation/deactivation. (i) In the closed state, the N-terminal tail does not directly interact with the gate. (ii) Upon depolarization, the VSD moves in the membrane into the activated state. This upward motion is coupled to opening of the gate via direct interactions of the S4–S5 and the S6 helix. (iii) With sustained depolarization, the VSD relaxes. Relaxation of the VSD is accompanied by a conformational change of the S4–S5 linker that causes it to dissociate from its binding site on the S6. The N-terminal tail then acts as an adaptor between the S4–S5 and S6 to couple VSD return during repolarization to closing of the gate.

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