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. V_0.5 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, V_0.5 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 (ΔG_{0, deact}) and activation (ΔG_{0, 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.