Role of the pH in state-dependent blockade of hERG currents

Figure 1. (a) Structural alignment of hERG homology models for closed (blue), open (green) and open-inactivated (light green) states and the Eag1 pore domain structure (PDB ID 5K7L). (b) RMSD per residue calculated between each of the different hERG model states and Eag1 subunit structure. RMSD coloured subunits for the different model states are shown in the inset. Sequence alignment for the pore region is shown at the top (53 % of identity and ~75% of similarity). (c) View of the internal cavity for the different model states aligned to the Eag1 pore structure.

Figure 2. (a) Reaction coordinate for binding free energy computations. The reaction coordinate is shown with a blue arrow. The reference position is the center of mass of the alpha carbon atoms of residues 623–628 in the filter (the part shown with red sticks). The initial and final location of dofetilide is shown in yellow and gray respectively. The gray lines represent the location of the head groups of the lipids. (b) Potentials of Mean Force for the binding of neutral (dark and light colors for the open and open-inactivated states, respectively) and cationic dofetilide forms (black and magenta for the open and open-inactivated states, respectively). Inset: Structure of neutral and cationic dofetilide:1-(4-methanesulfonamidophenoxy)-2-(N-(4-methanesulfonamidophenethyl)-N-methylamine)ethane .

Figure 3. State-dependent differences in binding of cationic dofetilide. (a) Potential of mean force for the movement of cationic dofetilide. Two energy wells were chosen from open (black) and open-inactivated (red) hERG. (b) Locations of dofetilide binding sites in hERG and interaction details were shown. All atoms within 3.9 Å of dofetilide were shown with sticks. Water molecules were shown as red balls and cyan sticks respond to the hydrogen bonds.

Figure 4. Conformation dynamics of bound dofetilide and coordinating residues in the hERG channel from free energy simulations. (a) Left: distance of benzene rings of cationic dofetilide in the inner binding site for the open (black) and open-inactivated (blue) channel and in the outer binding site for the open (red) and open-inactivated (green) channel. Middle: sketch map of the distance between two benzene rings. Right: distance of benzene rings of neutral dofetilide in the inner binding site for the open (black) and open-inactivated (red) channel. (b) Conformational changes of Y652 and F656 in the open (green) and open-inactivated (orange) channel.

Figure 5. Time and concentration-response relationships. (a) Amplitudes of hERG currents response to 100 nM dofetilide in control (pH7.2) and acidic (pH6.2) intracellular solutions during constant rate stimulation. (b) Concentration -response curves of dofetilide on hERG currents at different intracellular pH. At pH7.2 the IC50 was 0.041 μM, Hill’s coefficient 2.4; whereas pH6.2 IC50 was 0.015 μM, Hill’s coefficient 4.2. n = 3, 2, 2, 5 in pH7.2 and n = 2, 4, 2, 6, 7 in pH6.2 for concentrations tested. Panels c,d- The raw superimposed hERG current traces shown every 2 min after beginning superfusion with 100 nM dofetilide to 10 min at pH6.2 (c) and pH7.2 (d) intracellular pHs. The times (minutes) are shown at the end of the tail current traces.

Figure 6. Time-course of onset of use-dependent block (a,b). The superimposed hERG current traces before (dashed line at baseline) and for each pulse of use dependent block with 1 μM dofetilide (solid lines) in different intracellular pHs. (c) The time courses of cells of A and B. The insert showed the experiment protocol. (d) Decay time constants at 0.1 and 1 μM dofetilide in different intracellular pHs. Single exponential function was used to fit the decay time courses. N = 5, 4 in 0.1 μM and 1 μM dofetilide at pH of 6.2, 8.0.

Figure 7. Raw example hERG current traces recorded in the drug-free state at intracellular pH of 6.2 (a) and 8.0 (b) elicited by the pulse protocol shown in the insert. (c) Average g-V relationship of hERG currents at intracellular pH 6.2 versus 8.0. The average V1/2 were −2.5 +/− 1.9 mV in pH 6.2 n = 5 and 3.7 +/− 2.2 mV at pH 8.0 (n = 4, P < 0.05, t test). The slope factor were 9.2 +/− 0.4 and 8.2 +/− 0.3 respectively. (d) Average current density amplitudes at pH6.2 and pH8.0 (n = 9, 11).