Veratridine Can Bind to a Site at the Mouth of the Channel Pore at Human Cardiac Sodium Channel NaV1.5

Figures

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  Figure 1. The structure of veratridine (VTD) drawn in its protonated form with the individual rings and the dimethoxyphenyl (DMP) group as denoted in this manuscript labelled in red.

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  Figure 2. The structures of human NaV1.4 (PDB ID: 6AGF) bound to a detergent molecule at the “mouth site” (left), and the human NaV1.5 (PDB ID: 6LQA) bound to quinidine at the “pore site” (right). The ligand molecules are shown in orange for both structures. The approximate region of each binding site is marked with a black circle. The black bar represents the putative position of the membrane with the extracellular and cytosolic sides of the membrane labelled.

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  Figure 3. Automated patch clamp measurements of voltage-gated sodium current for representative HEK293 cells expressing wild-type SCN5A (A), E417R (B), I1466T (C), or L409S (D). Cells were treated with increasing concentrations of veratridine (inset) and subjected to a repeated voltage pulse (inset). All traces are normalized so that the peak inward current is at 100%. All three variants have a reduced response to veratridine.

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  Figure 4. The correlation between the measured VTD concentrations necessary for 10 % delayed late response the ΔΔG values calculated by Rosetta. The experimental concentrations corresponding to the L409S and I1466T mutations were considered to be 50 µM due to the inability to measure their effects at higher concentrations caused by their large responses.

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  Figure 5. The mouth site pose that had the best correlation factor viewed from different angles. (A) A side view of NaV1.5 showing the binding configuration of VTD at the mouth site (black circle), whereby the top section corresponds to the extracellular side and the bottom section corresponds to the cytosolic side; (B) VTD configuration at the mouth site with the residues reported to form interactions with VTD based on docking calculations labeled; and (C) VTD viewed from two different angles, whereby the residues whose mutations showed a significant (pink) and insignificant (purple) change based on the experimental results labeled. VTD is shown in light gray in all panels.