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Structural basis for inhibition of the cardiac sodium channel by the atypical antiarrhythmic drug ranolazine

Nature Cardiovascular Research volume 2, pages 587–594 (2023)Cite this article

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Abstract

Voltage-gated sodium (NaV) channels generate the upstroke of the cardiac action potential by activating rapidly in response to depolarization and conducting Na+ inward across the membrane1,2. NaV1.5 is the predominant NaV channel in the heart3,4. It is the molecular target for class I antiarrhythmic drugs (AADs), which often have unwanted side effects, including arrhythmias5,6. In contrast, the atypical AAD ranolazine is effective in treatment of atrial arrhythmias and angina pectoris, but with less proarrhythmia than traditional AADs7,8,9,10,11. Structures of NaV channels from prokaryotes12, skeletal muscle13, nerve14 and heart15 have been determined with AADs bound within the pore to physically block Na+ conductance12,15,16,17,18,19. Here we use electrophysiology and cryogenic electron microscopy to define the interaction of ranolazine with NaV1.5 at high resolution. We reveal ranolazine’s binding pose and elucidate distinct molecular interactions that might underlie its mechanism of action and high therapeutic index relative to traditional class I AADs.

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Fig. 1: Ranolazine binding and block of rNaV1.5c.
Fig. 2: The cryo-EM structure of ranolazine bound to rNaV1.5c.
Fig. 3: Functional effects of mutations of contact residues in the ranolazine binding site.
Fig. 4: The structures of ranolazine and other AADs bound to α and π conformations of transmembrane segment DIV-S6.

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Data availability

All data supporting the finding in this study are included in the main article and associated files. Structural data are available from the Protein Data Bank (PDB) under EMDB entry ID EMD-28887 and PDB entries ID 8F6P & 6UZ3. Source data are provided with this paper.

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Acknowledgements

This work was funded by National Institutes of Health Research grants K08 HL145630 (M.L.) and R01 HL112808-09 (W.A.C.) and by the Howard Hughes Medical Institue (N.Z.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors thank J. Quispe (Cryo-EM Facility, University of Washington) for support with cryo-EM data collection and J. Li (Pharmacology, University of Washington) for technical and editorial support.

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Authors and Affiliations

  1. Division of General Internal Medicine, Department of Medicine, University of Washington, Seattle, WA, USA

    Michael Lenaeus

  2. Department of Pharmacology, University of Washington, Seattle, WA, USA

    Michael Lenaeus, Tamer M. Gamal El-Din, Lige Tonggu, Ning Zheng & William A. Catterall

  3. Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA

    Ning Zheng

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Contributions

M.L, T.M.G.E.-D., L.T., N.Z. and W.A.C. designed the experiments; M.L. and L.T. carried out the cryo-EM experiments; T.M.G.E.-D. carried out the electrophysiology experiments; and M.L. carried out the molecular model building, refinement and comparison with previous data. All authors contributed to writing and revising the paper.

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Correspondence to Michael Lenaeus or William A. Catterall.

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Extended Data

Extended Data Fig. 1 The purification of rNav1.5c and preparation of samples for cryo-EM.

a, The size-exclusion chromatography profile is shown for the final step of purification of the rNav1.5c, FGF12B, calmodulin and ranolazine complex with red lines indicating the sample moved forward for cryo-EM analysis. This experiment was only performed once. b, Sample micrograph collected during cryo-EM data collection and representative 2D classifications as determined by Relion. Information on cryo-EM data collection and data processing is shown in Extended Data Fig. 2.

Extended Data Fig. 2 Cryo-EM data processing and 3D reconstruction.

a, Flowchart for EM data processing. b, Gold-standard FSC curve for the 3D reconstruction of rNav1.5c, FGF12B, calmodulin and ranolazine complex.

Extended Data Fig. 3 3D reconstruction of rNav1.5c, FGF12B, calmodulin, and ranolazine complex color coded for map resolution.

EM density is shown from below rNav1.5c as if one is inside the cell and looking outward at the plasma membrane. The map is colored by resolution with scale shown (blue = high resolution, red = lower). A close-up of the ranolazine site is shown, highlighting the quality of EM density at ranolazine’s binding site. A red circle is shown to identify the EM density corresponding to ranolazine.

Extended Data Fig. 4 Functional characterization of the Nav1.5c mutant Q372A and its effects on ranolazine binding.

a. G-V curves of Nav1.5c WT and Q372A mutant derived from I-V relationships. The voltages for half maximal activation and slopes are: WT V1/2 = -73 ± 0.8 mV, K = 5.6 ± 0.6, Q372A V1/2 = -69 ± 0.3 mV, K = 5.5 ± 0.3. Steady-state inactivation of Nav1.5c WT and Q372A mutant. Two pulses were applied: a 500-ms conditioning pulse to the indicated potentials followed by 50 ms test pulse to 0 mV. Nav1.5c WT Vh = -110 ± 0.4, K = 7.8 ± 0.5, Q372A Vh = -104 ± 0.6, K = 7.6 ± 0.5. WT activation and steady-state inactivation curves N = 4 distinct cells, Q372A GV and SSI curves N = 4 distinct cells. b. Dose-response curve for the tonic block of Q372A compared to Nav1.5c WT. Each concentration is an average of three different cells and each cell is used only once for each concentration. Error bars indicates s.e.m.

Extended Data Fig. 5 α-π transition of DIV S6 and π-helix dependent binding of ranolazine.

a, An overlay of DIV-S6 helices from the current structure (light green with pink π-helix highlighted), the apo structure of rNav1.5c (pdb 6uz3, grey), and the open structure of rNav1.5c (pdb 7fbs, cyan). The π-helical portion of DIV-S6 (pink) creates additional drug binding surface in the central cavity of rNav1.5c. b, A close-up of the ranolazine binding site with sticks shown for important residues F1762, V1765, and V1766. Coloring is as in part a and the side chain position of V1766 is shown for both the apo structure and the ranolazine-bound structure, with a black arrow highlighting the distance between these alpha carbon positions (2.7 Å). Yellow dashed lines show the strengthened van der waals contacts in the π-helical form of DIV-S6 found in the ranolazine structure (distance is 3.3 Å). c, A bubble diagram showing the local differences between π-helical (top) and α-helical (bottom) forms of DIV-S6. Residues are shown as bubbles with coloring dependent on the nearest approach of the side chain to the bound drug according to the color scale shown in the figure. The structure and the dimethylbenzyl moiety of ranolazine is shown for reference. d, Stick diagrams of ranolazine and the class IB AADs lidocaine, mexilitine, tocainide, and etidocaine are shown with a red line highlighting each molecule’s dimethylbenzyl moiety.

Extended Data Fig. 6 Effect of α-π transition of DIV S6 on the DIV-DI fenestration.

a. Cartoon models of DIV-S6 (green) and DI-S6 (pale yellow) as in earlier figures. Sticks are shown for DIV-S6 residue Y1769 (green, in π helix conformation), F403 (pale yellow) and the non-lidocaine portion of ranolazine that interacts with F403 by π-teeing (dashed line). Transparent sticks are shown in blue for the apo (α-helix) conformations of Y1769 and F403, illustrating the clash between the side chain of π-Y1769 and apo F403. b, Surface illustration of DIV/DI fenestration in α and π forms of DIV-S6. Domains are colored as in prior figures - DI pale yellow, DIII, pink, DIV, green, DIII/DIV linker (IFM) orange.

Extended Data Table 1 Statistical Parameters for cryo-EM data collection
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Extended Data Table 2 Statistical Parameters for Model Refinement
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Supplementary information

Source data

Source Data Fig. 3

The effects of mutations on ranolazine binding as measured by electrophysiology. This is a table of individual measurements used to create the data displayed in Fig. 3.

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Lenaeus, M., Gamal El-Din, T.M., Tonggu, L. et al. Structural basis for inhibition of the cardiac sodium channel by the atypical antiarrhythmic drug ranolazine. Nat Cardiovasc Res 2, 587–594 (2023). https://doi.org/10.1038/s44161-023-00271-5

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