Structural basis for antiarrhythmic drug interactions with the human cardiac sodium channel Phuong T. Nguyena,b, Kevin R. DeMarcoa,b, Igor Vorobyova,c, Colleen E. Clancya,c,1, and Vladimir Yarov-Yarovoya,1 aDepartment of Physiology and Membrane Biology, University of California, Davis, CA 95616; bBiophysics Graduate Group, University of California, Davis, CA 95616; and cDepartment of Pharmacology, University of California, Davis, CA 95616 Edited by Richard W. Aldrich, The University of Texas at Austin, Austin, TX, and approved January 9, 2019 (received for review October 10, 2018) – The human voltage-gated sodium channel, hNaV1.5, is responsible opening (7 9). This inactivation process plays critical roles in NaV for the rapid upstroke of the cardiac action potential and is target channel function and drug binding (10, 11). for antiarrhythmic therapy. Despite the clinical relevance of hNaV1.5- Forty years ago, Hille (12) proposed two distinct access path- targeting drugs, structure-based molecular mechanisms of promising ways for local anesthetics to the central binding site: the hydro- or problematic drugs have not been investigated at atomic scale to phobic pathway through the membrane and the hydrophilic inform drug design. Here, we used Rosetta structural modeling and pathway through the intracellular gate. Many antiarrhythmic and docking as well as molecular dynamics simulations to study the in- local anesthetic drugs are weak bases that exist in equilibrium teractions of antiarrhythmic and local anesthetic drugs with hNaV1.5. between both neutral and charged forms at physiological pH. These calculations revealed several key drug binding sites formed Neutral drugs may access the pore lumen binding site through within the pore lumen that can simultaneously accommodate up to both hydrophobic and hydrophilic pathways (13), but charged two drug molecules. Molecular dynamics simulations identified a drugs are much more likely to access the pore binding site through hydrophilic access pathway through the intracellular gate and a hy- the hydrophilic pathway, due to a large energetic penalty for drophobic access pathway through a fenestration between DIII and traversing a lipid membrane (14). Extensive electrophysiological DIV. Our results advance the understanding of molecular mecha- and site-directed mutagenesis experiments have identified a key nisms of antiarrhythmic and local anesthetic drug interactions with receptor site for antiarrhythmic and local anesthetic drugs within hNaV1.5 and will be useful for rational design of novel therapeutics. the eukaryotic NaV channel pore lumen (15–18). Mutations of two conserved aromatic residues in the DIV segment S6 (DIVS6) of BIOPHYSICS AND sodium channel | local anesthetics | antiarrhythmic drugs | docking | NaV channels, F1760 and Y1767 (hNaV1.5 numbering), signifi- COMPUTATIONAL BIOLOGY molecular dynamics cantly reduce antiarrhythmic and local anesthetic drug binding (15, 16). Other key residues for drug binding within the pore lu- men have been identified in DIS6 and DIIIS6 segments (17, 18). oltage-gated sodium channels (NaV) are transmembrane Vproteins that give rise to action potential generation and In addition, mutations within the NaV channel SF region can af- propagation in excitable cells. There are nine human Na (hNa ) fect drug binding, either through enhancement of slow in- V V – channel subtypes expressed in neuronal, cardiac, and muscle cells (1). activation or formation of alternative access pathway (19 21). Structural studies have advanced our mechanistic understanding The cardiac NaV channel (NaV1.5) plays a central role in congenital − and acquired cardiac arrhythmias and has been an important target of NaV channel drug interaction mechanisms. The first crystal structure of the bacterial Na channel Na Ab revealed open for antiarrhythmic drug development (2–4). Long-standing failures V V in drug treatment of heart rhythm disturbances and many other syndromes stem from a persistent failure to predict the effective or Significance harmful action of drugs. For example, the Cardiac Arrhythmia Suppression Trial (5) and Survival with Oral D-Sotalol (6) clinical Voltage-gated sodium channels play a central role in cellular ex- trials showed that common antiarrhythmic drugs, such as encainide citability and are key targets for drug development. Recent and flecainide, increased mortality and risk of sudden cardiac death breakthroughs in high-resolution cryo-electron microscopy pro- tein structure determination, Rosetta computational protein in patients. Thirty years later, there is still no effective preclinical structure modeling, and multimicrosecond molecular dynamics methodology to differentiate useful or potentially harmful drugs at simulations are empowering advances in structural biology to the molecular level. Mechanistic understanding of drug interactions study the atomistic details of channel−drug interactions. We used with NaV channels at the atomic scale is needed to develop and Rosetta structural computational modeling and molecular dy- screen novel drugs to reveal the mechanisms of drug failure or ef- namics simulations to study the interactions of antiarrhythmic ficacy for treatment of cardiovascular and other disorders and to and local anesthetic drugs with cardiac sodium channel. Our re- minimize side effects. sults provide crucial atomic-scale mechanistic insights into the NaV channels respond dynamically to changes in cell membrane channel–drug interactions, necessary for the rational design of voltage and adopt distinct conformational states: open (conducting), novel modulators of the human cardiac sodium channel to be closed (nonconducting), and inactivated (nonconducting). NaV used for the treatment of cardiac arrhythmias. channels contain four homologous domains (DI to DIV), with each domain consisting of a voltage-sensing domain (VSD) containing Author contributions: P.T.N., K.R.D., I.V., C.E.C., and V.Y.-Y. designed research; P.T.N., transmembrane segments S1 to S4 and a pore domain (PD) con- K.R.D., and I.V. performed research; P.T.N., K.R.D., I.V., C.E.C., and V.Y.-Y. analyzed data; and P.T.N., K.R.D., I.V., C.E.C., and V.Y.-Y. wrote the paper. taining transmembrane segments S5 and S6 connected by a loop The authors declare no conflict of interest. region with P1 and P2 helices forming selectivity filter (SF). Each VSD senses changes in membrane potential that lead to movement This article is a PNAS Direct Submission. This open access article is distributed under Creative Commons Attribution-NonCommercial- of its S4 segment which can, in turn, trigger channel activation (pore NoDerivatives License 4.0 (CC BY-NC-ND). opening) or channel deactivation (pore closing) at the intracellular 1To whom correspondence may be addressed. Email: [email protected] or yarovoy@ gate. The intracellular linker between DIII and DIV contains a ucdavis.edu. − − hydrophobic isoleucine phenylalanine methionine (IFM) motif, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. which contributes to a fast inactivation gating mechanism, resulting 1073/pnas.1817446116/-/DCSupplemental. + in rapid termination of Na conduction subsequent to the channel Published online February 6, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1817446116 PNAS | February 19, 2019 | vol. 116 | no. 8 | 2945–2954 Downloaded by guest on October 5, 2021 fenestrations within the PD (22), which supported the hypothesis that structural template for modeling of hNaV channels. The sequence drugs can access the binding site within the pore lumen through the identity between hNaV1.5 and eeNaV1.4 is ∼84% in the pore- hydrophobic pathway. Crystal structures of NaVMs and voltage-gated forming transmembrane region (SI Appendix,Fig.S1), which is calcium (CaV) Ab channels have been determined with drugs bound within an atomic-level accuracy homology modeling range (48), near the fenestration regions or in the pore lumen, suggesting the allowing us to generate an accurate model of hNaV1.5 in a par- possibility of similar drug binding receptor sites in eukaryotic NaV tially open and presumably inactivated state. channels (23, 24). The first high-resolution structures of eukaryotic The hNaV1.4 structure was published in September 2018 (9), NaV channels have recently been resolved using cryo-electron mi- when this study was already completed. The sequence identity be- croscopy (cryoEM). The American cockroach NaVPaS channel tween hNaV1.5 and hNaV1.4 is ∼87%, which is similar to the se- structures have been solved in a closed state (25, 26), and electric eel quence identity between hNaV1.5 and eeNaV1.4 (∼84%) and NaV1.4 (eeNaV1.4) channel structure has been solved in a partially suggests that eeNaV1.4 and hNaV1.4 structures are within the same open and presumably inactivated state (27). These structures have range of accuracy for modeling of hNaV channels. The overall root- unlocked new opportunities to study drug interactions with eukary- mean-square deviation (RMSD) between hNaV1.4 and eeNaV1.4 otic NaV channels at the atomic scale. structures is less than 1 Å (9) and RMSD over the PD segments The Rosetta computational modeling software (28, 29) has been S5 and S6 and P1 and P2 helices is less than 0.7 Å, which suggests used to study conformational changes in NaV, voltage-gated potassium very similar conformations of the PD structure—the main focus of (KV), CaV, and TRPV1 channels (30–33) and peptide toxin interac- this study. – tions with NaV,KV, and TRPV1 channels (34 36).