Open-state structure and pore gating mechanism of the cardiac sodium channel.

RESOURCE AVAILABILITY Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, William A. Catterall ( wcatt@uw.edu ).

Materials Availability

All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Data and Code Availability

Structural results are available from the Protein Data Bank and the EMData Resource. Other data and code are available upon request to the Lead Contact.

EXPERIMENTAL MODEL AND SUBJECT DETAILS Microbe strains E. coli

GC10 was cultured at 37°C in LB medium supplemented with 100μg/ml of ampicillin for plasmid DNA extraction. E. coli DH10Bac was cultured at 37°C in LB medium supplemented with 50 µg/mL kanamycin sulfate, 7 µg/mL gentamicin and 10 µg/mL tetracycline for bacmid production. Cell lines Sf9 ( Spodoptera frugiperda ) insect cells were maintained in Grace’s Insect Medium and supplemented with 10% FBS and penicillin/streptomycin at 27°C and passaged at 90% confluence for baculovirus production. HEK293S GnTI − ( Homo sapiens ) mammalian cells were maintained and infected in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and glutamine/penicillin/streptomycin at 37°C and 5% CO 2 for electrophysiology.

Determination of the Cryo-EM

Structure of Na V 1.5/QQQ Mammalian Na V s are large and structurally complicated transmembrane proteins, which are difficult to express in heterologous cells and purify to homogeneity. We previously overcame these difficulties by developing a fully functional core-construct of Na V 1.5 with truncations of unstructured regions of the intracellular loops and C-terminal domain (Na V 1.5 C ) ( Jiang et al., 2020 ). The IFM/QQQ mutation was introduced into this well-behaved protein construct. As expected from previous work ( West et al., 1992 ), this mutation completely blocked fast inactivation with no effect on the voltage dependence of activation ( Figure 1 , A-C). However, because the mutation abolishes fast inactivation, HEK293S cells were killed by the constant leak of Na + current after two days of over-expression of Na V 1.5/QQQ. We optimized expression conditions by testing several Na V channel blockers, and we finally maintained the HEK293S cells long enough for effective protein expression in the presence of the Na V -blocking antiarrhythmic drug propafenone ( Figure 1D - F ). The Na V 1.5/QQQ protein was solubilized in detergents and purified in a mono-disperse peak from size-exclusion chromatography ( Figure S1A ). Despite low yield, cryo-EM micrographs of the concentrated sample showed excellent quality, and the collected micrographs yielded sharp 2D averages with obvious Na V features ( Figure S1B ). A total of 287,306 particles were selected for calculating the final reconstruction map ( Figures S2 and S3A ), which fit the gold-standard Fourier Shell Correlation (FSC) curve at the criterion value of 0.143 with a resolution of 3.3 Å ( Figure S3B ). A local resolution map revealed the core region of Na V 1.5/QQQ at 3.0–3.5 Å ( Figure S3A ), illustrating the high quality of the cryo-EM map. Cryo-EM density is well distributed in spherical space, and an FSC curve comparing our model to the original cryo-EM map has overall resolution of 3.4 Å ( Figure S3C , D ).

METHOD DETAILS Electrophysiology

All experiments were performed at room temperature (21–24°C) as described previously ( Jiang et al., 2020 ). Human HEK293S GnTI − cells were maintained and infected on cell culture plates in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and glutamine/penicillin/streptomycin at 37°C and 5% CO 2 for electrophysiology. Unless otherwise mentioned, HEK293S GnTI − cells were held at −120 mV and 100-ms pulses were applied in 10 mV increments from −120 mV to +60 mV. A P/−4 holding leak potential was set to −120 mV. Extracellular solution contained in mM: 140 NaCl, 2 CaCl 2 , 2 MgCl 2 , 10 HEPES, pH 7.4. Intracellular solution contained: 35 NaCl, 105 CsF, 10 EGTA, 10 HEPES, pH 7.4. Glass electrodes had a resistance 1.5–3 MΩ. Currents resulting from applied pulses were filtered at 5 kHz with a low-pass Bessel filter, and then digitized at 20 kHz. Data were acquired using an Axopatch 200B amplifier (Molecular Devices), voltage commands were generated using Pulse 8.5 software (HEKA, Germany), and ITC18 analog-to-digital interface (Instrutech, Port Washington, NY). Protein Expression and Purification The IFM motif of rNa V 1.5 C was replaced by QQQ and the mutant gene was verified by DNA sequencing. Detailed expression and purification of rat Na V 1.5/QQQ was described in our previous study with modifications ( Jiang et al., 2020 ). In brief, Na V 1.5/QQQ was expressed in HEK293S GnTI − cells (ATCC) with 10 μM propafenone in the medium. The protein was extracted by 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace) and 0.2% (w/v) cholesteryl hemisuccinate Tris salt (CHS, Anatrace) in Buffer A containing 25 mM HEPES pH = 7.4, 150 mM NaCl, 10% glycerol, and 100 μM propafenone. After centrifugation, supernatant was agitated with anti-Flag M2-agarose resin (Sigma). Flag resin was washed in Buffer A supplemented with 0.06% glycol-diosgenin (GDN, Anatrace). The concentrated elution was loaded to Superose-6 column pre-equilibrated with buffer B containing 25 mM HEPES pH=7.4, 150 mM NaCl, 0.06% GDN, and 100 μM propafenone. Peak fractions of the first SEC were pooled and concentrated to ~ 0.5 ml, then was re-loaded to Superose-6 column pre-equilibrated with buffer C containing 25 mM imidazole pH=6.0, 150 mM NaCl, 0.006% GDN, and 100 μM propafenone. Finally, peak fractions were concentrated to 30 μl at 3.6 mg/ml.

CryoEM Grid Preparation and Data Collection

Three microliters of purified sample were applied to glow-discharged holey gold grids (UltraAuFoil, 300 mesh, R1.2/1.3), and blotted for 2.0 – 3.5 s at 100% humidity and 4 °C before being plunged frozen in liquid ethane cooled by liquid nitrogen using a FEI Mark IV Vitrobot. All data were acquired using a Titan Krios transmission electron microscope operated at 300 kV, a Gatan K2 Summit direct detector and Gatan Quantum GIF energy filter with a slit width of 20 eV. All movie stacks were automatically collected using Leginon ( Suloway et al., 2005 ) at a nominal magnification of 130,000× with a pixel size of0.528 Å (super-resolution mode). Defocus range was set between −1.0 and −2.4 μm. The dose rate was adjusted to 8 counts/pixel/s, two separate datasets for Na V 1.5/QQQ were collected, 2,352 movie stacks were collected for dataset 1, each stack was exposed for 7.0 s with 35 frames with a total dose of 50 e − / Å 2 . 2,833 movie stacks for dataset 2, each stack was exposed for 8.4 s with 42 frames with a total dose of 60 e − / Å 2 .

Cryo-EM Data Processing

All the movie stacks were motion-corrected with MotionCorr2 ( Zheng et al., 2017 ), binned 2- fold, and dose-weighted, yielding a pixel size of 1.056 Å. Defocus values of each aligned sum were estimated with Gctf ( Zhang, 2016 ). Particle picking, 2D classification, 3D classification and 3D auto-refine was performed in RELION3.0 ( Scheres, 2012 ). A detailed data processing diagram is presented in Figure S2 . The two data sets of Na V 1.5/QQQ were processed separately, and the last seven frames of each stack of the second dataset were discarded to keep the same total dose of 50 e − / Å 2 as dataset 1. Then the polished particles including 204,375 particles from dataset 1 and 328,465 particles from dataset 2 were combined and subjected a final round of multi-reference 3D classification. The best class containing 287,306 particles were subsequently auto-refined and sharpened in Relion 3.0. Local resolution was estimated by ResMap in Relion 3.0.

Model Building and Refinement

The structures of rat Na V 1.5 C (PDB code: 6UZ0) were manually fitted into the cryo-EM density map of Na V 1.5/QQQ using Chimera ( Pettersen et al., 2004 ), respectively. The models were manually checked and corrected in COOT ( Emsley et al., 2010 ) and subsequently refined in Phenix ( Adams et al., 2010 ). The model vs map FSC curves were calculated by Phenix.mtrage. Statistics for cryo-EM data collection and model refinement are summarized in Table S1 .

Molecular Dynamics Model

The cryo-EM structures of Na V 1.5 ( Jiang et al., 2020 ), Na V 1.5/LqhIII ( Jiang et al., 2021 ), and Na V 1.5/QQQ lacking D I- D II and D II- D III linkers are composed of three chains which correspond to D I, D II, and D III- D IV. The MODELLER software (ver. 9.22) was used to insert missing residues (including the missing QQQ motif) and sidechains within the polypeptide chains, followed by quick refinement using MD with simulated annealing ( Fiser and Sali, 2003 ). Neutral N- and C-termini were used for the three polypeptide chains in our refined model of Na V 1.5. N-termini from chains D II and D III-IV were acetylated, and a neutral amino terminus (-NH 2 ) was used for D I. Neutral carboxyl groups (-COOH) were used for all C-termini. Disulfide bonds linking residues 327–342, 909–918, and 1730–1744 were included in our models of Na V 1.5 and Na V 1.5/LqhIII as they were present in the cryo-EM structure; however, no glycans were added to the protein. In the case of Na V 1.5/QQQ, an additional disulfide bond linking residues 281–336 was included and the 1730–1744 bond was excluded as indicated in the cryo-EM structure. For the Na V 1.5/LqhIII model, charged N- and C-termini were used for LqhIII and disulfide bonds linking residues 12–65, 16–37, 23–47, and 27–49 were included.

Molecular Dynamics Simulations

Molecular models of Na V 1.5 structures were prepared using the input generator, Membrane Builder ( Jo et al., 2007 ; Jo et al., 2009 ; Lee et al., 2016 ; Lee et al., 2019 ; Wu et al., 2014 ) from CHARMM-GUI ( Jo et al., 2009 ). For each model, the channel was embedded in a hydrated DMPC bilayer, with approximately 150 mM NaCl. The protein was translated and rotated for membrane embedding using the PPM server ( Lomize et al., 2012 ). The lipid bilayer was assembled using the replacement method and solvent ions were added at random positions using a distance-based algorithm. A periodic rectangular cell with approximate dimensions of 14×14×13 nm was used, which comprised ~240,000 atoms. The CHARMM36 all-atom force field was used in conjunction with the TIP3P water model ( Best et al., 2012 ; Jorgensen et al., 1983 ; Klauda et al., 2010 ; MacKerell et al., 1998 ). Non-bonded fixes for backbone carbonyl oxygen atoms with Na + and lipid head groups with Na + were imposed ( Noskov and Roux, 2008 ; Venable et al., 2013 ). For rNa V 1.5/QQQ, an additional non-bonded fix for guanidinium nitrogen with carboxylate oxygen was imposed ( Huang et al., 2017 ). Electrostatic interactions were calculated using the particle-mesh Ewald algorithm ( Darden, 1993 ; Essmann, 1995 ) and chemical bonds were constrained using the LINCS algorithm ( Hess, 2008 ). The energy of the system was minimized with protein position restraints on the backbone (4000 kJ/mol/nm 2 ) and side chains (2000 kJ/mol/nm 2 ), as well as lipid position and dihedral restraints (1000 kJ/mol/nm 2 ) using 5000 steps of steepest descent. The simulation systems were then pre-equilibrated using multi-step isothermal-isovolumetric (NVT) and isothermal-isobaric (NPT) conditions while decreasing the restraints at each step (see Table S2a for parameters). “Production” simulations with and without harmonic restraints on backbone heavy atoms (500 kJ/mol/nm 2 ) were generated with a 2 fs time integration step. The equilibration time was decided based on RMSD analyses of Cα atoms (Figure S9) and average hydration of the gate over time (Figure S10). Details on production simulations equilibration time, and time used in analyses are indicated in Table S2b . All simulations were carried out using GROMACS version 2019.3 ( http://www.gromacs.org ).

Molecular Dynamics Simulation Analysis

In each snapshot of our simulations, atomic positions were translated and rotated by aligning the Cα atoms from pore transmembrane helices (S5 and S6 from all domains) of Na V 1.5 to the initial structure produced by CHARMM-GUI. The positions of all atoms were then centered in the xy-plane by the center of mass (CoM) of pore transmembrane helices and the z-axis by the CoM of Cα atoms from the DEKA motif in the SF. After performing the spatial transformations, the z-axis of the simulation box was used as the pore axis of Na V 1.5 and the transformed positions were used for subsequent analyses. The axial distribution of water was computed by counting the number of water O atoms within a cylindrical of radius 8.5 Å centered on the pore axis. The probability distribution of water was calculated for each replica by counting the number of water molecules in uniform cylindrical slices along the pore-axis and normalizing the counts by the slice with the highest number of water molecules (solvent slice). The average and SEM of the probability distribution was computed across replicas. Pore hydration analysis indicated a dehydrated region located at the ICAG (−2.8 nm < z < −1.5 nm). The number of water molecules in the gate was counted for each frame and normalized by the total number of frames to obtain the probability distribution. The average and SEM were computed across replicas. To measure the size of the intracellular activation gate, residues at the ends of the S6 helices were selected as described ( Jiang et al., 2021 ). The following residue number selections were used in rNa V 1.5: DI: 410–413, DII: 939–942, DIII: 1469–1472, and DIV: 1771–1774. The CoM of Cα-atoms from each selection was projected onto the xy-plane and the distances between opposing S6 tails were measured ( d 1 : DI-DIII and d 2 : DII-DIV). Analyses were performed using MDTraj ( McGibbon et al., 2015 ) and molecular visualizations were rendered using Visual Molecular Dynamics ( Humphrey et al., 1996 ).

QUANTIFICATION AND STATISTICAL ANALYSIS

For electrophysiological results, the data are presented as mean and standard error of the mean (SEM). Statistical significance was evaluated with Student’s t-test and ANOVA. For the molecular dynamics simulations, maximum likelihood values and statistical significance were estimated by bootstrapping.

DATA AND CODE AVAILABILITY

The cryo-EM map of the Na v 1.5QQQ/propafenone complex have been deposited in the Electron Microscopy Data Bank (EMDB) under accession code EMD-31519. The coordinates of the Na v 1.5QQQ/propafenone complex atomic model have been deposited in the Protein Data Bank (PDB) under accession code 7FBS. Any additional information required to reanalyze the data reported in this work paper is available from the Lead Contact upon request. ADDITIONAL RESOURCES No additional resources were generated in this work.

Materials Availability

All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

EXPERIMENTAL MODEL AND SUBJECT DETAILS Microbe strains E. coli

GC10 was cultured at 37°C in LB medium supplemented with 100μg/ml of ampicillin for plasmid DNA extraction. E. coli DH10Bac was cultured at 37°C in LB medium supplemented with 50 µg/mL kanamycin sulfate, 7 µg/mL gentamicin and 10 µg/mL tetracycline for bacmid production. Cell lines Sf9 ( Spodoptera frugiperda ) insect cells were maintained in Grace’s Insect Medium and supplemented with 10% FBS and penicillin/streptomycin at 27°C and passaged at 90% confluence for baculovirus production. HEK293S GnTI − ( Homo sapiens ) mammalian cells were maintained and infected in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and glutamine/penicillin/streptomycin at 37°C and 5% CO 2 for electrophysiology.

Determination of the Cryo-EM

Structure of Na V 1.5/QQQ Mammalian Na V s are large and structurally complicated transmembrane proteins, which are difficult to express in heterologous cells and purify to homogeneity. We previously overcame these difficulties by developing a fully functional core-construct of Na V 1.5 with truncations of unstructured regions of the intracellular loops and C-terminal domain (Na V 1.5 C ) ( Jiang et al., 2020 ). The IFM/QQQ mutation was introduced into this well-behaved protein construct. As expected from previous work ( West et al., 1992 ), this mutation completely blocked fast inactivation with no effect on the voltage dependence of activation ( Figure 1 , A-C). However, because the mutation abolishes fast inactivation, HEK293S cells were killed by the constant leak of Na + current after two days of over-expression of Na V 1.5/QQQ. We optimized expression conditions by testing several Na V channel blockers, and we finally maintained the HEK293S cells long enough for effective protein expression in the presence of the Na V -blocking antiarrhythmic drug propafenone ( Figure 1D - F ). The Na V 1.5/QQQ protein was solubilized in detergents and purified in a mono-disperse peak from size-exclusion chromatography ( Figure S1A ). Despite low yield, cryo-EM micrographs of the concentrated sample showed excellent quality, and the collected micrographs yielded sharp 2D averages with obvious Na V features ( Figure S1B ). A total of 287,306 particles were selected for calculating the final reconstruction map ( Figures S2 and S3A ), which fit the gold-standard Fourier Shell Correlation (FSC) curve at the criterion value of 0.143 with a resolution of 3.3 Å ( Figure S3B ). A local resolution map revealed the core region of Na V 1.5/QQQ at 3.0–3.5 Å ( Figure S3A ), illustrating the high quality of the cryo-EM map. Cryo-EM density is well distributed in spherical space, and an FSC curve comparing our model to the original cryo-EM map has overall resolution of 3.4 Å ( Figure S3C , D ).

METHOD DETAILS Electrophysiology

All experiments were performed at room temperature (21–24°C) as described previously ( Jiang et al., 2020 ). Human HEK293S GnTI − cells were maintained and infected on cell culture plates in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and glutamine/penicillin/streptomycin at 37°C and 5% CO 2 for electrophysiology. Unless otherwise mentioned, HEK293S GnTI − cells were held at −120 mV and 100-ms pulses were applied in 10 mV increments from −120 mV to +60 mV. A P/−4 holding leak potential was set to −120 mV. Extracellular solution contained in mM: 140 NaCl, 2 CaCl 2 , 2 MgCl 2 , 10 HEPES, pH 7.4. Intracellular solution contained: 35 NaCl, 105 CsF, 10 EGTA, 10 HEPES, pH 7.4. Glass electrodes had a resistance 1.5–3 MΩ. Currents resulting from applied pulses were filtered at 5 kHz with a low-pass Bessel filter, and then digitized at 20 kHz. Data were acquired using an Axopatch 200B amplifier (Molecular Devices), voltage commands were generated using Pulse 8.5 software (HEKA, Germany), and ITC18 analog-to-digital interface (Instrutech, Port Washington, NY). Protein Expression and Purification The IFM motif of rNa V 1.5 C was replaced by QQQ and the mutant gene was verified by DNA sequencing. Detailed expression and purification of rat Na V 1.5/QQQ was described in our previous study with modifications ( Jiang et al., 2020 ). In brief, Na V 1.5/QQQ was expressed in HEK293S GnTI − cells (ATCC) with 10 μM propafenone in the medium. The protein was extracted by 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace) and 0.2% (w/v) cholesteryl hemisuccinate Tris salt (CHS, Anatrace) in Buffer A containing 25 mM HEPES pH = 7.4, 150 mM NaCl, 10% glycerol, and 100 μM propafenone. After centrifugation, supernatant was agitated with anti-Flag M2-agarose resin (Sigma). Flag resin was washed in Buffer A supplemented with 0.06% glycol-diosgenin (GDN, Anatrace). The concentrated elution was loaded to Superose-6 column pre-equilibrated with buffer B containing 25 mM HEPES pH=7.4, 150 mM NaCl, 0.06% GDN, and 100 μM propafenone. Peak fractions of the first SEC were pooled and concentrated to ~ 0.5 ml, then was re-loaded to Superose-6 column pre-equilibrated with buffer C containing 25 mM imidazole pH=6.0, 150 mM NaCl, 0.006% GDN, and 100 μM propafenone. Finally, peak fractions were concentrated to 30 μl at 3.6 mg/ml.

CryoEM Grid Preparation and Data Collection

Three microliters of purified sample were applied to glow-discharged holey gold grids (UltraAuFoil, 300 mesh, R1.2/1.3), and blotted for 2.0 – 3.5 s at 100% humidity and 4 °C before being plunged frozen in liquid ethane cooled by liquid nitrogen using a FEI Mark IV Vitrobot. All data were acquired using a Titan Krios transmission electron microscope operated at 300 kV, a Gatan K2 Summit direct detector and Gatan Quantum GIF energy filter with a slit width of 20 eV. All movie stacks were automatically collected using Leginon ( Suloway et al., 2005 ) at a nominal magnification of 130,000× with a pixel size of0.528 Å (super-resolution mode). Defocus range was set between −1.0 and −2.4 μm. The dose rate was adjusted to 8 counts/pixel/s, two separate datasets for Na V 1.5/QQQ were collected, 2,352 movie stacks were collected for dataset 1, each stack was exposed for 7.0 s with 35 frames with a total dose of 50 e − / Å 2 . 2,833 movie stacks for dataset 2, each stack was exposed for 8.4 s with 42 frames with a total dose of 60 e − / Å 2 .

Cryo-EM Data Processing

All the movie stacks were motion-corrected with MotionCorr2 ( Zheng et al., 2017 ), binned 2- fold, and dose-weighted, yielding a pixel size of 1.056 Å. Defocus values of each aligned sum were estimated with Gctf ( Zhang, 2016 ). Particle picking, 2D classification, 3D classification and 3D auto-refine was performed in RELION3.0 ( Scheres, 2012 ). A detailed data processing diagram is presented in Figure S2 . The two data sets of Na V 1.5/QQQ were processed separately, and the last seven frames of each stack of the second dataset were discarded to keep the same total dose of 50 e − / Å 2 as dataset 1. Then the polished particles including 204,375 particles from dataset 1 and 328,465 particles from dataset 2 were combined and subjected a final round of multi-reference 3D classification. The best class containing 287,306 particles were subsequently auto-refined and sharpened in Relion 3.0. Local resolution was estimated by ResMap in Relion 3.0.

Model Building and Refinement

The structures of rat Na V 1.5 C (PDB code: 6UZ0) were manually fitted into the cryo-EM density map of Na V 1.5/QQQ using Chimera ( Pettersen et al., 2004 ), respectively. The models were manually checked and corrected in COOT ( Emsley et al., 2010 ) and subsequently refined in Phenix ( Adams et al., 2010 ). The model vs map FSC curves were calculated by Phenix.mtrage. Statistics for cryo-EM data collection and model refinement are summarized in Table S1 .

Molecular Dynamics Model

The cryo-EM structures of Na V 1.5 ( Jiang et al., 2020 ), Na V 1.5/LqhIII ( Jiang et al., 2021 ), and Na V 1.5/QQQ lacking D I- D II and D II- D III linkers are composed of three chains which correspond to D I, D II, and D III- D IV. The MODELLER software (ver. 9.22) was used to insert missing residues (including the missing QQQ motif) and sidechains within the polypeptide chains, followed by quick refinement using MD with simulated annealing ( Fiser and Sali, 2003 ). Neutral N- and C-termini were used for the three polypeptide chains in our refined model of Na V 1.5. N-termini from chains D II and D III-IV were acetylated, and a neutral amino terminus (-NH 2 ) was used for D I. Neutral carboxyl groups (-COOH) were used for all C-termini. Disulfide bonds linking residues 327–342, 909–918, and 1730–1744 were included in our models of Na V 1.5 and Na V 1.5/LqhIII as they were present in the cryo-EM structure; however, no glycans were added to the protein. In the case of Na V 1.5/QQQ, an additional disulfide bond linking residues 281–336 was included and the 1730–1744 bond was excluded as indicated in the cryo-EM structure. For the Na V 1.5/LqhIII model, charged N- and C-termini were used for LqhIII and disulfide bonds linking residues 12–65, 16–37, 23–47, and 27–49 were included.

Molecular Dynamics Simulations

Molecular models of Na V 1.5 structures were prepared using the input generator, Membrane Builder ( Jo et al., 2007 ; Jo et al., 2009 ; Lee et al., 2016 ; Lee et al., 2019 ; Wu et al., 2014 ) from CHARMM-GUI ( Jo et al., 2009 ). For each model, the channel was embedded in a hydrated DMPC bilayer, with approximately 150 mM NaCl. The protein was translated and rotated for membrane embedding using the PPM server ( Lomize et al., 2012 ). The lipid bilayer was assembled using the replacement method and solvent ions were added at random positions using a distance-based algorithm. A periodic rectangular cell with approximate dimensions of 14×14×13 nm was used, which comprised ~240,000 atoms. The CHARMM36 all-atom force field was used in conjunction with the TIP3P water model ( Best et al., 2012 ; Jorgensen et al., 1983 ; Klauda et al., 2010 ; MacKerell et al., 1998 ). Non-bonded fixes for backbone carbonyl oxygen atoms with Na + and lipid head groups with Na + were imposed ( Noskov and Roux, 2008 ; Venable et al., 2013 ). For rNa V 1.5/QQQ, an additional non-bonded fix for guanidinium nitrogen with carboxylate oxygen was imposed ( Huang et al., 2017 ). Electrostatic interactions were calculated using the particle-mesh Ewald algorithm ( Darden, 1993 ; Essmann, 1995 ) and chemical bonds were constrained using the LINCS algorithm ( Hess, 2008 ). The energy of the system was minimized with protein position restraints on the backbone (4000 kJ/mol/nm 2 ) and side chains (2000 kJ/mol/nm 2 ), as well as lipid position and dihedral restraints (1000 kJ/mol/nm 2 ) using 5000 steps of steepest descent. The simulation systems were then pre-equilibrated using multi-step isothermal-isovolumetric (NVT) and isothermal-isobaric (NPT) conditions while decreasing the restraints at each step (see Table S2a for parameters). “Production” simulations with and without harmonic restraints on backbone heavy atoms (500 kJ/mol/nm 2 ) were generated with a 2 fs time integration step. The equilibration time was decided based on RMSD analyses of Cα atoms (Figure S9) and average hydration of the gate over time (Figure S10). Details on production simulations equilibration time, and time used in analyses are indicated in Table S2b . All simulations were carried out using GROMACS version 2019.3 ( http://www.gromacs.org ).

Molecular Dynamics Simulation Analysis

In each snapshot of our simulations, atomic positions were translated and rotated by aligning the Cα atoms from pore transmembrane helices (S5 and S6 from all domains) of Na V 1.5 to the initial structure produced by CHARMM-GUI. The positions of all atoms were then centered in the xy-plane by the center of mass (CoM) of pore transmembrane helices and the z-axis by the CoM of Cα atoms from the DEKA motif in the SF. After performing the spatial transformations, the z-axis of the simulation box was used as the pore axis of Na V 1.5 and the transformed positions were used for subsequent analyses. The axial distribution of water was computed by counting the number of water O atoms within a cylindrical of radius 8.5 Å centered on the pore axis. The probability distribution of water was calculated for each replica by counting the number of water molecules in uniform cylindrical slices along the pore-axis and normalizing the counts by the slice with the highest number of water molecules (solvent slice). The average and SEM of the probability distribution was computed across replicas. Pore hydration analysis indicated a dehydrated region located at the ICAG (−2.8 nm < z < −1.5 nm). The number of water molecules in the gate was counted for each frame and normalized by the total number of frames to obtain the probability distribution. The average and SEM were computed across replicas. To measure the size of the intracellular activation gate, residues at the ends of the S6 helices were selected as described ( Jiang et al., 2021 ). The following residue number selections were used in rNa V 1.5: DI: 410–413, DII: 939–942, DIII: 1469–1472, and DIV: 1771–1774. The CoM of Cα-atoms from each selection was projected onto the xy-plane and the distances between opposing S6 tails were measured ( d 1 : DI-DIII and d 2 : DII-DIV). Analyses were performed using MDTraj ( McGibbon et al., 2015 ) and molecular visualizations were rendered using Visual Molecular Dynamics ( Humphrey et al., 1996 ).

Supplementary Material 1 Figure S1.

Purification and Cryo-EM

Analysis of Na V 1.5/QQQ Related to Figure 1 (A) Purification of Na V 1.5/QQQ by size-exclusion chromatography. Red dash lines indicate peak fractions collected for structural study. SDS-PAGE gel image of peak fractions was stained by Coomassie blue. (B) Selected 2D classification averages. 2 Figure S2. Cryo-EM data processing of Nav1.5/QQQ Related to Figure 2 The flowchart for processing of the two Na V 1.5/QQQ datasets. 3 Figure S3. Resolution of the Cryo-EM Data and the Refined Cryo-EM Map Related to Figure 3 (A) Distribution of local resolution in the reconstruction map. High resolution to low resolution colored from blue to red. (B) The gold-standard Fourier Shell Correlation (FSC) curve of the final Na V 1.5/QQQ reconstruction. Red arrow indicates resolution at 3.3 Å. (C) Particle angular distribution of the final reconstruction. (D) The Fourier Shell Correlation curve of the model versus the map used for Na V 1.5/QQQ refinement. Red arrow indicates the model fit to 3.4 Å at FSC=0.5. 4 Figure S4. Structures and Cryo-EM Density Maps of the Voltage Sensor and Pore Module Related to Figures 3 and 4 (A) Comparison of the four VSs of Na V 1.5/QQQ and Na V 1.5 C (grey). Hydrophobic constriction site (HCS) of each VS was indicated by a yellow bar. A key Tyr or Phe residue in the HCS and the gating charge residues are shown in sticks. (B) The cryo-EM density for the S6 segments of D I, D II, D III and D IV, respectively. Side chains of the residues are shown in sticks. (C) The cryo-EM density for two putative detergent or lipid molecules located near intracellular activation gate presented from intracellular view and side view, respectively. 5 Figure S5. Structural Fluctuations of S6 Helices in MD Simulations of Na V 1.5/QQQ and Na V 1.5. Related to Figure 6 . (A) The free energy of diagonal distances of opposing S6 helix tails ( d 1 : D I and D III, d 2 : D II and D IV) was computed from (top) unrestrained and (bottom) backbone-restrained MD simulations of Na V 1.5, Na V 1.5/LqhIII, and Na V 1.5/QQQ (see Methods ). The conformations of cryo-EM structures of Na V 1.5 (PDB ID: 6UZ3; red +), Na V 1.5/LqhIII (PDB ID: 7K18; orange ×), and Na V 1.5/QQQ (green ●) are indicated. Contour lines are shown every 0.5 kcal/mol from 0 to 4 kcal/mol. The cryo-EM structure of Na V 1.5/QQQ was wider [( d 1 , d 2 ) = (16.6 Å, 18.1 Å)] than Na V 1.5 [( d 1 , d 2 ) = (16.2 Å, 17.3 Å)] or Na V 1.5/LqhIII [( d 1 , d 2 ) = (16.6 Å, 17.2 Å)]. The conformations observed by cryo-EM are approximately 0.5 to 2 kcal/mol higher in free energy than the asymmetric free energy minimum. In the restrained simulations, fluctuations of the channel are limited by design to the close vicinity of the initial cryo-EM structure. (B) The free energy of diagonal distances of opposing S6 helix tails (grayscale, d 1 : D I and D III, d 2 : D II and D IV) was computed from (top) unrestrained and (bottom) backbone-restrained MD simulations of Na V 1.5, Na V 1.5/LqhIII, and Na V 1.5/QQQ (see Methods ). Contour lines are shown every 0.5 kcal/mol from 0 to 4 kcal/mol. A scatterplot (viridis color-scale) of the number of water molecules in the gate ( N water ) at the corresponding d 1 , d 2 for each timestep. Points with higher N water are shown at the topmost layer of the scatterplot. Only points with N water ≥ 8 are shown for clarity. N water increases with small increases in d 1 and d 2 . 6 Figure S6. Na + Permeation Through the Intracellular Gate of Na V 1.5/QQQ Related to Figure 6 . (A) Side-view representations of the gate with protein backbone (orange ribbons; D I and D III) shown together with selected pore-facing residues (cyan licorice) as well as Na + ions (blue spheres) and water molecules (red and white licorice) contained in a cylindrical radius of 8.5 Å centered on the pore axis. (B) Timeseries of Na + movement along the pore axis ( z ) depicting one of 7 spontaneous translocation events through the gate observed in structurally restrained simulations for a total of 2.5 μs. Each blue/green line represents the position of a single Na + within a cylindrical radius of 8.5 Å. Regions of the selectivity filter (SF; gold), central cavity (CC), and intracellular activation gate (ICAG; grey) are indicated. In this trajectory, a single Na + enters the gate from below, traverses the hydrated gate, and arrives in the CC within 0.2 ns. 7 Figure S7. Time Evolution of the Protein Root-Mean-Square Deviation (RMSD) and the Average Hydration of the Activation Gate in MD Simulations of rNa V 1.5 Related to Figure 6 . (A) The average RMSD was calculated over time relative to the initial cryo-EM structures in unrestrained MD simulations of (top) Na V 1.5, (middle) Na V 1.5/LqhIII, and (bottom) Na V 1.5/QQQ. The RMSD of backbone Cα atoms for all residues (black), LqhIII only (orange), transmembrane segments (TMS) of rNa V 1.5 (black), as well as TMS from the pore module (PM; red) and voltage sensing domains (VS) from domains I ( D I, blue), II ( D II, cyan), III ( D III, green), and IV ( D IV, purple) are shown. Based on PM TMS and LqhIII RMSD values, simulations equilibrate at approximately 100 ns. (B) The average number of water molecules at the gate (⟨ N water ⟩; red line) over time was computed across replicas in unrestrained and restrained simulations of Na V 1.5, Na V 1.5/LqhIII, and Na V 1.5/QQQ. Magenta line shading corresponds to the standard error of the mean (SEM). Data from the last 50 ns of simulations (grey shaded region) was combined across simulation repeats and used to calculate N water ± SEM of each system, as indicated on the top right of each subplot. 8