Pulsed Electric Fields Can Create Pores in the Voltage Sensors of Voltage-Gated Ion Channels.pdf

Biophysica Biophysical Society Article Pulsed Electric Fields Can Create Pores in the Voltage Sensors of Voltage-Gated lon Channels ABSTpACTnpeoeccieasnanocreaengyluednmecneotansentyncaeasahecolmembranepemeabthyva that membrane proteinsare affected as well,particularly ed ion char els(VGICs).The m olecular mechanisms s by take exposing them to fields mimicking electropora that fieldscaninduc pores in domains(VDs)of s and tha CaiedcompiexporesnthSainp anticpate tha such SDs become dysfunctional and unabletorespond ochan es in transmembrane voltage.which is in agree ent tra 1e0 eaoyeeaaoewhakaeetacaEgahaegnscaeae GICs by SIGNIFICANCE Pulsed electric fields are often used for treatment of excitable cells,e.g.,for gene delivery into skeletal muscles.ablation of the heart muscle.or brain tumors.Voltage-gated ion chann elsGiCs)undertiegenerationand the molecular mec ns by whic VGIC estions that have been a by ele hat make the prone to protins.thus future investigations.Finally.we propose a mechanismor ong-ived memrane permeability after pulse treatment,which to date remains poorly understoo INTRODUCTION of ger nhenomenon called electr The integrity of the cell membrane.although essential for the life of any biological cell,presents a barrier that needs tion.Thanks to insights from molecular dynamics (MD) we now und rstand that on to be trans cules 10 in medicine to achie increase in cell membrane permeability.Examples are However.experimental evidence suggests that membrane Proteins.particularly votagon channels GICs) d b drugs and gene therapy techniques for intracel are cla of tran voltage (TMV)with ch Submined November 1.2019.and aceepted for publication May 15.2020. rangements that lead to opening or closure of an ion-selec tive pore.They play crucial roles in the generation and Co espondence:.se propagation c acton potentials in elect rically excitable 16bp2020.05.030 This is an open ess article under the CC BY-NC-ND icese(http/ creanivecommons.org/licenses/by-nc-nd/4.0/) 190 Biophysical Joumal 119.190-205.July 7.2020

Article Pulsed Electric Fields Can Create Pores in the Voltage Sensors of Voltage-Gated Ion Channels Lea Rems,1 Marina A. Kasimova,1 Ilaria Testa,1 and Lucie Delemotte1, * 1 Science for Life Laboratory, Department of Applied Physics, KTH Royal Institute of Technology, Solna, Sweden ABSTRACT Pulsed electric fields are increasingly used in medicine to transiently increase the cell membrane permeability via electroporation to deliver therapeutic molecules into the cell. One type of event that contributes to this increase in membrane permeability is the formation of pores in the membrane lipid bilayer. However, electrophysiological measurements suggest that membrane proteins are affected as well, particularly voltage-gated ion channels (VGICs). The molecular mechanisms by which the electric field could affects these molecules remain unidentified. In this study, we used molecular dynamics simulations to unravel the molecular events that take place in different VGICs when exposing them to electric fields mimicking electroporation conditions. We show that electric fields can induce pores in the voltage-sensor domains (VSDs) of different VGICs and that these pores form more easily in some channels than in others. We demonstrate that poration is more likely in VSDs that are more hydrated and are electrostatically more favorable for the entry of ions. We further show that pores in VSDs can expand into socalled complex pores, which become stabilized by lipid headgroups. Our results suggest that such complex pores are considerably more stable than conventional lipid pores, and their formation can lead to severe unfolding of VSDs from the channel. We anticipate that such VSDs become dysfunctional and unable to respond to changes in transmembrane voltage, which is in agreement with previous electrophysiological measurements showing a decrease in the voltage-dependent transmembrane ionic currents after pulse treatment. Finally, we discuss the possibility of activation of VGICs by submicrosecond-duration pulses. Overall, our study reveals a new, to our knowledge, mechanism of electroporation through membranes containing VGICs. INTRODUCTION The integrity of the cell membrane, although essential for the life of any biological cell, presents a barrier that needs to be transiently disrupted to deliver therapeutic molecules into the cell. High-intensity pulsed electric fields are increasingly used in medicine to achieve such a transient increase in cell membrane permeability. Examples are cancer treatment for enhanced delivery of chemotherapeutic drugs and gene therapy techniques for intracellular delivery of genetic material (1). The applied electric field induces a phenomenon called electroporation or electropermeabilization. Thanks to insights from molecular dynamics (MD) simulations, we now understand that one type of events that takes place in the cell membrane is the formation of pores in the membrane lipid bilayer (2,3). However, experimental evidence suggests that membrane proteins, particularly voltage-gated ion channels (VGICs), could be affected as well (4). VGICs are a class of transmembrane proteins that respond to changes in the transmembrane voltage (TMV) with conformational rearrangements that lead to opening or closure of an ion-selective pore. They play crucial roles in the generation and propagation of action potentials in electrically excitable Submitted November 11, 2019, and accepted for publication May 15, 2020. *Correspondence: lucied@kth.se Editor: Vasanthi Jayaraman. SIGNIFICANCE Pulsed electric fields are often used for treatment of excitable cells, e.g., for gene delivery into skeletal muscles, ablation of the heart muscle, or brain tumors. Voltage-gated ion channels (VGICs) underlie generation and propagation of action potentials in these cells, and consequently are essential for their proper function. Our study reveals the molecular mechanisms by which pulsed electric fields directly affect VGICs and addresses questions that have been previously opened by electrophysiologists. We analyze the characteristics of VGICs that make them prone to electroporation, including hydration and electrostatic properties. This analysis is easily transferable to other membrane proteins, thus opening directions for future investigations. Finally, we propose a mechanism for long-lived membrane permeability after pulse treatment, which to date remains poorly understood. 190 Biophysical Journal 119, 190–205, July 7, 2020 https://doi.org/10.1016/j.bpj.2020.05.030 2020 Biophysical Society. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/)

cells, including neurons and muscle cells. All VGICs share a common architecture: each of the four protein domains contains six transmembrane segments (S1–S6) and a pore loop between segments S5 and S6. Segments S1–S4 act as the voltage sensor, whereas S5, S6, and the pore loop serve as the pore-forming module (Fig. 1, a and b; (5)). Segment S4 contains positively charged residues and has the ability to respond to changes in TMV. The movement of S4 then acts on S5 and S6 to open or close the channel pore depending on the direction of the electric field. Because VGICs are sensitive to changes in TMV and because electroporative pulses induce a TMV of several hundreds of millivolts, far beyond the physiological resting voltage or voltage generated during action potentials, one can speculate that these channels become perturbed by pulsed electric fields. Indeed, by using advanced patch-clamp techniques, electrophysiologists have demonstrated that high-intensity pulses with submicrosecond duration can decrease the ionic currents mediated by different VGICs during action potentials. Nesin et al. (6) studied the effects of 300 or 600 ns pulses on Nav and Cav channels using murine pituitary (GH3) and murine neuroblastoma-rat glioma hybrid (NG108) cells. Electrophysiological measurements revealed a decrease in Nav and Cav currents for pulse amplitudes above 1.5–2 kV/cm. The results from a follow-up study (7) suggested that the decrease in Nav current was not mediated by Naþ leakage across the electropermeabilized membrane or downregulation of the Nav channels by a calcium-dependent mechanism. The authors thus proposed as the mechanism either an electroconformational change or a calcium-independent downregulation of the Nav channels (e.g., caused by alteration of the lipid bilayer). Decrease in Nav channel current was also observed by Yang et al. (8) when they exposed adrenal chromaffin cells to even shorter 5 ns, 50–100 kV/cm pulses. Their analysis suggested that the decrease in Nav current was not due to a change in either the steady-state inactivation or activation of the Nav channels but instead was associated with a decrease in maximal Naþ conductance. This decrease could be observed immediately after the pulse (within 0.5 s, earliest time measured), suggesting a direct effect of the electric field on Nav channels. No effect was observed on Kv channels, whereas a decrease in conductance could also be observed for Cav channels and/or calcium-dependent potassium channels. Although less explored, a decrease in VGIC current has been also reported when using longer millisecond pulses. Chen et al. (9,10) measured up to 40% decrease in Nav and delayed rectifier Kv currents after subjecting voltage-clamped frog skeletal muscle cells to hyperpolarizing 4 ms, 0.5 V pulses. In all the above-discussed studies, the effects depended on the pulse amplitude, with lower amplitude resulting in milder decrease in channel currents. Importantly, all studies also reported that the decrease was not reversible within the observation time (50 min in (10), 10–15 min in (6,8)). Apart from decreased current through VGICs, there have been other electric-field effects observed as well. Submicrosecond pulses have been shown to activate specific VGICs (11,12). However, it remains unclear whether the electric field during such short pulses is able to directly + + + + S1 S2 S3 S5 S6 S4 VSD pore NavMs NavPaS HCN1 a b c FIGURE 1 Structure of the voltage-gated ion channels (VGICs) investigated in this study. (a) All VGICs share an overall architecture that includes four subunits, each containing six membrane-spanning segments denoted S1–S6. (b) Side view and extracellular view of the NavMs channel, indicating the segments of a single subunit: VSD (S1–S4) colored in red, S5 together with S4–S5 linker colored in green, pore helix colored in blue, and S6 colored in ochre. (c) Simulation box of the investigated systems: NavMs, NavPaS, and HCN1. Ion channels are shown as ribbons colored as in (a). Water molecules are represented as transparent volume, whereas phosphorus atoms of lipid headgroups, Naþ ions, and Cl ions are shown as gold, yellow, and green spheres, respectively. To see this figure in color, go online. Mechanistic Insights into the Modulation Biophysical Journal 119, 190–205, July 7, 2020 191

move the VSDs or whether the channel activation is indirectly triggered by postpulse membrane depolarization, which is caused by nonselective ion leakage across the electropermeabilized membrane. Furthermore, this activation of VGICs has been found to participate in postpulse membrane depolarization (13,14) and intracellular calcium increase (15–17), both of which are important signals that can initiate cell death, proliferation, or differentiation, depending on their spatiotemporal profile (18,19). Understanding the effects of pulsed electric fields on VGICs is thus also interesting for new applications in wound healing and tissue engineering. Electropermeabilization is associated with a complex set of events, including oxidative lipid damage, disruption of the cytoskeleton network, and its association with the plasma membrane, as well as lipid scrambling, which could all affect the function of membrane proteins (2). Yet, electrophysiological measurements suggest that pulsed electric fields induce some electroconformational change of VGICs during the pulse. However, electrophysiological measurements are not able to directly determine whether and how pulsed electric fields can affect VGICs on the molecular level. To fill this gap, we used MD simulations and unraveled the molecular events that take place in different VGICs when exposing them to an electric field that mimics electroporation conditions. Because experimental studies consistently reported a decrease in Nav current, we performed simulations of two Nav channels for which the structure had been resolved by x-ray crystallography and cryo-electron microscopy: a bacterial Nav from Magnetococcus marinus (NavMs) (20) and an eukaryotic Nav from Periplaneta americana (NavPaS) (21). Eukaryotic Nav channels are composed of a single polypeptide containing four homologous but nonidentical domains connected by intracellular linkers; bacterial Navs, on the other hand, are comprised of four identical domains, each being analogous to a single domain of their eukaryotic counterparts, and can thus be used as simple models of eukaryotic channels (22,23). In addition, we performed simulations of the human hyperpolarizationactivated cyclic nucleotide-gated (HCN1) channel (24), which is a nonselective voltage-gated cation channel that is responsible for generation of rhythmic activity in heart and brain. Unlike Nav channels, HCN1 activates under hyperpolarizing TMV. The effects on HCN1 are of interest giving the rapid development of pulsed-electric- field-based cardiac ablation for treatment of heart arrhythmias (25). We found that the three tested channels, NavMs, NavPaS, and HCN1, responded differently to electric pulses, which we could relate to the channels’ structural differences resulting in considerably different hydration and electrostatic profiles along their VSDs. Comparing their response thus enabled us to gain an atomistic level insight into the biophysical mechanisms governing their interaction with an electric field and to relate their propensity to be porated with their biophysical characteristics. METHODS Systems preparation The computational systems with NavMs (Protein Data Bank, PDB: 5HVX), NavPaS (PDB: 5X0M), and HCN1 (PDB: 5U6O) were built using the CHARMM-GUI webserver (26). Briefly, each protein was embedded into a 1-palmytoyl-2-oleoyl-phosphatidylcholine bilayer and solvated with 150 mM NaCl solution. The CHARMM36 force field (27,28) was used for proteins, lipids, and ions and the TIP3P model (29) for water. The composition of each system is reported in Table S1. MD simulations All simulations were performed in GROMACS 2016 (30,31). Each system was first minimized using the steepest descent algorithm. The equilibration and production run was then carried out using a leap-frog integrator with a time step of 2.0 fs, Nose-Hoover thermostat (t ¼ 0.4 ps, T ¼ 300 K) (32,33), and Parrinello-Rahman barostat (t ¼ 5 ps, P ¼ 1 bar, semiisotropic coupling) (34,35). During the first 100 ns, the protein atoms were restrained to their initial positions, after which the simulations were continued for 400–1200 ns without restraints (400 ns for NavMs, 1200 ns for NavPaS, and 700 ns for HCN1). The long-range electrostatic interactions were calculated using the particle mesh Ewald method (36), together with a Fourier grid spacing of 0.15 nm and a cutoff of 1.2 nm. A switching function was used between 0.8 and 1.2 nm to smoothly bring the short-range electrostatic interactions and the van der Waals interactions to 0 at 1.2 nm. The chemical bonds were constrained to their equilibrium values using the LINCS algorithm (37). Periodic boundary conditions were applied. To simulate the exposure to an electric pulse, we added a force qEz to every atom carrying a charge q (2,3). This method induces a TMV that is approximately equal to the product of the imposed electric field and the simulation box length, TMV ¼ Ez Lz. The Ez was chosen based on the following considerations. Previous simulations on pure lipid bilayers showed that formation of a lipid pore occurs faster with increasing electric-field magnitude; to observe lipid pores within few tens of nanoseconds, simulations have been typically conducted by applying an electric field resulting in TMV above 3V(38,39). Experimentally, however, the TMV that can be built on the membrane is limited because the membrane starts discharging through pores after it becomes electroporated. According to measurements performed using pulses with duration of 60 ns and longer together with voltage-sensitive dyes (40,41) and microelectrodes (42), the TMV of 1.5 V is the largest that a cell membrane can sustain before discharging. Thus, we chose the value of the electric field such that it resulted in a TMV of about 51.5 V. The chosen value is a compromise between trying to observe an effect of the electric field within reasonable simulation time and at the same time staying within realistic experimental TMV values. These simulations were performed in an NVT ensemble (constant number of atoms, volume, and temperature), which kept the simulation box size constant. This ensured that the voltage applied across the membrane remained constant. We also performed additional control simulations on the NavMs channel by using an electric field in an NPT ensemble but did not observe any considerable differences from NVT simulations that would affect the results and conclusions presented in the work (Supporting Materials and Methods, Section S4.1; Figs. S6 and S10; Table S4). In addition, we performed control simulations by applying a constant charge imbalance instead of an external electric field (Supporting Materials and Methods, Section S4.2; Fig. S7; Table S5). The charge imbalance was achieved by 1) separating the water bath into two parts by adding a vacuum layer and 2) transferring Rems et al. 192 Biophysical Journal 119, 190–205, July 7, 2020

Mechanistic Insights into the Modulation more hydrated and expands enough to conduct ion e charge im ance was kept cons the first h a VSD.we call such an ion-conductive pathway the "VSD on by r setu pore cribed in( stabili 2)AVSD pore forms,begins to expand,and becomes zed by lipic groups cal ned in VSDs,inclu ng thei ionic c ty VSD and then induces mieration of a le wo lipid head groups toward the membrane center.Field-induced expan .Ine type pore that fo Analysis the re ultsobtained for all channels (different type e visualiz are indicated by different markers as shown in the o the p bars in ind ion pa iven n nd the be ith y ius to R ng both ntially More detailed is given in the following sections.For all simulations,the eader can also find in Figs.S1-S4 SD ng sition of t e,the pro helical structure.and the change in the protein's solvent-accessible surface area. 10h 05.19 Electroporation of the NavMs channel d at the We first performed a simulation of NavMs channel under hyperpolarizing TMV of-1.5 V.The VSDs of NavMs ly.we I the average and standard devia already hy rated in the abs ence f an exteral electric of RESULTS ins de ted as VSD4.the S3 helix om S General observations S2.and 4.which further promoted hydration of the do 3 c).Compared to the other helice to the res of the volt larized.whereas the part facing the negative electrode can form and second it has only one salt-bridge intera becomes depolarized Thus,for each of the three investi with S4.unlike.for instance.S2.which has two such sal gated channels(NavMs,NavPaS,and HCN1).we generated bridges we that this s under hyperpo zIng and d The simulations revealed that electric field promotes cre by the first Cl-ion (Fi which was followed by the pas ation of different types of ion-conducting transmembrane sage of the first Na+ion at~130 ns.We will further refer to aqueous pathways. which are schematic such condu thway as the pore During the lined by lipid s the we y hich f d the lipid bilayer next to the rotein and has been previousl dle of the membrane ( observed also in pure lipid bilayers(2.3).We now report that gestions in the literature (4950).we will refer to such lipid-st zed structure as th With expan ion of the complex pore,the Its secondary Biophysical Journal 119.190-205.July 7.2020 193

a certain number of positive and negative ions across the membrane, which created the desired initial TMV. The charge imbalance was kept constant despite the transport of ions through transmembrane pores throughout the simulation by replenishing the charge imbalance using a Monte Carlo setup (43). The box size was allowed to expand in the x and y directions by using surface-tension coupling, as described in (44). Simulations in which we characterized the complex pores formed in VSDs, including their growth dynamics, ionic conductance, stability in the absence of an applied electric field, and perturbation of the VSD secondary structure (Figs. 8 and 9; Supporting Materials and Methods, Section S8), were carried out in the NPT ensemble. Analysis Trajectories were visualized with VMD (45). Ion passage through pores formed in the system was determined by a custom MATLAB (The MathWorks, Natick, MA) code based on the positions of ions extracted from the trajectories. The code is available at https://github.com/ delemottelab. The radius of pores was determined by the CHAP tool (46), which uses the same concept as HOLE (47), i.e., using a Monte Carlo simulated annealing procedure to find the best route for a sphere with variable radius to squeeze through the pore cavity. Secondary structure and solvent-accessible surface area were determined with the GROMACS functions gmx do_dssp and gmx sasa, respectively. Free energy estimates for water molecules along VSDs were determined based on kernel density estimate of the probability distribution of the positions of water molecules extracted from the trajectories at 0 V (see also Supporting Materials and Methods, Section S5). To calculate electrostatic profiles along the VSDs, additional 2-ns-long simulations under an electric field were performed while keeping the position of the protein heavy atoms restrained. These short simulations were carried out starting from 10 different configurations extracted from the last 100 ns of the trajectories without applied electric field. For each of these short trajectories, we determined the three-dimensional electrostatic potential using the VMD tool PMEpot (options: ewaldfactor 0.5, grid 1.0 A˚ ) (48). From each of the 10 three-dimensional profiles, we determined a one-dimensional (1D) electrostatic profile along the VSD by averaging the potential along a cylinder with a diameter of 0.5 nm centered at the center of mass of the VSD. Finally, we computed the average and standard deviation of the 10 1D profiles. RESULTS General observations When a cell is exposed to an electric field, the part of its membrane facing the positive electrode becomes hyperpolarized, whereas the part facing the negative electrode becomes depolarized. Thus, for each of the three investigated channels (NavMs, NavPaS, and HCN1), we generated up to 600-ns-long trajectories under hyperpolarizing and depolarizing TMV of 51.5 V. The simulations revealed that electric field promotes creation of different types of ion-conducting transmembrane aqueous pathways, which are schematically depicted in Fig. 2 a. One of possible pathways is the well-known hydrophilic lipid pore, lined by lipid headgroups, which forms in the lipid bilayer next to the protein and has been previously observed also in pure lipid bilayers (2,3). We now report that ion-conductive pathways can also form within the VSDs of VGICs, whereby two scenarios can occur: 1) A VSD becomes more hydrated and expands enough to conduct ions but not much more. As soon as the first ion passes through a VSD, we call such an ion-conductive pathway the ‘‘VSD pore.’’ 2) AVSD pore forms, begins to expand, and becomes stabilized by lipid headgroups. We call this pathway a ‘‘complex pore’’ and define it as a pore that initiates in a VSD and then induces migration of at least two lipid headgroups toward the membrane center. Field-induced expansion of such complex pore can lead to severe unfolding of the VSD. The type of pore that forms preferentially depends on the type of ion channel and the hydration and electrostatic profile of the VSDs. The graphs in Fig. 2 b summarize the results obtained for all channels (different types of pores are indicated by different markers as shown in the figure legend). The horizontal bars in Fig. 2 b indicate the length of each simulation. The markers show the time of the first ion passage through a given pore (orange color for Na, green color for Cl, circles for lipid pores, triangles for VSD pores). Note that unlike lipid pores, which start conducting both Na and Cl ions practically simultaneously, VSD pores usually start conducting one type of ions preferentially. More detailed description of individual simulations is given in the following sections. For all simulations, the reader can also find additional results in Figs. S1–S4, including the number of ions that passed each pore, the progression of the pore radius, the change in the number of protein residues in helical structure, and the change in the protein’s solvent-accessible surface area. Electroporation of the NavMs channel We first performed a simulation of NavMs channel under a hyperpolarizing TMV of 1.5 V. The VSDs of NavMs were already hydrated in the absence of an external electric field (Fig. 3 a). After the onset of the electric field, more water molecules entered the VSDs (Fig. 3 b). In one of these domains, denoted as VSD4, the S3 helix moved away from S1, S2, and S4, which further promoted hydration of the domain’s interior (Fig. 3 c). Compared to the other helices, S3 is weakly connected to the rest of the voltage sensor. First, it is a short helix, which limits the number of interactions it can form, and second, it has only one salt-bridge interaction with S4, unlike, for instance, S2, which has two such salt bridges; we anticipate that this weak connection resulted in the S3 helix being detached first under an excessive electric field. Shortly after S3 moved, at 90 ns, VSD4 was crossed by the first Cl ion (Fig. 3 d), which was followed by the passage of the first Naþ ion at 130 ns. We will further refer to such conductive pathway as the ‘‘VSD pore.’’ During the simulation, this VSD pore expanded and eventually became stabilized by lipid headgroups that migrated toward the middle of the membrane (Fig. 3, e–g). Following previous suggestions in the literature (49,50), we will refer to such lipid-stabilized structure as the ‘‘complex pore.’’ With expansion of the complex pore, the S4 helix lost its secondary Mechanistic Insights into the Modulation Biophysical Journal 119, 190–205, July 7, 2020 193

structure, and VSD4 began to unfold, as shown in Fig. 3, h–i (and Fig. S1; Videos S1 and S2). In this simulation, another VSD pore was formed in VSD1 at 120 ns, which allowed the passage of a single Cl ion. To verify that these results are reproducible, we performed a second 200-ns-long simulation under the same conditions. Again, we observed formation of a VSD pore followed by its expansion into a complex pore, but this time in VSD3, with the first passage of a Cl ion occurring at 110 ns. Subsequently, a second complex pore was formed in VSD4, suggesting that multiple such pores can be formed in the same voltage-gated ion channel (Fig. S9). The NavMs has four identical VSDs; therefore, it is expected that pores can be formed randomly in any of them with equal probability. We further performed simulations under a depolarizing TMV of 1.5 V. In this case, we also observed formation of VSD pores, but they did not expand into complex ones, even though we prolonged the simulations to 400 ns. In the first out of two simulations, VSD pores were formed in VSD2 and VSD4. In the second simulation, a single Naþ and a single Cl ion passed through VSD2, confirming that the formation of complex pores is more difficult under depolarizing compared to hyperpolarizing TMV. In all simulations, we also observed passage of Naþ through the central pore of the channel (Fig. 4; see also results from control simulations in Supporting Materials and Methods, Section S4.3; Fig. S8). The structure of NavMs was presumed to be in an open state (20), although in simulations, we observed that the channel pore became fully hydrated and conductive only upon application of a strong (beyond physiological values) electric field. Furthermore, the pore stopped conducting as soon as we either lowered the electric field by 50% or completely turned it off, although in both cases, the channel pore still became fully hydrated occasionally, similarly as already shown in Fig. 4 a at 2 ns. By counting the number of Naþ ions that passed through the channel pore within 150 ns at 51.5 V, we a b FIGURE 2 Summary of the simulation results. (a) A schematic representation of the types of ion-conductive transmembrane pathways induced by an electric field that were observed in the simulations. (b) Graphs show the time of the first Naþ and Cl ion passages through a given pore (see legend) in simulations for all channels. Black dots and black stars show the time at which a lipid pore and a VSD pore, respectively, became stabilized by lipid headgroups (i.e., the time when at least two lipid headgroups were found within 0.5 nm from the middle of the membrane). If a VSD pore became stabilized by lipid headgroups, such pore was called a ‘‘complex pore.’’ The horizontal bars correspond to the simulation length. The text next to each horizontal bar gives the index of the VSD in which the VSD pore or complex pore was formed. The data presented in this figure are also tabulated in Table S2. The scheme on the bottom right shows how ions redistribute around the cell membrane upon exposure to an electric field; on the side facing the positive electrode (anode), the membrane becomes hyperpolarized, whereas on the opposite side, it becomes depolarized. To see this figure in color, go online. Rems et al. 194 Biophysical Journal 119, 190–205, July 7, 2020