Rems eta move the VSDs or whether the channel activation is indi- propensity to be porated with their biophysical rectly triggered by postpulse membrane depolarization characteristics. which is cat sed by no selective ion leakage cross the ele ane.Furne METHODS (157).both of whichare important signals that can initiate Systems preparation The ns with NavMs (Protein Data Bank,PDB:5HVX .depend ing on esting for new applications in wound healing and tissue 150 mM NaCl solution.The CHARMM36 force field (27,2)was engineering. nd it mamembrane.as well as lipid scrambling.which could MD simulations all affect the function of membrane proteins(2).Yet.elec GROMACS 2016(3 using the The trophysiological measurements suggest that pul VGICS Hoo measurements not able to directly det ne whethe 35).D ring the firs 100 and how pulsed electric fields can affect VGICs on the mo 40 lecular le the sing the pe electroporation conditions.Because exp mental studies consistently reported a decrease in Nav cun we per 2 n equ formed simulations of two ed by channels for which x-ray cry Magn cr NavMs)020)and an u Nav from Periplaneta americana(NavPaS)(21).Eukary TMV Na ne composed of single ng Navs,on the other hand,are compr sed of four identical d by apply domains,each being analogous to a single domain of their is li P the act vated cyclic nucleotide-gated (HCN1 channel (24) which is a ctive channe ha TM under hy olarizing TMV.The effects o HCNI interes giving the rapid developm ent of pulsed-elec field-based eardiac ablation for treatment of heart arrhyth that the th spono the nble but did ferences resulting in considerably different hydration and electrostatic profile along their VSD ring si harge i resp nabledu to gain a )The ch nd to harge imng chieved b 192 Biophysical Joumal 119.190-205.July 7.2020 move the VSDs or whether the channel activation is indi￾rectly triggered by postpulse membrane depolarization, which is caused by nonselective ion leakage across the elec￾tropermeabilized 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 ef￾fects of pulsed electric fields on VGICs is thus also inter￾esting 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, elec￾trophysiological 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 mo￾lecular 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 per￾formed 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). Eukary￾otic Nav channels are composed of a single polypep￾tide 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 per￾formed simulations of the human hyperpolarization￾activated 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 arrhyth￾mias (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 dif￾ferences 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 equilibra￾tion and production run was then carried out using a leap-frog integrator with a time step of 2.0 fs, Nos
e-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, semi￾isotropic 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 interac￾tions to 0 at 1.2 nm. The chemical bonds were constrained to their equi￾librium 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 elec￾tric-field magnitude; to observe lipid pores within few tens of nanoseconds, simulations have been typically conducted by applying an electric field re￾sulting 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 dis￾charging. 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 mem￾brane 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 con￾trol 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