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 193a 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 second￾ary 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 Math￾Works, 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 struc￾ture and solvent-accessible surface area were determined with the GRO￾MACS 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 Support￾ing 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 hyperpo￾larized, whereas the part facing the negative electrode becomes depolarized. Thus, for each of the three investi￾gated channels (NavMs, NavPaS, and HCN1), we generated up to 600-ns-long trajectories under hyperpolarizing and de￾polarizing TMV of 51.5 V. The simulations revealed that electric field promotes cre￾ation 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 hydro￾philic 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 be￾comes 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 head￾groups toward the membrane center. Field-induced expan￾sion 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 electro￾static 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 con￾ducting both Na and Cl ions practically simultaneously, VSD pores usually start conducting one type of ions prefer￾entially. 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 pro￾gression of the pore radius, the change in the number of pro￾tein 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 wa￾ter molecules entered the VSDs (Fig. 3 b). In one of these do￾mains, denoted as VSD4, the S3 helix moved away from S1, S2, and S4, which further promoted hydration of the do￾main’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 pas￾sage 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 mid￾dle of the membrane (Fig. 3, e–g). Following previous sug￾gestions in the literature (49,50), we will refer to such lipid-stabilized structure as the ‘‘complex pore.’’ With expan￾sion of the complex pore, the S4 helix lost its secondary Mechanistic Insights into the Modulation Biophysical Journal 119, 190–205, July 7, 2020 193