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

Mechanistic Insights into the Modulation 42n5 100 Time (ns) VSD and this VSD by lipi .(g)As mplex por in gold,and s vely.B 实9 ine TMV (all at fomulation under depopi in the lipid bilav 27C)CTable S2).The estimated conductance rs lowe nel.as illustrated in.In the second simulation.a than the experimental value (33 pS at 2C)(51).Overall. that the channel str we used in ou y in d the pore expand hat the led by the high in the electric field,which promoted the hydration of the hydro perturbation are expected.Ion passage through the centra phobic gate along the channel pore. at the functional stat ctu to electrophysiologi Electroporation of the NavPaS channel To verify that the formation of VSD pores and complex pores is not obs in NavMs we studied the cukaryo Electroporation of the HCN1 channe chanf two under depolarizn TMV.In the first simula prone to poration than those of NavMs and NavPaS (Figs and S )In one ut of two simulatio did not th VSD2 exp in both simulations.we also observed formation of a lipid of the VSD from the channel,similarly as in NavMs com- pore in the lipid bilayer surrounding the protein,suggesting plex pores (Fig. s S3 and S4).In the second sim that formation of pores within the ntical ing TMV.a VSD por e was Ior han latic pore.However,the complex pore did not continue to expand TMV,we observed no pores within the entire 600 ns run. within the VSD.Instead,the pore expanded into the bilayer. The structure of this channel is expected to be in the closed secondary structure (Fig.S2).In the first Biophysical Journal 119.190-205.July 7.2020 195

estimated its conductance to be 17 and 2 pS in the two simulations under hyperpolarizing TMV and 17 and 15 pS in the two simulations under depolarizing TMV (all at 27C) (Table S2). The estimated conductance appears lower than the experimental value (33 pS at 22C) (51). Overall, we speculate that the channel structure that we used in our simulations does not correspond to a fully open state and that the passage of Naþ ions was only enabled by the high electric field, which promoted the hydration of the hydro￾phobic gate along the channel pore. Electroporation of the NavPaS channel To verify that the formation of VSD pores and complex pores is not observable only in NavMs, we studied also the eukaryotic NavPaS channel. The same as for NavMs, we performed two simulations for NavPaS under hyperpola￾rizing and two under depolarizing TMV. In the first simula￾tion under a hyperpolarizing field, VSD pores were formed in VSD1 and VSD2 at 233 ns and 249 ns, respectively. Whereas the pore in VSD1 did not expand considerably and mainly enabled the transport of Cl ions, that in VSD2 expanded into a complex pore that led to unfolding of the VSD from the channel, similarly as in NavMs com￾plex pores (Fig. 5 a; Videos S3 and S4). In the second simu￾lation under hyperpolarizing TMV, a VSD pore was formed in VSD2 at 120 ns, which further expanded into a complex pore. However, the complex pore did not continue to expand within the VSD. Instead, the pore expanded into the bilayer, as shown in Fig. 5 b. This limited the number of residues that lost their helical secondary structure (Fig. S2). In the first simulation under depolarizing TMV, a VSD pore was formed in VSD1 at 80 ns. In addition, at 190 ns a lipid pore was formed in the lipid bilayer surrounding the chan￾nel, as illustrated in Fig. 5 c. In the second simulation, a VSD pore was again formed in VSD1 and also in VSD2, but only in VSD2 did the pore expand into a complex one, similarly as in Fig. 5 a. Importantly, unlike NavMs, NavPaS has four different VSDs; therefore, differences in their perturbation are expected. Ion passage through the central pore was not observed in any of the simulations. It should be noted that the functional state of the NavPaS structure is unclear because it was not possible to electrophysiologi￾cally characterize this channel (21). Electroporation of the HCN1 channel In addition to Nav channels, we performed simulations on HCN1. The VSDs of HCN1 turned out to be much less prone to poration than those of NavMs and NavPaS (Figs. 2 and S3). In one out of two simulations performed under hyperpolarizing TMV, we observed the passage of two Naþ ions through VSD4 at 497 and 525 ns. However, in both simulations, we also observed formation of a lipid pore in the lipid bilayer surrounding the protein, suggesting that formation of pores within the identical VSDs is energet￾ically less favorable than that of lipid pores (Videos S5 and S6). In the single simulation performed at depolarizing TMV, we observed no pores within the entire 600 ns run. The structure of this channel is expected to be in the closed state, and in none of the simulations did we observe passage of ions through the channel pore. ab c d i ef g h FIGURE 3 Formation of a complex pore in VSD4 of NavMs channel. (a) VSD was hydrated already in the absence of electric field. (b and c) Upon electric field application, more water molecules entered the VSD. (d) The first Cl ion passed through the VSD at 90 ns after the onset of the electric field. (e and f) More water and ions entered the VSD, and this VSD pore became stabilized by lipid headgroups forming the so-called complex pore. (g) As the complex pore expanded, the VSD began to unfold from the channel. (h) Unfolded VSD viewed from the extracellular side. In (a)–(h) the VSD is colored in red, water in cyan, lipid phosphorus atoms in gold, and sodium and chloride ions in yellow and green, respectively. Black arrows mark the first Cl ion within VSD and the first lipid headgroup moving into the pore. (i) Disruption of the VSD’s secondary structure. To see this figure in color, go online. Mechanistic Insights into the Modulation Biophysical Journal 119, 190–205, July 7, 2020 195

Rems et al I pore of t table bridge n color,go online s and omplex pores form more easily in ules VSDs that electrostatically more favorable for the entry of nels,we plotted the height of the free ene rbarrier versus ions or CI)through The results presented above showed he that t bles.c to be perturbed by the electric field ().Consider ate this hypoth simulations ormed,we observed conduc tion In additior s4 VSDs of N n of reported in Tables S4 and S5).In NavPaS.which has four shown in Fig.6a.We then performed simulations of the c conduction only through mutant HCNI under hyperpolarizing TMV.In one out of ons, nd formed a c a VSD pore was for he follow sage of two sodium ions YSD of the mutant HCNIn be more easily observe the th ough VSD4 in one out o the th e simulations,where lipid pore was form our hypothesis thes VSD is and VSD that make it ne to n We thu 6 fomed more poration.The simulations showed that in NavMs.com sized that VSD pores and complex pore bores could only be formed under hyp erpolarizing tmv easily in VSDs more hydrated This hypoth yer the 7 omplex pos th e lipid c52).as well as on th tha t in lipid bilayer,a pore is formed more easily if this bilayer is VSDI was considerably nder depolarizing TMV. We hypoth that the pre-embedded with vater molecules (53) asymr ry in nd con To inve e hyd SD ion of thei ition along the VSDs by lyzing the ability distribution of these molecules in the abse alons one of the VSDs of NavMs (all four VSDs nce of an field (Fig.6 a).The free similar profile:Fig.S19)and VSDI and VSD2 of NavPaS VSD in the e ers in th potential in the VSDs of NavMs and in VSDI and VSD2 of NavPaS ak at th ide tracts Cl but repels Nat The transport of ions is The water probability distribution in the two other VSDs of ded under for NavPaS and the four which we SDs of HCNI has hydrophobic gap ores,bu iplex pore also in con y mg al i energy vPa oth por 196 Biophysical Joumal 119.190-205.July 7.2020

VSD pores and complex pores form more easily in VSDs that are more hydrated and are electrostatically more favorable for the entry of ions The results presented above showed that it is possible to observe conduction of ions through one or more VSDs in all three channels. However, not all VSDs are equally likely to be perturbed by the electric field (Fig. 2). Considering all simulations performed, we observed ionic conduction through all of the identical VSDs of NavMs. In addition, in NavMs we observed the expansion of VSD pores into complex ones in all VSDs (see also the control simulations reported in Tables S4 and S5). In NavPaS, which has four different VSDs, we observed ionic conduction only through VSD1 and VSD2. Moreover, VSD2 formed a complex pore, whereas VSD1 did not. In HCN1, formation of a lipid pore was more favorable compared to a VSD pore, as we could only observe the passage of two sodium ions through VSD4 in one out of the three simulations, whereas a lipid pore was formed in two of these simulations. Overall, these differences suggest that there should be some features of the VSD that make it more prone to porate. We thus hypothe￾sized that VSD pores and complex pores are formed more easily in VSDs that are more hydrated. This hypothesis is based on previous MD simulations of pure lipid bilayers showing the crucial role of water molecules in formation of lipid pores (52), as well as on the observation that in a lipid bilayer, a pore is formed more easily if this bilayer is pre-embedded with water molecules (53). To investigate the hydration profile of individual VSDs, we estimated the free energy of water molecules as a func￾tion of their position along the VSDs by analyzing the prob￾ability distribution of these molecules in the absence of an external electric field (Fig. 6 a). The free energy profiles indeed show the lowest barriers in the VSDs in which we observed formation of VSD pores and complex pores, i.e., in the VSDs of NavMs and in VSD1 and VSD2 of NavPaS. The water probability distribution in the two other VSDs of NavPaS and the four VSDs of HCN1 has hydrophobic gaps, resulting in considerably higher free energy barriers (see also two-dimensional images showing the position of water molecules in Fig. 6 c; Supporting Materials and Methods, Section S5). Furthermore, for all VSDs from all the chan￾nels, we plotted the height of the free energy barrier versus the time of the first ion passage (either Naþ or Cl) through the VSD. The graph in Fig. 6 b shows that there is a positive correlation between these two variables, confirming that hy￾dration of the VSD is an important feature that contributes to VSD’s propensity for poration. To corroborate this hypoth￾esis further, we considered a mutant’s HCN1 VSD that con￾tained three mutations of nonpolar into polar residues (54). The mutations increased the hydration of the VSDs and decreased the free energy barrier for water molecules, as shown in Fig. 6 a. We then performed simulations of the mutant HCN1 under hyperpolarizing TMV. In one out of two simulations, a VSD pore was formed at 250 ns after the onset of the electric field, followed by its transformation into a complex pore; this observation indicates that the VSDs of the mutant HCN1 can be more easily porated compared with the wild-type channel and goes in line with our hypothesis (Fig. S4; Table S2). The electrostatic potential inside the VSD is another candidate feature that contributes to VSDs’ propensity for poration. The simulations showed that in NavMs, complex pores could only be formed under hyperpolarizing TMV. In NavPaS, we observed formation of a complex pore in VSD2 regardless of the TMV polarity, whereas in VSD1 we only observed formation of a VSD pore. Moreover, the pore in VSD1 was considerably more selective for Cl ions under depolarizing TMV. We hypothesized that the observed asymmetry in pore formation and conduction is due to the asymmetric distribution of charges along the VSDs. Fig. 7 compares the electrostatic potential profiles along one of the VSDs of NavMs (all four VSDs show a similar profile; Fig. S19) and VSD1 and VSD2 of NavPaS. Note that the electrostatic potential was determined under an applied electric field before poration. The NavMs VSD has a high positive peak at the extracellular side, which at￾tracts Cl ions but repels Naþ. The transport of ions is thus impeded under depolarizing TMV, for which we observed formation of VSD pores, but not complex pores. In VSD2 of NavPaS, in which a complex pore was formed under both hyperpolarizing and depolarizing TMV, the a b FIGURE 4 Passage of Naþ ions through the cen￾tral pore of the NavMs channel. (a) Before electric- field application, the bottom half of the pore is dehy￾drated. After the application, a stable water bridge is formed in the bottom half of the channel pore, and the number of water molecules hydrating the bottom half gradually increases, allowing the transport of Naþ ions. (b) The cumulative sum of the number of ions that passed through the channel pore in sim￾ulations under hyperpolarizing (blue shades) and depolarizing TMV (red shades). To see this figure in color, go online. Rems et al. 196 Biophysical Journal 119, 190–205, July 7, 2020

Mechanistic Insights into the Modulation FIGURE S Formation of ores in the NavPas (a)For of n of th d pore se o. om row sho ws the of t electrostatic profile is similar at either side of the membrane. tion.The NavPaS complex pore was crossed by 104 Na DI of NavPaS the ions under hyperpolarization and by 9 Na sitive pea f CI un ization. comple VSD under denolarizing tmy allowing passa A similar prefe ce for cl-ions has heen ohserved in ions compared with only I Naion(see Table S2).Interest- lipid pores.in which this selectivity could be explained yet comp pores wer only in which nects the ectrosltaicfoy VSDs that influence nore formation such as for instane in the direction of Clion movement (56). the salt-bridge connec salt-bridge The size of complex pores depends on the TM cou asily the 8gnascaneonauatleaspretereniayn Comiebabedicieheapietelectncieid (and th eby the TMV).This is simila to what has been for poro nlex n s in NavMs and NavPaS.The pores were eated at TMVof: at a TMV to prevent the 0 ized ionic transport through such stabilized po eg535 TMV of +15 V the As expected,the conduction of ions through NavMs com crease.For a TMVof1.0 V,the pore radius also pore w to incr but at a ugh thec ndent on TMV polarity (Fig.S20).More specifically helices and linid headgrouns and have an irregular shape within 100 ns of the simulation.the NavMs complex pore The pore radius.determined here as the radius of the largest sphere than can be pushed through the pore,is thus a simpli and 52 Cl ions under ied rep or the pore s12 Biophysical Journal 119.190-205.July 7.2020 197

electrostatic profile is similar at either side of the membrane. In VSD1 of NavPaS, the electrostatic potential also shows a positive peak at the extracellular side. Accordingly, we observed selective conduction of Cl ions through this VSD under depolarizing TMV, allowing passage of 9 Cl ions compared with only 1 Naþ ion (see Table S2). Interest￾ingly, the height of the peaks for VSD1 and VSD2 are similar, yet complex pores were formed only in VSD2. This suggests that there are additional features of the VSDs that influence pore formation such as, for instance, the salt-bridge connections. VSD2 has fewer salt-bridge connections between S1–S4 helices than VSD1 (3 vs. 4; see Table S7), which could be the reason why it unfolds more easily. Complex pores can conduct ions preferentially in one direction To investigate further the asymmetric ion conduction through complex pores, we performed additional simula￾tions of NavMs and NavPaS with these pores under a 3 lower electric field, i.e., resulting in TMV z Ez Lz ¼ 0.5 V (55). We reduced the electric field to prevent the complex pores from further expansion, and we character￾ized ionic transport through such stabilized pores (53,54). As expected, the conduction of ions through NavMs com￾plex pore was considerably larger under hyperpolarizing TMV, especially for Cl ions, whereas the conduction through the NavPaS complex pore (in VSD2) was less dependent on TMV polarity (Fig. S20). More specifically, within 100 ns of the simulation, the NavMs complex pore was crossed by 37 Naþ and 195 Cl ions under hyperpolar￾ization and by 20 Naþ and 52 Cl ions under depolariza￾tion. The NavPaS complex pore was crossed by 104 Naþ and 276 Cl ions under hyperpolarization and by 99 Naþ and 189 Cl ions under depolarization. Both complex pores were less conductive to Naþ compared to Cl ions. A similar preference for Cl ions has been observed in lipid pores, in which this selectivity could be explained by lower bulk mobility of Naþ ions and their binding to lipid headgroups, which also affects the electrostatic envi￾ronment inside the pore and induces an electroosmotic flow in the direction of Cl ion movement (56). The size of complex pores depends on the TMV When maintaining the TMV at 1.5 V, complex pores continue to expand in size. However, the size of the pores can be stabilized by reducing the applied electric field (and thereby the TMV). This is similar to what has been observed before for pores in pure lipid bilayers (44,57). Fig. 8 shows the progression of the radius of example com￾plex pores in NavMs and NavPaS. The pores were created at a TMV of 51.5 V, and then the simulation was continued either at a TMV of 51.5 Vor at a lower electric field, result￾ing in TMV of 51.0 V or 50.5 V. The behavior of both NavMs and NavPaS complex pores was similar. For a TMV of 51.5 V, the pore radius continued to rapidly in￾crease. For a TMVof 51.0 V, the pore radius also continued to increase but at a slower rate. For a TMV of 50.5 V, the pore radius somewhat decreased and became stabilized. Note that complex pores are lined both by transmembrane helices and lipid headgroups and have an irregular shape. The pore radius, determined here as the radius of the largest sphere than can be pushed through the pore, is thus a simpli- fied representation of the pore size. ab c FIGURE 5 Formation of pores in the NavPaS system. (a) Formation of a complex pore in VSD2. Expansion of the pore continues to unfold the VSD. (b) Formation of a complex pore initiates in the VSD and then expands into the bilayer. (c) Formation of a lipid pore close to, but not associated with, the protein. Top row shows the side view of the VSD. Bottom row shows the extracellular view of the protein. Representation of atoms is the same as in Figs. 1 and 3. To see this figure in color, go on￾line. Mechanistic Insights into the Modulation Biophysical Journal 119, 190–205, July 7, 2020 197

Rems et al. Estimated free energy of water along VSDs NavMs NavPaS HCN1 wt HCN1 mut 人 Correlation ition of water along VSDs of NavPaS VSD1 VSD2 VSD () r(nm r(nm) height of the gy barrier and the f the first ion passag either Na*or for this last 200ns of trajctory.To this figure Complex pores are more stable than lipid pores Pores created by electric fields in pure 1-palmytoyl-2-oleoyl- phosphatidylcholine bilayers have been reported to close on ulations did we observe complete recovery of the VSD's sec ondary structure:all simulations suggested that formationof complex pores ex pore we cond in Fig. 3.in the of the pore at .100.and 1000ns after switching off the ele Salt-bridge reorganization in VSDs e but remainec by pass thup The ability of VSDs to respond to cha in TMV is tested the sbility of complex pores ented in Fis These pores also remained opened and stabilized by lipid dues are primary elements to sense an applied electric field ation (Sup- pect that the time for whicha ever, n eectric-ficlda open depends on the size that it reached during the elc move along its direction,causing the disruption of existing comple pore,which ions,application o lectric field me some ex es wer idges wer 198 Biophysical Joumal 119.190-205.July 7.2020

Complex pores are more stable than lipid pores Pores created by electric fields in pure 1-palmytoyl-2-oleoyl￾phosphatidylcholine bilayers have been reported to close on average within 50 ns (38). To investigate the postpulse sta￾bility of complex pores, we conducted a 1-ms-long simulation of the NavMs complex pore, presented in Fig. 3, in the absence of an electric field. Fig. 9 a shows the configuration of the pore at 0, 100, and 1000 ns after switching off the elec￾tric field. The pore reduced in size but remained stabilized by lipid headgroups even after 1000 ns. Ions were able to enter and pass through the pore by diffusion (Fig. 9 b). We further tested the stability of complex pores presented in Fig. 8. These pores also remained opened and stabilized by lipid headgroups at the end of the 400-ns-long simulation (Sup￾porting Materials and Methods, Section S8). However, we expect that the time for which a complex pore remains open depends on the size that it reached during the elec￾tric-field exposure and the extent by which it deformed the VSD. We observed that in a NavMs complex pore, which barely met our criterion for a complex pore and became sta￾bilized by only two lipids, the lipids returned to their default orientation 200 ns after turning off the electric field (Fig. S22). Nevertheless, in none of the abovementioned sim￾ulations did we observe complete recovery of the VSD’s sec￾ondary structure; all simulations suggested that formation of complex pores leads to persistent perturbation of the VSD structure, especially the S4 helix (Figs. S21–S23). Salt-bridge reorganization in VSDs The ability of VSDs to respond to changes in TMV is granted by positively charged residues of the S4 segment. Being embedded into a low dielectric medium, these resi￾dues are primary elements to sense an applied electric field. Inside a VSD, they interact with negative counterparts com￾ing from the remaining S1–S3 segments through salt bridges. Upon electric-field application, the S4 residues move along its direction, causing the disruption of existing salt bridges and formation of new ones (58–60). In our sim￾ulations, application of the electric field modified the salt￾bridge connections within the investigated VSD: some ex￾isting salt bridges were broken, and new salt bridges were a b c FIGURE 6 Hydration of VSDs. (a) Free energy profiles estimated from probabilities of water distribution along the VSD principal axis (z) of NavMs, NavPaS, wild-type HCN1, and mutant HCN1, averaged over 200 ns. Note that the free energy barriers are cut at 6 kcal/mol. (b) Correlation between the height of the free energy barrier and the time of the first ion passage (either Naþ or Cl) through a VSD. Ion passage times are taken from all simulations reported in Table S2. Gray dots represent VSDs that were not porated in any of the simulations. The outlier at (600 ns, 2.3 kcal/mol) corresponds to VSD2 of HCN1 mutant; note that we performed only two simulations for this channel. (c) Positions of water molecules in each VSD of NavPaS projected along the VSD radius (r) and the VSD principal axis (z). The VSDs’ center of mass is located at (0,0). Blue circles show all positions extracted from 200 frames of the last 200 ns of the equilibration trajectory. To see this figure in color, go online. Rems et al. 198 Biophysical Journal 119, 190–205, July 7, 2020

Mechanistic Insights into the Modulation NavMs VSD4 NavPaS VSD1 NavPaS VSD2 Na +Na 2 Na 05 VSD com Distance from VSD com (nm) side ide s.charge (neg.charge) side is on the left.Tose this figure formed.An example is depicted in Fig.10 a and Video S7, Comparison with the charge imbalance method which show how salt bridges were perturbed in VSD2 of the ore appnc ets of event negatively charged residues on the S1-S3 helices(Fig.10 as in simulations with the external electric-field method a).Upon an ele nd depolai ction with asp81 Afte off th .the VSD pore formed in then expanded into a complex pore.Under depolarizing ble for at least I us (last time tested).Two other connections, and ARG109-ASP81.that existed be TMV,the VSD pore formed in VSD3 at 104 ns,whereas and fo of salt bridge also observed in other NavMs VSDs and in other channels. important difference between the charg external electric-field simulations.In charge imbalance as shown in Tables S6-S8.It is interesting to note that the entry of ions nt IO kage. fe to red Biophysical Journal 119.190-205.July 7.2020 199

formed. An example is depicted in Fig. 10 a and Video S7, which show how salt bridges were perturbed in VSD2 of the NavMs channel under hyperpolarizing TMV (note that VSD2 was not porated in this simulation). Before applica￾tion of the electric field, there were four connections formed by positively charged arginine residues on the S4 helix and negatively charged residues on the S1–S3 helices (Fig. 10 a). Upon an electric-field application, all these salt bridges were broken, and ARG106 shifted such that it formed a new connection with ASP81. After turning off the electric field, this new connection, ARG106-ASP81, remained sta￾ble for at least 1 ms (last time tested). Two other connections, ARG103-ASP49 and ARG109-ASP81, that existed before the electric-field application were reformed (Figs. S24– S27). Similar breakage and formation of salt bridges was also observed in other NavMs VSDs and in other channels, as shown in Tables S6–S8. It is interesting to note that the entry of ions into the VSDs was not necessary for the breakage of salt bridges, but it could facilitate this breakage, as shown in Fig. 10 b. Comparison with the charge imbalance method Finally, we performed additional simulations for the NavMs system, in which we mimicked electroporation conditions by establishing a charge imbalance across the membrane. With these simulations we observed similar sets of events as in simulations with the external electric-field method: both under hyperpolarizing and depolarizing TMV, firstly Naþ ions started to pass through the central channel pore, and then a VSD pore formed (Fig. S11). Under hyperpola￾rizing TMV, the VSD pore formed in VSD1 at 29 ns and then expanded into a complex pore. Under depolarizing TMV, the VSD pore formed in VSD3 at 104 ns, whereas this pore was not able to expand into a complex pore within the 200-ns-long simulation. However, we observed an important difference between the charge imbalance and external electric-field simulations. In charge imbalance, the TMV kept dropping though the simulation, even though the charge imbalance was kept constant. This is due to redis￾tribution of ions during simulation (some of the ions move ab c FIGURE 7 Electrostatic profiles along different VSDs under hyperpolarizing (blue lines) and depolarizing TMV (red lines). (a) VSD4 of NavMs. (b) VSD1 of NavPaS. (c) VSD2 of NavPaS. Corresponding profiles in the absence of applied electric field are given in Fig. S18. The line thickness corresponds to standard deviation of 10 1D profiles. The gray area at the back of each graph shows the mass density profile of the VSD. Images below graphs show the position of charged residues on each VSD: positively charged ARG, HIS, and LYS are colored green, negatively charged residues ASP and GLU are colored magenta. Intracellular side is on the left. To see this figure in color, go online. Mechanistic Insights into the Modulation Biophysical Journal 119, 190–205, July 7, 2020 199