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 199formed. 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