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

Rems et al unfolded VSDs become dysfunctional and unable to regt NavMs late the opening and closure of the channel pore.A dysfunc 1.5V 1.0 tional ion channel cannot respond to changes in TMV in electrophys measuremen -0.5V 0. t0.5 the channel got stuck in an intermediate open state)can 200 ich would be inter 250 300 2 +1.5V NavPas +1.0V simulations offer a m hanism that ca explain the experimentally measured decrease in ion chan -0.5V nels'currents 0.5 +0.5V ions also revealed that. ling on the hy nd ele ated than others This offers a possible explanatio 400 450 of why some channels appear to be affected at weaker elec Time(ns fields than others (8).Furthermore,our simulation FIGURE8 Progre of the radiu ted at hyperpo mechanism of how the decrease in channel conductance de ulting in TMV ±10o ends on the polarity of the TMV (9).Because different spe expre hannels with en uences d-med en d alson ion type specific. into the central chann el pore or VSD por e or complex pores) and thus contrbu than at the egin x pores:a type of long- the TMy dropped in absolute value y pore The mechanisms by which cell membranes remain perme (Fig.11).The complex pore thus did not continue to expand. but its size became stabilized at a radius of0.8 nm.We able for seconds or minutes after application of electric pulses being discussed (4).Li do no Indeed.the pore radius same time,the TMV dro eral microseconds as measured experimentally on pur lipid bilayers(61.62)or a few tens to hundreds of nanosee V,although only a VSD onds in t por lve int and membrane proteins（4g,0 DISCUSSION observed in our simulations are indeed stabilized by lipid atenoreo For x pores is o adgroups VSD helic an have ne ex ong lifetime obs rved exp within a lus lon perimentally simulation.This is on hadTepiedforclosueoflpidporesinNMDsimu Electrophysiological measurements showed that electropor- lations (38.63).Although we cannot infer from our simula ative submicre ectrc pulses can cause a decrease in tions whethe such complex pores could remain open fo potcia5eEniioue if a VSD un was suggested to involve electroconformational changes of not spo ontaneously refold back.thus leaving a long-lived the channels(6-8).Our simulations showed that electric permeable defect in the membrane until pr fields ormation of VSD pores that can n by I's quality control machine y(64 el Wres.le ogical measu 200 Biophysical Joumal 119.190-205.July 7.2020

into the central channel pore or VSD pore or complex pores) and thus contribute differently to the TMV than at the begin￾ning of the simulation. Under hyperpolarizing conditions, the TMV dropped in absolute value from 2 to 0.5 V (Fig. 11). The complex pore thus did not continue to expand, but its size became stabilized at a radius of 0.8 nm. We tested whether we could increase the pore size by increasing the charge imbalance once a complex pore is formed. Indeed, the pore radius expanded to 1.2 nm, but at the same time, the TMV dropped to 0.6 V. Under hyperpola￾rizing conditions, the TMV also kept dropping throughout the simulation from 2 to 1.3 V, although only a VSD pore was formed in this simulation. DISCUSSION Formation of complex pores is one explanation for the decrease in ionic current through VGICs observed experimentally Electrophysiological measurements showed that electropor￾ative submicrosecond electric pulses can cause a decrease in ionic currents through Nav and Cav channels during action potentials in different excitable cells, whereby this decrease was suggested to involve electroconformational changes of the channels (6–8). Our simulations showed that electric fields can induce the formation of VSD pores that can expand into lipid-stabilized complex pores, leading to un￾folding of the VSD from the channel. We expect that such unfolded VSDs become dysfunctional and unable to regu￾late the opening and closure of the channel pore. A dysfunc￾tional ion channel cannot respond to changes in TMV anymore, which in electrophysiological measurements can indeed be detected as reduced current during action potentials. The complex pores (and the channel pore, if the channel got stuck in an intermediate open state) can only contribute to the ‘‘leak’’ current, which would be inter￾preted as the current flowing through lipid pores or other permeable defects caused by pulsed electric field. Overall, our simulations offer a mechanism that can potentially explain the experimentally measured decrease in ion chan￾nels’ currents. Our simulations also revealed that, depending on the hy￾dration and electrostatic properties, some VSDs can be more easily porated than others. This offers a possible explanation of why some channels appear to be affected at weaker elec￾tric fields than others (8). Furthermore, our simulations showed that some VSDs are more easily porated at hyperpo￾larizing (or depolarizing) TMV, which offers a possible mechanism of how the decrease in channel conductance de￾pends on the polarity of the TMV (9). Because different spe￾cies express channels with different sequences, we anticipate that the electric-field-mediated effects on ion channels should to some extent be species and also cell￾type specific. VSD-associated complex pores: a type of long￾lived metastable pores? The mechanisms by which cell membranes remain perme￾able for seconds or minutes after application of electric pulses are still being discussed (4). Lipid pores do not seem to be stable long enough to account for this long-lived permeability as suggested based on their average lifetime: several microseconds as measured experimentally on pure lipid bilayers (61,62) or a few tens to hundreds of nanosec￾onds in the absence of external electric fields as estimated using MD (38,63). It has been proposed that lipid pores could evolve into metastable pores involving both lipids and membrane proteins (49,50). The complex pores observed in our simulations are indeed stabilized by lipid headgroups and VSD helices and can have a long lifetime: after turning off the electric field, we could not observe the pore closure within a 1-ms-long simulation. This is longer than reported for closure of lipid pores in MD simu￾lations (38,63). Although we cannot infer from our simula￾tions whether such complex pores could remain open for seconds or minutes after the pulse, it seems reasonable to anticipate that if a VSD unfolds to a major extent, it would not spontaneously refold back, thus leaving a long-lived permeable defect in the membrane until protein internaliza￾tion by cell’s quality control machinery (64). Recent electro￾physiological measurements on wild-type HEK cells and HEK cells expressing Cav1.3 channels indeed showed about FIGURE 8 Progression of the radius of complex pores in NavMs and NavPaS. Arrows mark the time at which the simulation was continued either at 51.5 V or at a lower electric field, resulting in TMV of 51.0 or 50.5 V. Images on the right show extracellular views of the NavPaS com￾plex pore at times indicated by Roman numbers. The topmost image shows the NavPaS VSD before electric-field application. Scale bar, 2 nm. VSD is shown in red, the rest of the protein in gray, and the lipids in gold. To see this figure in color, go online. Rems et al. 200 Biophysical Journal 119, 190–205, July 7, 2020

Mechanistic Insights into the Modulation FIGURE 9 Postpulse stability of a in Na ntation of ate The bla that are cose to the VSD pore and within 5 m he VSD hat ic fiel VSD Do still stabilized by lipids.Th botom graph shows the progression of the radius of h tw fold greate increase in th postpulse membr submicrosecond pulses (65).Interestingy despite the all increase Activation of Nav channels by nanosecond current through Cav channels became pulsed electric fields decreas exposure Its sup of excitable cells at the ial ome dysfunctional and unable to respond to changes in and is used for various purposes,for example,artificial stimulation S pores ca on.su pores could therefore contribute to the not-well-understood in clectroporation-hased teatments that utilize micro mechanism of asymmetric cell membrane electropermeabi- second-or millisecond-long pulses (69).High-intensity lization,whereby exposure to electric pulses makes the with nano-and picosecond duration and sure to high-in sufficiently long to activate VGICs by moving the VSDs we expect that tensityofVGIC cousorinp during the pulse or whether the activation is caused by post- pulse membrar depolarization resulting from ionic leakage sure on: morane that c the mu 0.7 V(this simulation was initially intended for a different toring as the increase of intracellular Ca? and has been study(54)),we obser observed in bovine chromaffin cells exposed toa5ns.50 cthat VSD nd 2080 ulses bryoni 6 gTMV showing pos ARG10 ASP49 G109-GLU5 and ARG10 81.broke du on facili()An e of ho 200ns n ion fo go onlin Biophysical Journal 119.190-205.July 7.2020 201

two-fold greater increase in the postpulse membrane conductance of Cav-expressing cells after treatment with submicrosecond pulses (65). Interestingly, despite the over￾all increase in the membrane conductance, the voltage￾dependent Ca2þ current through Cav channels became decreased after exposure. These experimental results sup￾port our predictions that VGICs can act as (additional) sites of poration, whereas at the same time, such porated channels become dysfunctional and unable to respond to changes in TMV. It is further noteworthy that formation and ionic con￾duction of complex pores can be asymmetric with respect to TMV polarity. Such or similar protein-associated complex pores could therefore contribute to the not-well-understood mechanism of asymmetric cell membrane electropermeabi￾lization, whereby exposure to electric pulses makes the membrane more extensively permeabilized either on anodic (hyperpolarized) or cathodic (depolarized) side (66–68). Although our simulations mimicked exposure to high-in￾tensity submicrosecond electric pulses, we expect that similar perturbation of VGICs could also occur during expo￾sure to longer, more conventional microsecond or milli￾second pulses with lower intensity. In one simulation of the mutant HCN1 channel under a two-fold lower TMV of 0.7 V (this simulation was initially intended for a different study (54)), we observed formation of a complex pore at 4 ms after the onset of the electric field (Fig. S28). Thus, we expect that VSD pores and complex pores can form also at lower TMV, just at a lower rate, similarly as observed for lipid pores (38). Activation of Nav channels by nanosecond pulsed electric fields Depolarization of the membrane of excitable cells by external electric fields is known to trigger action potentials and is used for various purposes, for example, artificial pacemaking, heart defibrillation, deep brain stimulation, and functional electrical stimulation. Stimulation of periph￾eral nerves is also an expected, though undesired, side effect in electroporation-based treatments that utilize micro￾second- or millisecond-long pulses (69). High-intensity pulses with nano- and picosecond duration can activate VGICs and trigger action potentials as well (15,70,71). However, it remains unclear whether such short pulses are sufficiently long to activate VGICs by moving the VSDs during the pulse or whether the activation is caused by post￾pulse membrane depolarization resulting from ionic leakage across permeabilized membrane that disrupts the transmem￾brane ionic gradients. Stimulation of excitable cells by nanosecond pulses has been characterized by optical moni￾toring as the increase of intracellular Ca2þ and has been observed in bovine chromaffin cells exposed to a 5 ns, 50 kV/cm pulse (16); adult rat cardiomyocytes exposed to 4 ns, 20–80 kV/cm pulses (15); and rat embryonic a b FIGURE 9 Postpulse stability of a complex pore. (a) Configuration of a complex pore in NavMs at 0, 100, and 1,000 ns after turning off the electric field. Representation of atoms is the same as in Fig. 3; wa￾ter is represented as a transparent surface. (b) The cumulative sum of the number of Naþ and Cl that passed through the VSD pore (yellow and green line, respectively); the passage of ions is here medi￾ated by diffusion and can occur either from intracel￾lular to extracellular side or vice versa. The black line shows the number of lipid phosphorus atoms that are close to the VSD pore and within 0.5 nm of the z-position of the lipid bilayer’s center of mass. The number of lipids that stabilize the VSD pore decreases with time but stabilizes above zero, meaning that even at 1 ms after turning off the electric field, the VSD pore is still stabilized by lipids. The bottom graph shows the progression of the radius of the complex pore. To see this figure in color, go online. a b FIGURE 10 Reorganization of salt bridges in VSDs. (a) An example from the first simulation of NavMs under hyperpolarizing TMV showing posi￾tively charged (green) and negatively charged (magenta) residues in VSD2. All four salt-bridge connections, ARG103-ASP49, ARG106-ASP49, ARG109-GLU59, and ARG109-ASP81, broke dur￾ing the pulse, and one new connection was formed: ARG106-ASP81. (b) An example showing how a Cl ion facilitates breakage of a salt bridge between ARG109 and GLU59 in VSD4 of NavMs. Other res￾idues are omitted from representation for clarity. Frames were taken from the second simulation un￾der hyperpolarizing TMV. To see this figure in color, go online. Mechanistic Insights into the Modulation Biophysical Journal 119, 190–205, July 7, 2020 201

Rems et al VSDs of the channel and mainly involves formation of jus hyp 34 g one new salt bridge.An additional in-depth study is required 1.5 dep 34g to confirm or exclude the possibility that the TMV in ced hyp 44g by high-intensity nanosecond pulses can activate VGICs 0.5 Comment on the difference between the external electric-field and charge imbalance methods Our nal electric-fiel thod.the TMV k dropping throughout the simulation becaus of the for tion and expansion of pores.However,neither of the two models a realistic time course 50 150 the membrane is er scale by the charg e(ns) and discharging process of the entire cell membrane.More FIGURE 11 Cha mulations T pecifically,the TMV in experin ents is governed by the uch that it (4e is the of th TMV E TMVa rmittivity of the extracellular and intracellular solutions 44 un (72).Thus,the TMV does not change much by formation of a single por (although formation o num s pores a Vs the of a pathway w field.To TMV).The time course of TMVdurin exposure to electrp porative pulses with microsecond duration and nanosecond luration has been predicted 1 by models (73,74)and exp ah the through cay channels which were activated indirectly by for nanos ond pulses have been challenging h cause of un postpulse membrane depolarization. The second study certain calibration of the voltage-sensitive dye (41).With (15)also that Nav and urther sin expenmental mea the third 17 of TMV in MD sin increase in Ca could be ob erved below the threshold for detectable membrane electropermeabilization CONCLUSIONS (63 kV/cm). sof three VGIC rescent dve (1).This study reported that action pot entials fields.The following conclusions can be drawn from our bilization esults Pulsed ectric fields create conductive pores in hresh was possibl Nav th s.which pore of the VSD f or le meabilization,was also suggested in experiments on pe prone to poration,depending on their hydration and el ripheral nerve (12) trostatic profile:the more hydrated the VSD is and the VGIC involves the mo nore e entry of ions. the during which the ement of $4 helix sily it po of Na charged residues on SI-S3 helices.Our analysis revealed observed in experiments.The differences in poration of application of elec ffecte at Iov his omly in pores an ep 202 Biophysical Joumal 119.190-205.July 7.2020

cardiomyocytes exposed to a 10 ns, R36 kV/cm pulse (17). The first study (16) suggested that Ca2þ entered the cells through Cav channels, which were activated indirectly by postpulse membrane depolarization. The second study (15) also suggested that Nav and Cav channels became activated by postpulse membrane depolarization. Finally, the third study (17) reported that a Cav-mediated increase in intracellular Ca2þ could be observed below the threshold for detectable membrane electropermeabilization (63 kV/cm). More recently, action potential generation in response to a 200 ns, R1.9 kV/cm pulse has been monitored in E18 rat hippocampal neurons by voltage-sensitive fluo￾rescent dye (11). This study reported that action potentials could only be triggered above the electropermeabilization threshold; however, it was possible that Nav channels were activated directly by the electric field. Direct activation of Nav channels by 12 ns pulses, not mediated by electroper￾meabilization, was also suggested in experiments on a pe￾ripheral nerve (12). Activation of a VGIC involves the movement of S4 helix, during which the positively charged residues on S4 break existing connections and form new ones with negatively charged residues on S1–S3 helices. Our analysis revealed that a high TMV, which can build on the membrane during application of electroporative pulses, can induce breakage and formation of salt bridges in the VSD on a nanosecond timescale. However, this occurs quite randomly in different VSDs of the channel and mainly involves formation of just one new salt bridge. An additional in-depth study is required to confirm or exclude the possibility that the TMV induced by high-intensity nanosecond pulses can activate VGICs. Comment on the difference between the external electric-field and charge imbalance methods Our results showed that in contrast to external electric-field method, in the charge imbalance method, the TMV keeps dropping throughout the simulation because of the forma￾tion and expansion of pores. However, neither of the two simulation methods models a realistic time course of TMV on a cell membrane. When exposing a cell to an elec￾tric field in experiments, the TMV that establishes itself on the membrane is controlled on a larger scale by the charging and discharging process of the entire cell membrane. More specifically, the TMV in experiments is governed by the capacitance and conductance of the entire cell membrane, the geometry of the cell, and the conductivity and dielectric permittivity of the extracellular and intracellular solutions (72). Thus, the TMV does not change much by formation of a single pore (although formation of numerous pores over a large cell membrane area does influence the mem￾brane conductance considerably and affects the induced TMV). The time course of TMV during exposure to electro￾porative pulses with microsecond duration and nanosecond duration has been predicted by models (73,74) and measured experimentally using voltage-sensitive fluores￾cent membrane dyes (41,66), although the measurements for nanosecond pulses have been challenging because of un￾certain calibration of the voltage-sensitive dye (41). With further progress in experimental measurements, it will be interesting in the future to simulate different possible real￾istic time courses of TMV in MD simulations. CONCLUSIONS We performed MD simulations of three different VGICs under conditions mimicking exposure to pulsed electric fields. The following conclusions can be drawn from our results. Pulsed electric fields create conductive pores in the VSDs of VGICs, which can evolve into complex pores stabilized by lipid headgroups accompanied by unfolding of the VSD from the channel. VSDs are more or less prone to poration, depending on their hydration and elec￾trostatic profile: the more hydrated the VSD is and the more electrostatically favorable for the entry of ions, the more easily it porates. Poration of VSDs is one explanation for the decreased conductance of Nav and Cav channels observed in experiments. The differences in poration of different VSDs could explain why some channels are affected at lower pulse amplitude than others. Moreover, formation of VSD pores and complex pores, as well as their ionic conduction, can depend on the polarity of the TMV. FIGURE 11 Charge imbalance simulations. Two main simulations were performed, in which the charge imbalance was 34qe (qe is the elementary charge), such that it resulted in hyperpolarizing or depolarizing TMV. For hyperpolarizing TMVa short additional simulation was performed in which the charge imbalance was increased to 44qe after a complex pore was formed under 34qe. Graphs show the absolute value of the TMV and the pore radius in all three simulations. Note that a complex pore formed under hyperpolarizing TMV and a VSD pore formed under depolarizing TMV. The gray horizontal line shows the average radius of a pathway within a VSD in the absence of an electric field. To see this figure in color, go online. Rems et al. 202 Biophysical Journal 119, 190–205, July 7, 2020

Mechanistic Insights into the Modulation cel membrane with respect to its anodie and cathodic side.We speculate that VSD-associated complex pores that s to ngo SD from the protein coul during the secc REFERENCES nsnort of s nds. es-long cell membrane resealing Our study opens several new research directions.First we hope that our sults will stimulate further experiment nance energy transfer.Furthermore.it will be interesting to nvestigate whethe our findings are translatable to other prote ur are mainly Re Bioplys.48:63-91. which can be straighforwardly perform d on other mem. brane proteins.In this regard,a recent MD study on a hu- pore e through this prot unlik closed within 2 ns after nin off the ele field A.Gi.Pakh (75.76).In our work.we additionally reported c onduction of ions throug the pore in the NavMs .G.L Cr c.2017 to inve ate elec ation of the different fune states of this and other channels.Finally.an interesting e cases,particu ently st table for elect oration:for。p forma tion of lipid pores is facilitated in lipids with shorter tails or in peroxidized lipids (77,78).Therefore,it will be 11.Pa the 20 S.2 0.2017 ormlimegsdics me ane proteins in up- SUPPORTING MATERIAL 13.Burke.R.C..S.M.Bardet. ...R.P.O'Connor.2017.Na be found oine t 14.D.R.C AUTHOR CONTRIBUTIONS P T.Ver D.an revised the ACKNOWLEDGMENTS 12 48218212 efor Life Labo Ce Biophysical Journal 119.190-205.July 7.2020 203

Hence, such pores could participate in the not completely understood asymmetric electropermeabilization of the cell membranes with respect to its anodic and cathodic side. We speculate that VSD-associated complex pores that lead to unfolding of the VSD from the protein could act as long-lived permeable defects, allowing enhanced transport of species during the seconds- or minutes-long cell membrane resealing. Our study opens several new research directions. First, we hope that our results will stimulate further experimental investigations of electroporation-mediated membrane pro￾tein perturbation by methods such as NMR or Fo¨rster reso￾nance energy transfer. Furthermore, it will be interesting to investigate whether our findings are translatable to other membrane proteins. Our conclusions are mainly built on analyses of protein hydration and electrostatic profiles, which can be straightforwardly performed on other mem￾brane proteins. In this regard, a recent MD study on a hu￾man aquaporin has revealed the formation of a transient electric-field-induced pore through this protein; unlike the complex pores observed in our simulations, this pore closed within 20 ns after turning off the electric field (75,76). In our work, we additionally reported conduction of ions through the central channel pore in the NavMs structure, which was presumably solved in an open state. Another possible research direction, therefore, would be to investigate electroporation of the different functional states of this and other channels. Finally, an interesting observation from our study is that in some cases, particu￾larly in HCN1, lipid pores formed in addition to, or instead of, VSD pores. It is known that different lipids are differ￾ently suspectable for electroporation; for example, forma￾tion of lipid pores is facilitated in lipids with shorter tails or in peroxidized lipids (77,78). Therefore, it will be interesting to investigate how the lipid environment affects poration of VGICs and other membrane proteins in up￾coming studies. SUPPORTING MATERIAL Supporting Material can be found online at https://doi.org/10.1016/j.bpj. 2020.05.030. AUTHOR CONTRIBUTIONS L.R., M.A.K., I.T., and L.D. designed the research. L.R. performed simula￾tions and analyzed data. L.R., M.A.K., I.T., and L.D. interpreted the results. L.R. wrote the manuscript. M.K., L.D., and I.T. revised the manuscript. ACKNOWLEDGMENTS The authors thank Koushik Choudhury, Sergio Perez Conesa, and Damijan Miklavcic for useful comments to the manuscript. This work was supported by grants from the Science for Life Laboratory and a synergy postdoc grant to L.D. and I.T. from KTH Royal Institute of Technology. The simulations were performed on resources provided by the Swedish National Infrastructure for Computing at parallelldatorcen￾trum (PDC) Centre for High Performance Computing and at High Perfor￾mance Computing Center North (HPC2N). REFERENCES 1. Yarmush, M. L., A. Golberg, ., D. Miklavcic. 2014. Electroporation￾based technologies for medicine: principles, applications, and chal￾lenges. Annu. Rev. Biomed. Eng. 16:295–320. 2. Tieleman, D. P. 2004. The molecular basis of electroporation. BMC Biochem. 5:10. 3. Delemotte, L., and M. Tarek. 2012. Molecular dynamics simulations of lipid membrane electroporation. J. Membr. Biol. 245:531–543. 4. Kotnik, T., L. Rems, ., D. Miklavcic. 2019. Membrane electropora￾tion and electropermeabilization: mechanisms and models. Annu. Rev. Biophys. 48:63–91. 5. Bezanilla, F. 2008. How membrane proteins sense voltage. Nat. Rev. Mol. Cell Biol. 9:323–332. 6. Nesin, V., A. M. Bowman, ., A. G. Pakhomov. 2012. Cell permeabi￾lization and inhibition of voltage-gated Ca(2þ) and Na(þ) channel cur￾rents by nanosecond pulsed electric field. Bioelectromagnetics. 33:394–404. 7. Nesin, V., and A. G. Pakhomov. 2012. Inhibition of voltage-gated Na(þ) current by nanosecond pulsed electric field (nsPEF) is not medi￾ated by Na(þ) influx or Ca(2þ) signaling. Bioelectromagnetics. 33:443–451. 8. Yang, L., G. L. Craviso, ., N. Leblanc. 2017. Nanosecond electric pulses differentially affect inward and outward currents in patch clamped adrenal chromaffin cells. PLoS One. 12:e0181002. 9. Chen, W., Y. Han, ., D. Astumian. 1998. Electric field-induced func￾tional reductions in the Kþ channels mainly resulted from supramem￾brane potential-mediated electroconformational changes. Biophys. J. 75:196–206. 10. Chen, W., Z. Zhongsheng, and R. C. Lee. 2006. Supramembrane poten￾tial-induced electroconformational changes in sodium channel pro￾teins: a potential mechanism involved in electric injury. Burns. 32:52–59. 11. Pakhomov, A. G., I. Semenov, ., S. Xiao. 2017. Neuronal excitation and permeabilization by 200-ns pulsed electric field: an optical mem￾brane potential study with FluoVolt dye. Biochim. Biophys. Acta Bio￾membr. 1859:1273–1281. 12. Casciola, M., S. Xiao, and A. G. Pakhomov. 2017. Damage-free periph￾eral nerve stimulation by 12-ns pulsed electric field. Sci. Rep. 7:10453. 13. Burke, R. C., S. M. Bardet, ., R. P. O’Connor. 2017. Nanosecond pulsed electric fields depolarize transmembrane potential via voltage￾gated Kþ, Ca2þ and TRPM8 channels in U87 glioblastoma cells. Bio￾chim. Biophys. Acta Biomembr. 1859:2040–2050. 14. Dermol-Cerne, J., D. Miklav cic, ., R. O’Connor. 2018. Plasma mem￾brane depolarization and permeabilization due to electric pulses in cell lines of different excitability. Bioelectrochemistry. 122:103–114. 15. Wang, S., J. Chen, ., M. Valderra´bano. 2009. Cardiac myocyte exci￾tation by ultrashort high-field pulses. Biophys. J. 96:1640–1648. 16. Craviso, G. L., S. Choe, ., P. T. Vernier. 2010. Nanosecond electric pulses: a novel stimulus for triggering Ca2þ influx into chromaffin cells via voltage-gated Ca2þ channels. Cell. Mol. Neurobiol. 30:1259–1265. 17. Semenov, I., C. Zemlin, ., A. G. Pakhomov. 2015. Diffuse, non-polar electropermeabilization and reduced propidium uptake distinguish the effect of nanosecond electric pulses. Biochim. Biophys. Acta. 1848:2118–2125. 18. Blackiston, D. J., K. A. McLaughlin, and M. Levin. 2009. Bioelectric controls of cell proliferation: ion channels, membrane voltage and the cell cycle. Cell Cycle. 8:3527–3536. Mechanistic Insights into the Modulation Biophysical Journal 119, 190–205, July 7, 2020 203