Electroporation is a well-established technique that induces transient pores in cell membranes through intense electric fields. While previous studies have focused primarily on lipid bilayers, the role of membrane proteins in electroporation remains poorly understood. This study investigates the impact of high-intensity electric fields on the TRPV4 ion channel using molecular dynamics simulations to elucidate protein involvement in electroporation processes. The research examines two conformational states of human TRPV4 (hTRPV4, closed and inactivated) embedded in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipid bilayers in a hydrated system under electric (E-)field exposure of 50, 70, and 80 MV/m. A novel algorithm was developed to precisely identify and analyze two distinct water populations: interfacial water near membrane surfaces and channel-confined water within the TRPV4 channel. Key findings reveal fundamentally different responses between these water populations. These findings provide new mechanistic insights into protein-mediated electroporation, highlighting its potential role in electroporation-driven membrane permeabilization, and suggest opportunities for developing targeted therapeutic strategies that exploit the presence and functional state of specific transmembrane proteins in membrane permeabilization processes.
KotnikT, RemsL, TarekM, et al.Membrane electroporation and electropermeabilization: Mechanisms and models. Annu Rev Biophys, 2019; 48(1):63–91; doi: 10.1146/annurev-biophys-052118-115451
2.
TeissieJ. Mechanistic description of membrane electropermeabilization. Handbook of Electroporation, 2017; 1:451–472; doi: 10.1007/978-3-319-32886-7_39
3.
RakoczyK, KisielewskaM, SędzikM, et al.Electroporation in clinical applications—The potential of gene electrotransfer and electrochemotherapy. Applied Sciences, 2022; 12(21):10821; doi: 10.3390/APP122110821
4.
ScuderiM, Dermol-ČerneJ, Amaral da SilvaC, et al.Models of electroporation and the associated transmembrane molecular transport should be revisited. Bioelectrochemistry, 2022; 147:108216; doi: 10.1016/j.bioelechem.2022.108216
5.
Batista NapotnikT, PolajžerT, MiklavčičD. Cell death due to electroporation—A review. Bioelectrochemistry, 2021; 141:107871; doi: 10.1016/j.bioelechem.2021.107871
6.
KotnikT, FreyW, SackM, et al.Electroporation-based applications in biotechnology. Trends Biotechnol, 2015; 33(8):480–488; doi: 10.1016/j.tibtech.2015.06.002
7.
Ruiz-FernándezAR, CamposL, Gutierrez-MaldonadoSE, et al.Nanosecond Pulsed Electric Field (nsPEF): Opening the biotechnological Pandora’s box. Int J Mol Sci, 2022; 23(11):6158; doi: 10.3390/IJMS23116158
8.
CampeloSN, HuangP-H, BuieCR, et al.Recent advancements in electroporation technologies: From bench to clinic. Annu Rev Biomed Eng, 2023; 25(1):77–100; doi: 10.1146/annurev-bioeng-110220-023800
9.
ŠmercR, MiklavčičD, Mahnič-KalamizaS. An experimentally validated numerical model of pH changes in surrogate tissue induced by electroporation pulses. Electrochim Acta, 2025; 511:145363; doi: 10.1016/j.electacta.2024.145363
10.
DenziA, Della ValleE, EspositoG, et al.Technological and theoretical aspects for testing electroporation on liposomes. Biomed Res Int, 2017; 2017:5092704–5092710; doi: 10.1155/2017/5092704
11.
TielemanDP. The molecular basis of electroporation. BMC Biochem, 2004; 5:10–12; doi: 10.1186/1471-2091-5-10
12.
FreemanSA, WangMA, WeaverJC. Theory of electroporation of planar bilayer membranes: Predictions of the aqueous area, change in capacitance, and pore-pore separation. Biophys J, 1994; 67(1):42–56; doi: 10.1016/S0006-3495(94)80453-9
13.
MarracinoP, CaramazzaL, LibertiM, et al.Electroporation Mechanisms: The Role of Lipid Orientation in the Kinetics of Pore Formation. In: 2020 42nd Annual International Conference of the IEEE Engineering in Medicine & Biology Society (EMBC) IEEE; 2020; pp. 2235–2238; doi: 10.1109/EMBC44109.2020.9175706
14.
BöckmannRA, de GrootBL, KakorinS, et al.Kinetics, statistics, and energetics of lipid membrane electroporation studied by molecular dynamics simulations. Biophys J, 2008; 95(4):1837–1850; doi: 10.1529/biophysj.108.129437
15.
MarracinoP, CaramazzaL, MontagnaM, et al.Electric-driven membrane poration: A rationale for water role in the kinetics of pore formation. Bioelectrochemistry, 2022; 143:107987; doi: 10.1016/j.bioelechem.2021.107987
16.
LevineZA, VernierPT. Life cycle of an electropore: Field-Dependent and field-independent steps in pore creation and annihilation. J Membr Biol, 2010; 236(1):27–36; doi: 10.1007/s00232-010-9277-y
17.
Wayne AlbersRRW, SiegelGJ. Cell membrane structures and functions. Basic neurochemistry: Principles of molecular, cellular, and medical neurobiology: Eighth Edition. ScienceDirect.com, 2012:26–39; doi: 10.1016/B978-0-12-374947-5.00002-X
18.
KotnikT, KramarP, PuciharG, et al.Cell membrane electroporation-Part 1: The phenomenon. IEEE Electr Insul Mag, 2012; 28(5):14–23; doi: 10.1109/MEI.2012.6268438
19.
PhillipsR, UrsellT, WigginsP, et al.Emerging roles for lipids in shaping membrane-protein function. Nature, 2009; 459(7245):379–385; doi: 10.1038/nature08147
20.
Ruiz-FernándezAR, CamposL, VillaneloF, et al.Protein-Mediated electroporation in a cardiac voltage-sensing domain due to an nsPEF stimulus. Int J Mol Sci, 2023; 24(14):11397; doi: 10.3390/ijms241411397
21.
MuratoriC, PakhomovAG, GianulisE, et al.Activation of the phospholipid scramblase TMEM16F by Nanosecond Pulsed Electric Fields (nsPEF) facilitates its diverse cytophysiological effects. J Biol Chem, 2017; 292(47):19381–19391; doi: 10.1074/jbc.M117.803049
22.
BezanillaF. How membrane proteins sense voltage. Nat Rev Mol Cell Biol, 2008; 9(4):323–332; doi: 10.1038/nrm2376
23.
RemsL, MiklavčičD. Tutorial: Electroporation of cells in complex materials and tissue. J Appl Phys, 2016; 119(20); doi: 10.1063/1.4949264
24.
RemsL, KasimovaMA, TestaI, et al.Pulsed electric fields can create pores in the voltage sensors of voltage-gated ion channels. Biophys J, 2020; 119(1):190–205; doi: 10.1016/j.bpj.2020.05.030
25.
BernardiM, MarracinoP, LibertiM, et al.Controlling ionic conductivity through transprotein electropores in human aquaporin 4: A non-equilibrium molecular-dynamics study. Phys Chem Chem Phys, 2019; 21(6):3339–3346; doi: 10.1039/C8CP06643D
26.
MarracinoP, BernardiM, LibertiM, et al.Transprotein-Electropore characterization: A molecular dynamics investigation on human AQP4. ACS Omega, 2018; 3(11):15361–15369; doi: 10.1021/acsomega.8b02230
27.
BernardiM, MarracinoP, GhaaniMR, et al.Human aquaporin 4 gating dynamics under axially oriented electric-field impulses: A non-equilibrium molecular-dynamics study. J Chem Phys, 2018; 149(24):245102; doi: 10.1063/1.5044665
28.
ZhangM, MaY, YeX, et al.TRP (Transient Receptor Potential) ion channel family: Structures, biological functions and therapeutic interventions for diseases. Signal Transduct Target Ther, 2023; 8(1):261; doi: 10.1038/s41392-023-01464-x
29.
Sánchez-HernándezR, Benítez-AngelesM, Hernández-VegaAM, et al.Recent advances on the structure and the function relationships of the TRPV4 ion channel. Channels (Austin), 2024; 18(1):2313323; doi: 10.1080/19336950.2024.2313323
30.
NiliusB, VoetsT. The puzzle of TRPV4 channelopathies. EMBO Rep, 2013; 14(2):152–163; doi: 10.1038/embor.2012.219
31.
Hantute-GhesquierA, HaustrateA, PrevarskayaN, et al.TRPM family channels in cancer. Pharmaceuticals (Basel), 2018; 11(2):58; doi: 10.3390/PH11020058
32.
YueL, XuH. TRP channels in health and disease at a glance. J Cell Sci, 2021; 134(13):jcs258372; doi: 10.1242/jcs.258372
33.
WaterhouseA, BertoniM, BienertS, et al.SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Res, 2018; 46(W1):W296–W303; doi: 10.1093/nar/gky427
34.
WebbB, SaliA. Comparative protein structure modeling using MODELLER. Curr Protoc Bioinformatics, 2016; 54(1):5.6.1–5.6.37; doi: 10.1002/CPBI.3
35.
DengZ, PaknejadN, MaksaevG, et al.Cryo-EM and X-ray structures of TRPV4 reveal insight into ion permeation and gating mechanisms. Nat Struct Mol Biol, 2018; 25(3):252–260; doi: 10.1038/s41594-018-0037-5
36.
LeeJ, ChengX, SwailsJM, et al.CHARMM-GUI input generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM simulations using the CHARMM36 additive force field. J Chem Theory Comput, 2016; 12(1):405–413; doi: 10.1021/acs.jctc.5b00935
37.
JorgensenWL, ChandrasekharJ, MaduraJD, et al.Comparison of simple potential functions for simulating liquid water. J Chem Phys, 1983; 79(2):926–935; doi: 10.1063/1.445869
38.
Van Der SpoelD, LindahlE, HessB, et al.GROMACS: Fast, flexible, and free. J Comput Chem, 2005; 26(16):1701–1718; doi: 10.1002/jcc.20291
BerendsenHJC, PostmaJPM, Van GunsterenWF, et al.Molecular dynamics with coupling to an external bath. J Chem Phys, 1984; 81(8):3684–3690; doi: 10.1063/1.448118
41.
HessB, BekkerH, BerendsenHJC, et al.LINCS: A linear constraint solver for molecular simulations. J Comput Chem, 1997; 18(12):1463–1472; doi: 10.1002/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H
DienH, DeaneCM, KnappB. Gro2mat: A package to efficiently read gromacs output in MATLAB. J Comput Chem, 2014; 35(20):1528–1531; doi: 10.1002/jcc.23650
44.
NadezhdinKD, TalyzinaIA, ParthasarathyA, et al.Structure of human TRPV4 in complex with GTPase RhoA. Nat Commun, 2023; 14(1):3733; doi: 10.1038/s41467-023-39346-z
45.
SmartOS, NeduvelilJG, WangX, et al.HOLE: A program for the analysis of the pore dimensions of ion channel structural models. J Mol Graph, 1996; 14(6):354–360, 376; doi: 10.1016/S0263-7855(97)00009-X
46.
VernierPT, LevineZA, WuY-H, et al.Electroporating fields target oxidatively damaged areas in the cell membrane. Rubinsky B. ed. PLoS One, 2009; 4(11):e7966; doi: 10.1371/journal.pone.0007966
47.
RemsL, TangX, ZhaoF, et al.Identification of electroporation sites in the complex lipid organization of the plasma membrane. Elife, 2022; 11:e74773; doi: 10.7554/eLife.74773
48.
CasciolaM, KasimovaMA, RemsL, et al.Properties of lipid electropores I: Molecular dynamics simulations of stabilized pores by constant charge imbalance. Bioelectrochemistry, 2016; 109:108–116; doi: 10.1016/j.bioelechem.2016.01.006
49.
DelemotteL, KleinML, TarekM. Molecular dynamics simulations of voltage-gated cation channels: Insights on voltage-sensor domain function and modulation. Front Pharmacol, 2012; 3:97; doi: 10.3389/FPHAR.2012.00097
50.
VernierPT, SunY, GundersenMA. Nanoelectropulse-driven membrane perturbation and small molecule permeabilization. BMC Cell Biol, 2006; 7(1):37; doi: 10.1186/1471-2121-7-37
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
