Molecular simulations reveal the free energy landscape and transition state of membrane electroporation

Abstract

The formation of pores over lipid membranes by the application of electric fields, termed membrane electroporation, is widely used in biotechnology and medicine to deliver drugs, vaccines, or genes into living cells. Continuum models for describing the free energy landscape of membrane electroporation have been proposed decades ago, but they have never been tested against spatially detailed atomistic models. Using molecular dynamics (MD) simulations with a recently proposed reaction coordinate, we computed potentials of mean force of pore nucleation and pore expansion in lipid membranes at various transmembrane potentials. Whereas the free energies of pore expansion are compatible with previous continuum models, the experimentally important free energy barrier of pore nucleation is at variance with established models. We trace the discrepancy to previously incorrect assumptions on the geometry of the transition state; previous continuum models assumed the presence of a membrane-spanning defect throughout the process whereas, according to the MD simulations, the transition state of pore nucleation is typically passed before a transmembrane defect has formed. A modified continuum model is presented that qualitatively agrees with the MD simulations. Using kinetics of pore opening together with transition state theory, our free energies of pore nucleation are in excellent agreement with previous experimental data.

Equivalence of Charge Imbalance and External Electric Fields during Free Energy Calculations of Membrane Electroporation

“…In qualitative agreement with previous simulations of spontaneous pore formation under nonequilibrium conditions ,,,− and with theories of electroporation, application of electric fields leads to a large stabilization of the pore in a voltage- and radius-dependent manner. The physical implications of the PMFs will be discussed elsewhere . As a key finding of this study, Figure demonstrates that the PMFs based on charge imbalance reveal excellent agreement with the PMFs based on external electric fields.…”

Pore formation in complex biological membranes: torn between evolutionary needs

“…The shape of the PMF and the structural mechanisms involved in pore nucleation and expansion agree qualitatively with results obtained previously for simpler model membranes. 20,39,40 The linear regime of the PMF is compatible with classical nucleation theory 45,46 that describes the free energy of larger pores by ∆G CNT = 2πRγ − πR 2 σ, where γ denotes the line tension along the pore rim of length 2πR. The second term models the relief of surface tension σ owing to pore expansion.…”

“…The shape of the PMF and the structural mechanisms involved in pore nucleation and expansion agree qualitatively with results obtained previously for simpler model membranes. 20,39,40…”

“…We hypothesize that the lipid composition of biological membranes has evolved to strictly avoid the formation of long-living pores, underlining that uncontrolled leakage of protons or other ions would be fatal for living cells. Thus, to form long-living pores even in biological membranes, external perturbations are required such as the application of large transmembrane potentials during electroporation experiments 20,27 or the presence of membrane-active agents including channel-forming toxins, 5 proteins involved in regulated cell death, 7,48 polymers, 49 peptides, 50,51 or organic solvents such as DMSO. 39,52 The outer leaflet of the plasma membrane is the main protection layer against pore formation Next, we examined how lipid asymmetry influences pore formation in the plasma membrane and in the inner and outer mitochondrial membranes.…”

“…[15][16][17] In absence of external stressors, pore formation involves considerable energetic costs owing to the initial water penetration into the hydrophobic membrane core as well as owing to membrane bending and lipid tilting deformations along the pore rim. 15,16,[18][19][20][21] Previous experimental and computational studies highlighted the effects of membrane thickness 22,23 and cholesterol content 24,25 on increased membrane stability against pore formation. On the contrary, the addition of anionic 16:0-18:1-phosphatidylglycerol (POPG) into 16:0-18:1-phospatidylcholine (POPC) membranes has been shown to reduce resistance against pore formation.…”