1. 1 MOSIS, Information Sciences Institute, Viterbi School of Engineering (VSoE), University of Southern California (USC), Marina del Rey, USA 2 Department of Physics and Astronomy, College of Letters, Arts, and Sciences, USC, Los Angeles, USA 3 Ming Hsieh Department of Electrical Engineering, VSoE, USC, Los Angeles, USA, Lipid Bilayer Electropore Modulation using Calcium, Phosphatidylserine, and Temperature Zachary A. Levine 1,2 and P. Thomas Vernier 1,3 Introduction Methods Ca 2+ and PS simulations were performed using GROMACS version 3.3.3 whereas temperature simulations were performed using version 4.0.5. Bilayers consisted of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipids. Each system contained a total of 128 lipids and at least 4480 water molecules. Mixed bilayers were obtained by replacing 20 neutral phosphatidylcholine (PC) molecules on one leaflet with 20 negatively charged phosphatidylserine (PS) molecules and then equilibrating until the total area per lipid was constant. Pore life cycles were determined using custom Perl scripts. Pore creation was defined as the period from the application of an external electric field to the appearance of a mature pore (a pore which contains at least 10 cathode phosphorus atoms at a distance of no more than 1.2 nm [2] from anode phosphorus atoms). Pore annihilation begins with the removal of the electric field and ends with the permanent separation of water groups, as indicated from density profiles across the membrane interior. Pore creation and annihilation are divided into stages based on the connections of water and phosphorus groups. For pore creation in the presence of calcium we used the GROMACS function ‘genion’ to place calcium ions in bulk water, after which the system was equilibrated for 150 ns. For calcium effects on pore annihilation the calcium was inserted at the same time that the external electric field was removed. Molecular-scale details of the mechanism of electric field-driven pore formation in lipid bilayers are not well understood, in part because experiments are unable to access the nanoscopic domain at which individual pore formation occurs. Models can help to fill this void. We use molecular dynamics (MD) simulations to characterize the life cycle of an electropore, from the formation of an initial water defect to the restoration of the intact bilayer. We apply this analysis to systems which were electroporated under various conditions such as those containing Ca 2+, negatively-charged lipid mixtures (such as phosphatidylcholine:phosphatidylserine (PC:PS) systems) and to pure lipid bilayers at various temperatures to understand their respective contributions to pore formation and annihilation, both directly and indirectly. Systems containing Ca 2+ and/or PS are more difficult to electroporate than pure PC systems, and have shorter pore lifetimes. This effect becomes minimal at very high electric fields, where pore formation times are nearly identical for all systems investigated. We find that the temperature at which electroporation occurs does not greatly affect the pore formation time, though pore annihilation time is affected by temperature. Finally, we report binding isotherms for Ca 2+ and PC:PS bilayers and compare them to experimental values [1], an additional metric for the validity of phospholipid bilayer simulations containing calcium. Pore creation time is inversely dependent on the bilayer internal electric field (below). This functional relation may facilitate reconciliation of molecular and continuum models and experiments. Each data point is averaged over three trials. 300 MV/m 400 MV/m 500 MV/m 600 MV/m 0 PS: 0 Ca 0 PS: 100 Ca 20 PS: 0 Ca 20 PS: 100 Ca Conclusions Identification of the stages in the life cycle of an electropore provides an objective scheme for characterizing the mechanisms of pore creation and annihilation. Calcium and PS increase pore creation time. PS decreases pore annihilation time. Temperature does not significantly modify pore creation time, though pore annihilation times are reduced at higher temperatures. Pore creation time is an inverse function of the bilayer internal electric field. MD simulations of calcium binding are consistent with experimental data. Pore annihilation time appears to be independent of the pore- initiating electric field and depends instead on the structure and composition of the pore. In these simulations calcium does not have a large effect on pore annihilation, though mixed PC:PS bilayers produce shorter pore annihilation times than pure PC bilayers for all electric fields observed. Pore creation time depends on the externally applied electric field. We also observe longer pore creation times for systems containing calcium and/or mixed PC:PS bilayers. At higher external electric fields the effects of PS and calcium are less evident. Internal Electric Field and Calcium Binding Isotherm 320 MV/m 400 MV/m 600 MV/m 0 PS: 0 Ca0 PS: 100 Ca20 PS: 0 Ca20 PS: 100 Ca 0 PS: 0 Ca0 PS: 100 Ca20 PS: 0 Ca20 PS: 100 Ca 0 PS: 0 Ca0 PS: 100 Ca20 PS: 0 Ca20 PS: 100 Ca PS and Ca 2+ — Pore Annihilation Values averaged over five simulations PS and Ca 2+ — Pore Creation Values averaged over three simulations T = 0 C T = 10 C T = 20 C T = 30 CT = 40 C T = 50 C T = 0 C T = 10 C T = 20 C T = 30 CT = 40 C T = 50 C POPC Lipid Bilayers at Various Temperatures Values averaged over five simulations Pore Creation Pore Annihilation Though more data is needed at lower temperatures, each stage of pore annihilation is temperature dependent, occurring more quickly at higher temperatures. Additionally, the pore annihilation time as a whole is reduced at higher temperatures. Pore creation time as a whole is not affected by varying temperature, though the maturation and construction stages during pore creation are longer at lower temperatures. E = 400 MV/m Calcium binding to the bilayer interface in our simulations (right) resembles the 1:2 Langmuir binding isotherm established by experiment [1]. Values calculated after the system has equilibrated for 150 ns. This observation shows the operational validity of the calcium ion model in GROMACS. Computation for this work was supported by the University of Southern California Center for High-Performance Computing and Communications (www.usc.edu/hpcc). Special thanks to MOSIS for providing additional resources and funding for this project.www.usc.edu/hpcc [1] Sinn, C. G., M. Antonietti, and R. Dimova. 2006. Binding of calcium to phosphatidylcholine-phosphatidylserine membranes. Colloids and Surfaces A -Physicochemical and Engineering Aspects 282:410-419. [2] Sengupta, D., H. Leontiadou, A. E. Mark, and S. J. Marrink. 2008. Toroidal pores formed by antimicrobial peptides show significant disorder. Biochimica et Biophysica Acta-Biomembranes 1778:2308-2317. Destabilization