European Biophysics Journal

, Volume 40, Issue 8, pp 947–957 | Cite as

Plasma membrane charging of Jurkat cells by nanosecond pulsed electric fields

  • Jody A. White
  • Uwe Pliquett
  • Peter F. Blackmore
  • Ravindra P. Joshi
  • Karl H. Schoenbach
  • Juergen F. Kolb
Original Paper

Abstract

The initial effect of nanosecond pulsed electric fields (nsPEFs) on cells is a change of charge distributions along membranes. This first response is observed as a sudden shift in the plasma transmembrane potential that is faster than can be attributed to any physiological event. These immediate, yet transient, effects are only measurable if the diagnostic is faster than the exposure, i.e., on a nanosecond time scale. In this study, we monitored changes in the plasma transmembrane potential of Jurkat cells exposed to nsPEFs of 60 ns and amplitudes from 5 to 90 kV/cm with a temporal resolution of 5 ns by means of the fast voltage-sensitive dye Annine-6. The measurements suggest the contribution of both dipole effects and asymmetric conduction currents across opposite sides of the cell to the charging. With the application of higher field strengths the membrane charges until a threshold voltage value of 1.4–1.6 V is attained at the anodic pole. This indicates when the ion exchange rates exceed charging currents, thus providing strong evidence for pore formation. Prior to reaching this threshold, the time for the charging of the membrane by conductive currents is qualitatively in agreement with accepted models of membrane charging, which predict longer charging times for lower field strengths. The comparison of the data with previous studies suggests that the sub-physiological induced ionic imbalances may trigger other intracellular signaling events leading to dramatic outcomes, such as apoptosis.

Keywords

nsPEF Plasma membrane charging Membrane poration Real-time fluorescence imaging Annine-6 

Notes

Acknowledgments

This study was funded by an AFOSR DOD MURI grant on “Subcellular Response to Narrow Band and Wide Band Radio Frequency Radiation” administered by Old Dominion University. We would also like to thank Peter Fromherz (Max Planck Institute for Biochemistry) and Bernd Kuhn (Max Planck Institute for Medical Research) for their valuable advice and assistance in discussions regarding the Annine-6 dye.

References

  1. Beebe SJ, Schoenbach KH (2005) Nanosecond pulsed electric fields: a new stimulus to activate intracellular signaling. J Biomed Biotechnol 4:297–300. doi: 10.1155/JBB.2005.297 CrossRefGoogle Scholar
  2. Beebe SJ, Fox PM, Rec LJ, Willis EL, Schoenbach KH (2003) Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells. FASEB J 17:1493–1495. doi: 10.1096/fj.02-0859fje PubMedGoogle Scholar
  3. Beebe SJ, White J, Blackmore PF, Deng Y, Somers K, Schoenbach KH (2004a) Diverse effects of nanosecond pulsed electric fields on cells and tissues. DNA Cell Biol 22:785–796. doi: 10.1089/104454903322624993 CrossRefGoogle Scholar
  4. Beebe SJ, Blackmore PF, White J, Joshi RP, Schoenbach KH (2004b) Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms. Physiol Meas 25:1077–1109. doi: 10.1088/0967-3334/25/4/023 PubMedCrossRefGoogle Scholar
  5. Benz R, Zimmermann U (1980) Pulse lenght dependence of the electrical breakdown in lipid bilayer membranes. Biochim Biophys Acta 597:637–642PubMedCrossRefGoogle Scholar
  6. Böckmann RA, de Groot BL, Kakorin S, Neumann E, Grubmüller H (2008) Kinetics, statistics, and energetics of lipid membrane electroporation studied by molecular dynamics simulations. Biophys J 95:1837–1850. doi: 10.1529/biophysj.108.129437 PubMedCrossRefGoogle Scholar
  7. Bowman AM, Nesin OM, Pakhomova ON, Pakhomov AG (2010) Analysis of plasma membrane integrity by fluorescent detection of Tl+ uptake. J Membrane Biol 236:15–26. doi: 10.1007/s00232-010-9269-y CrossRefGoogle Scholar
  8. Chen N, Schoenbach KH, Kolb JF, Swanson RJ, Garner AL, Yang J, Joshi RP, Beebe SJ (2004) Leukemic cell intracellular responses to nanosecond electric fields. Biochem Biophys Res Commun 317:421–427. doi: 10.1016/j.bbrc.2004.03.063 PubMedCrossRefGoogle Scholar
  9. Deng J, Schoenbach KH, Buescher ES, Hair PS, Fox PM, Beebe SJ (2003) The effects of intense submicrosecond electrical pulses on cells. Biophys J 84:2709–2714. doi: 10.1016/S0006-3495(03)75076-0 Google Scholar
  10. Escoffre J-M, Portet T, Wasungu L, Teissié J, Dean D, Rols M-P (2009) What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues. Mol Biotechnol 41:286–295. doi: 10.1007/s12033-008-9121-0 PubMedCrossRefGoogle Scholar
  11. Fischer JK, von Bruning DM, Labhart H (1976) Lightmodulation by electrochromism. Appl Opt 15:2812–2816PubMedCrossRefGoogle Scholar
  12. Frey W, White JA, Price RO, Blackmore PF, Joshi RP, Nuccitelli RL, Beebe SJ, Schoenbach KH, Kolb JF (2006) Plasma membrane voltage changes during nanosecond pulsed electric field exposure. Biophys J 90:3608–3615. doi: 10.1529/biophysj.105.072777 PubMedCrossRefGoogle Scholar
  13. Gowrishankar TR, Esser AT, Vasilkoski Z, Smith KC, Weaver JC (2006) Microdosimetry for conventional and supra-electroporation in cells with organelles. Biochem Biophys Res Commun 341:1266–1276. doi: 10.1016/j.bbrc.2006.01.094 PubMedCrossRefGoogle Scholar
  14. Hu Q, Joshi RP, Schoenbach KH (2005a) Simulations of nanopore formation and phosphatidylserine externalization in lipid membranes subjected to a high-intensity, ultrashort electric pulse. Phys Rev E 72:031902(10 pp). doi: 10.1103/PhysRevE.72.031902
  15. Hu Q, Viswanadham S, Joshi RP, Schoenbach KH, Beebe SJ, Blackmore PF (2005b) Simulations of transient membrane behavior in cells subjected to a high-intensity ultrashort electric pulse. Phys Rev E 71:031914. doi: 10.1103/PhysRevE.71.031914 CrossRefGoogle Scholar
  16. Hu Q, Sridhara V, Joshi RP, Kolb JF, Schoenbach KH (2006) Molecular dynamics analysis of high electric pulse effects on bilayer membranes containing DPCC and DPSS. IEEE Trans Plasma Sci 34:1405–1411. doi: 10.1109/TPS.2006.876501 CrossRefGoogle Scholar
  17. Joshi RP, Hu Q, Schoenbach KH (2004) Modeling studies of cell response to ultrashort, high-intensity electric fields–implications for intracellular manipulation. IEEE Trans Plasma Sci 32:1677–1688. doi: 10.1109/TPS.2004.830971 CrossRefGoogle Scholar
  18. Knisley SB, Blitchington TF, Hill BC, Grant AO, Smith WM, Pilkington TC, Ideker RE (1993) Optical measurements of transmembrane potential changes during electric field stimulation of ventricular cells. Circ Res 72:255–270PubMedGoogle Scholar
  19. Kotnik T, Miklavcic D (2006) Theoretical evaluation of voltage inducement on internal embranes of biological cells exposed to electric fields. Biophys J 90:480–491PubMedCrossRefGoogle Scholar
  20. Krassowska W, Neu JC (1994) Response of a single cell to an external electric field. Biohpys J 66:1768–1776. doi: 10.1016/S0006-3495(94)80971-3 Google Scholar
  21. Kuhn B, Fromherz P (2003) Annellated hemicyanine dyes in a neuron membrane: molecular stark effect and optical voltage recording. J Phys Chem B 107:7903–7913CrossRefGoogle Scholar
  22. Kuhn B, Fromherz F, Denk W (2004) High sensitivity of Stark-shift voltage-sensing dyes by one- or two-photon excitation near the red spectral edge. Biophys J 87:631–639. doi: 10.1529/biophysj.104.040477 PubMedCrossRefGoogle Scholar
  23. Lojewska Z, Farkas DL, Ehrenberg B, Loew LM (1989) Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields. Biophys J 56:121–128. doi: 10.1016/S0006-3495(89)82657-8 PubMedCrossRefGoogle Scholar
  24. Marrink JS, de Vries Tieleman DP AH, Tieleman DP (2009) Lipids on the move: simulations of membrane pores, domains, stalks and curves. Biochim Biophys Acta 1788:149–168. doi: 10.1016/j.bbamem.2008.10.006 PubMedCrossRefGoogle Scholar
  25. Nuccitelli R, Pliquett U, Chen X, Ford W, Swanson RJ, Beebe SJ, Kolb JF, Schoenbach KH (2006) Nanosecond pulsed electric fields cause melanomas to self-destruct. Biochem Biophys Res Commun 343:351–360. doi: 10.1016/j.bbrc.2006.02.181 PubMedCrossRefGoogle Scholar
  26. Nuccitelli R, Chen X, Pakhomov AG, Baldwin WH, Sheikh S, Pomicter JL, Ren W, Osgood C, Swanson RJ, Kolb JF, Beebe SJ, Schoenbach KH (2009) A new pulsed electric field therapy for melanoma disrupts the tumor’s blood supply and causes complete remission without recurrence. Int J Cancer 125:438–445. doi: 10.1002/ijc.24345 PubMedCrossRefGoogle Scholar
  27. Pakhomov AG, Phinney A, Ashmore J, Walker K III, Kolb JF, Kono S, Schoenbach KH, Murphey MR (2004) Characterization of the cytotoxic effect of high-intensity, 10 ns duration electrical pulses. IEEE Trans Plasma Sci 32:1579–1586. doi: 10.1109/TPS.2004.831773 CrossRefGoogle Scholar
  28. Scarlett SS, White JA, Blackmore PF, Schoenbach KH, Kolb JF (2009) Regulation of intracellular calcium concentrations by nanosecond pulsed electric fields. Biochim Biophys Acta 1788:1168–1175. doi: 10.1016/j.bbamem.2009.02.006 Google Scholar
  29. Schoenbach KH, Beebe SJ, Buescher ES (2001) Intracellular effect of ultrashort electrical pulses. Bioelectromagnetics 22:440–448. doi: 10.1002/bem.71 PubMedCrossRefGoogle Scholar
  30. Schoenbach KH, Joshi RP, Kolb JF, Chen N, Stacey M, Blackmore PF, Buescher ES, Beebe SJ (2004) Ultrashort electrical pulses open a new gateway into biological cells. Proc IEEE 90:1122–1137. doi: 10.1109/JPROC.2004.829009 CrossRefGoogle Scholar
  31. Schwan HP (1957) Electrical properties of tissue and cell suspension. Adv Biol Med Phys 5:147–209Google Scholar
  32. Simcic S, Bobanovic F, Kotnik V, Vodovnik L (1997) Local changes in membrane potential intensify neutrophil oxidative burst. Phsiol. Chem Phys Med NMR 29:39–50Google Scholar
  33. Smith KC, Weaver JC (2008) Active mechanisms are needed to describe cell responses to submicrosecond, megavolt-per-meter pulses: cell models for ultrashort pulses. Biophys J 95:1547–1563. doi: 10.1529/biophysj.107.121921 PubMedCrossRefGoogle Scholar
  34. Tarek M (2004) Membrane electroporation: a molecular dynamics simulation. Biophys J 88:4045–4053. doi: 10.1529/biophysj.104.050617 CrossRefGoogle Scholar
  35. Teissié J, Eynard N, Vernhes MC, Benichou A, Ganeva V, Galutzov B, Cabanes PA (2002a) Recent biotechnological developments of electropulsation. A prospective review. Bioelectrochemistry 55:107–112. doi: 10.1016/S1567-5394(01)00138-4 Google Scholar
  36. Teissié J, Golzio M, Rols MP (2002b) Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of?) knowledge. Biochim Biophys Acta 1724:270–280. doi: 10.1016/j.bbagen.2005.05.006 Google Scholar
  37. Tekle E, Astumian RD, Chock PB (1990) Electro-permeabilization of cell membranes: effect of the resting membrane potential. Biochem Biophys Res Commun 172:282–287PubMedCrossRefGoogle Scholar
  38. Tekle E, Astumian RD, Friauf WA, Chock PB (2001) Asymmetric pore distribution and loss of membrane lipid in electroporated DOPC vesicles. Biophys J 91:960–968. doi: 10.1016/S0006-3495(01)75754-2 CrossRefGoogle Scholar
  39. Tieleman DP, Leontiadou H, Mark AE, Marrink S-J (2003) Simulation of pore formation in lipid bilayers by mechanical stress and electric fields. J Am Chem Soc 125:6382–6383. doi: 10.1021/ja029504i PubMedCrossRefGoogle Scholar
  40. Tsong TY (1991) Electroporation of cell membranes. Biophys J 60:297–306. doi: 10.1016/S0006-3495(91)82054-9 PubMedCrossRefGoogle Scholar
  41. Vernier PT, Ziegler MJ (2007) Nanosecond field alignment of head group and water dipoes in electroporating phospholipid bilayers. J Phys Chem B 111:12993–12996. doi: 0.1021/jp077148q PubMedCrossRefGoogle Scholar
  42. Vernier PT, Sun Y, Marcu L, Craft CM, Gundersen MA (2004a) Nanoelectropulse-induced phosphatidylserine translocation. Biophys J 86:4040–4048. doi: 10.1529/biophysj.103.037945 PubMedCrossRefGoogle Scholar
  43. Vernier PT, Sun Y, Marcu L, Craft CM, Gundersen MA (2004b) Nanosecond pulsed electric fields perturb membrane phospholipids in T lymphoblasts. FEBS Letters 572:103–108. doi: 10.1016/j.febslet.2004.07.021 PubMedCrossRefGoogle Scholar
  44. Vernier PT, Ziegler MJ, Sun Y, Gundersen MA, Tieleman DP (2006a) Nanopore-facilitated, voltage-driven phosphatidylserine translocation in lipd bilayers–in cells and in silicio. Phys Biol 3:233–247. doi: 10.1088/1478-3975/3/4/001 PubMedCrossRefGoogle Scholar
  45. Vernier PT, Sun Y, Gundersen MA (2006b) Nanoelectropulse-driven membrane perturbation and small molecule permeabilization. BMC Cell Biology 7:37. doi: 10.1186/1471-2121-7-37 PubMedCrossRefGoogle Scholar
  46. White JA, Blackmore PF, Schoenbach KH, Beebe SJ (2004) Stimulation of capacitative calcium entry in HL-60 cells by nanosecond pulsed electric fields. J Biol Chem 279:22964–22972PubMedCrossRefGoogle Scholar
  47. Zhang J, Blackmore PF, Hargrave BY, Xiao S, Beebe SJ, Schoenbach KH (2008) The characteristics of nanosecond pulsed electrical field stimulation on platelet aggregation in vitro. Arch Biochem Biophys 471:240–248PubMedGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2011

Authors and Affiliations

  • Jody A. White
    • 1
  • Uwe Pliquett
    • 1
  • Peter F. Blackmore
    • 2
  • Ravindra P. Joshi
    • 3
  • Karl H. Schoenbach
    • 1
  • Juergen F. Kolb
    • 1
    • 3
  1. 1.Frank Reidy Research Center for BioelectricsOld Dominion UniversityNorfolkUSA
  2. 2.Department of Physiological SciencesEastern Virginia Medical SchoolNorfolkUSA
  3. 3.Department of Electrical and Computer EngineeringOld Dominion UniversityNorfolkUSA

Personalised recommendations