ID: 2201

  • Title:
    Role of resting membrane potential in Ca2+ influx following exposure to intense electrical pulse

    Fujita,Kaito-Kumamoto University
    Matsunaga,Kazuya-Kumamoto University
    Katsuki,Sunao-Kumamoto University

    1. Introduction

    Electropermeabilization is used to introduce drugs and genes into cells and to extract intracellular components [1-2]. In particular, the calcium ion (Ca2+) influx following PEF exposure is the promising event for cancer therapy [3]. The Ca2+ influx following PEF exposure can last for from milliseconds to several minutes, which is dependent on the PEF parameters including field strength and pulse duration. The Ca2+ influx through the electropermeabilized membrane can be governed by diffusion, field-driven drift due to applied PEF and/or resting membrane potential (RMP), as well as biochemical activities such as ion transporters. However, there are few studies showing clearly which is dominant and how it depends on the PEF parameters. This study focuses on the dominant physical mechanisms of the Ca2+ influx following PEF exposure, and the dependence on PEF parameters.

    2. Materials and Methods

     We used artificial cells (giant unilamellar vesicles: GUVs) consisting only of a DOPC membrane to exclude biochemical activities. W/o emulsion method was used to prepare GUVs with potassium (K+) ions inside. In order to simulate the RMP of live cells, membrane potentials were generated using a K+ ionophore, valinomycin, to drain the internal K+ through a concentration gradient. A fluorescent probe for Ca2+, Fluo-8, contained only in the GUVs, allowed detection of Ca2+ influx following the PEF exposure.

    3. Result and Discussion

    We successfully controlled the RMP up to 50 mV by changing the initial K+ concentration in the GUVs. The fluorescent microscopy for the Ca2+ uptake to the GUVs following the single 10 us-long PEF exposure indicated the significant Ca2+ uptake was observed in the GUVs with the membrane potential and the amount was proportional to the potential, whereas only slight Ca2+ increase was detected even in the GUVs without the membrane potential. This indicates that the RMP plays significant role in the Ca2+ influx following the 10 us-long PEF exposure. The significance of the RMP in the Ca2+ influx depends on the PEF parameters, in particular, pulse duration. For sub-microsecond PEF the significance of RMP was increased, whereas it was decreased for hundreds microseconds PEF. These results provide knowledge to better understanding of kinetics of the Ca2+ influx following PEF exposure and to find optimal parameters to Ca2+ influx. 

    Pulsed electric field, Giant Unilamellar Vesicle, Electroporation, Electropermeabilization, Ca2+ influx

    (1) K.H. Schoenbach, F.E. Peterkin, R.W. Alden, S.J. Beebe: IEEE Trans. Plasma Sci. 25 (1997) 284 (2) D.E. Clapham: Calcium signaling, Cell 80 (1995) 259 (3) S.K. Frandsen, M. Vissing, Julie Gehl: Cancers 12 (2020) 290

    Topic 1:
    2. Biophysics and biochemistry of interaction mechanisms

    Topic 2:
    1. Biological responses (molecular, subcellular, cellular and intercellular)

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