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. 2008 Sep 15;95(6):2837-48.
doi: 10.1529/biophysj.108.135541. Epub 2008 Jun 6.

Kinetics of transmembrane transport of small molecules into electropermeabilized cells

Gorazd Pucihar  1 , Tadej Kotnik, Damijan Miklavcic, Justin Teissié
Affiliations

Kinetics of transmembrane transport of small molecules into electropermeabilized cells

Gorazd Pucihar et al. Biophys J. .

Abstract

The transport of propidium iodide into electropermeabilized Chinese hamster ovary cells was monitored with a photomultiplier tube during and after the electric pulse. The influence of pulse amplitude and duration on the transport kinetics was investigated with time resolutions from 200 ns to 4 ms in intervals from 400 micros to 8 s. The transport became detectable as early as 60 micros after the start of the pulse, continued for tens of seconds after the pulse, and was faster and larger for higher pulse amplitudes and/or longer pulse durations. With fixed pulse parameters, transport into confluent monolayers of cells was slower than transport into suspended cells. Different time courses of fluorescence increase were observed during and at various times after the pulse, reflecting different transport mechanisms and ongoing membrane resealing. The data were compared to theoretical predictions of the Nernst-Planck equation. After a delay of 60 micros, the time course of fluorescence during the pulse was approximately linear, supporting a mainly electrophoretic solution of the Nernst-Planck equation. The time course after the pulse agreed with diffusional solution of the Nernst-Planck equation if the membrane resealing was assumed to consist of three distinct components, with time constants in the range of tens of microseconds, hundreds of microseconds, and tens of seconds, respectively.

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Figures

FIGURE 1
FIGURE 1
Schematic of the imaging system.
FIGURE 2
FIGURE 2
Noise analysis. (Left column) Noise in solution of 100 μM PI. (Right column) Noise in suspension of cells and 100 μM PI. The measurements were performed at (A) 117 kHz, (B) 39 kHz, (C) 7.7 kHz, and (D) 3.5 kHz bandwidth of the amplifier. The background fluorescence of the solution was subtracted by the differential acquisition of the signal.
FIGURE 3
FIGURE 3
Time response of the imaging system on a step change of the light from a green LED. Durations of the LED signal were set to (A) 100 μs, and (B) 10 μs. Bandwidths of the amplifier: (1) 117 kHz, (2) 39 kHz, (3) 7.7 kHz, and (4) 3.5 kHz.
FIGURE 4
FIGURE 4
Time course of fluorescence measured from a single cell. (A) The influence of pulse amplitude, and (B) pulse duration on the time course of fluorescence measured from a single cell. CHO cells in dilute suspension containing 100 μM of PI were exposed either to a single rectangular 1 ms pulse with an amplitude of 350, 500, 650, and 800 V or a single rectangular 500 V pulse with a duration of 0.1, 0.5, 1, and 3 ms. The excitation light was focused on a single cell, and fluorescence from this cell was detected with a PM tube. The background fluorescence was subtracted by the differential acquisition of the signal. Each curve shows a single measurement, as there were no large differences between the three repetitions.
FIGURE 5
FIGURE 5
Influence of pulse amplitude on the time course of fluorescence during and after electropermeabilization. The changes in fluorescence were monitored on a (A) 2 ms, (B) 80 ms, and (C) 8 s acquisition interval. CHO cells in suspension containing 100 μM of PI were exposed to a single rectangular 1 ms pulse with amplitudes of 350, 500, 650, and 800 V. The dashed line in A denotes the end of the pulse, and the horizontal dashed line in C is the baseline. Results shown on different acquisition intervals were obtained from different experiments. The background fluorescence was subtracted by the differential acquisition of the signal. Note different scale on the y axes.
FIGURE 6
FIGURE 6
Influence of pulse duration on the time course of fluorescence during and after electropermeabilization. The changes in fluorescence were monitored on a (A) 2 ms, (B) 80 ms, and (C) 8 s acquisition interval. CHO cells in suspension containing 100 μM of PI were exposed to a single rectangular 500 V pulse with durations of 0.1, 0.5, 1, and 3 ms. Results in different acquisition intervals were obtained from different experiments. The dashed line in C is the baseline. The background fluorescence was subtracted by the differential acquisition of the signal. Note different scale on y axes.
FIGURE 7
FIGURE 7
Onset of the transport of PI. (A) The time course of fluorescence increase obtained from three independent experiments. (B) Average of the three signals in A. CHO cells in suspension containing 1 mM PI were exposed to a single rectangular 1 ms, 800 V pulse. PM tube was set to the highest sensitivity, where all dynodes were activated. The background fluorescence was subtracted by the differential acquisition of the signal.
FIGURE 8
FIGURE 8
Time course of fluorescence for confluent monolayers of cells and suspended cells. Signals were measured on confluent monolayers of cells (black curves) and suspended cells (gray curves) in 80 ms (left column) and 8 s (right column) time intervals. Cells were electropermeabilized with a single 1 ms pulse with amplitudes (A) 350 V, (B) 500 V, (C) 650 V, and (D) 800 V. The gray curve in figure D2 was cut off because of the saturation of the amplifier. Results shown on different acquisition intervals were obtained from different experiments. The background fluorescence was subtracted by the differential acquisition of the signal.
FIGURE 9
FIGURE 9
Theoretically calculated solution of Eq. 5 for different functions SP(t).
FIGURE 10
FIGURE 10
Time course of the fraction of permeable structural defects fP(t) in the membrane after electropermeabilization. Cells were exposed to a 1 ms, 800 V pulse, and fP was calculated from fP(t) = SP(t)/SC, where SP(t) = SP1 exp(−t/τ1) + SP2 exp(−t/τ2) + SP3 exp(−t/τ3) and SC is the total surface of the cell membrane. The parameters SP1, SP2, SP3, τ1, τ2, τ3 were obtained from fitting Eq. 5 to the experimental data shown in Fig. 9: SP1 = 4.5 × 10−15 m2, SP2 = 6.3 × 10−15 m2, SP3 = 7.2 × 10−14 m2, τ1 = 14 s, τ2 = 380 ms, τ3 = 12 ms.
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References

    1. Kinosita, K., and T. Y. Tsong. 1977. Voltage-induced pore formation and hemolysis of human erythrocytes. Biochim. Biophys. Acta. 471:227–242. - PubMed
    1. Tsong, T. Y. 1991. Electroporation of cell membranes. Biophys. J. 60:297–306. - PMC - PubMed
    1. Barnett, A., and J. C. Weaver. 1991. Electroporation: a unified, quantitative theory of reversible electrical breakdown and rupture. Bioelectroch. Bioener. 25:163–182.
    1. Teissié, J., N. Eynard, B. Gabriel, and M. P. Rols. 1999. Electropermeabilization of cell membranes. Adv. Drug Deliver. Rev. 35:3–19. - PubMed
    1. Neumann, E., S. Kakorin, and K. Toensing. 1999. Fundamentals of electroporative delivery of drugs and genes. Bioelectroch. Bioener. 48:3–16. - PubMed

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