Electroporation increases the permeability of the plasma membrane, allowing us to introduce or extract desired substances into/from the cell that would not normally pass through the plasma membrane. This technique has various medical applications such as gene therapy, tumor treatment, and drug delivery. The success of electroporation is based on achieving a balance between increasing the permeability of the plasma membrane and cell survival. A crucial role in cell survival is played by the cell's ability to restore the resting transmembrane potential and establish cellular homeostasis.
In the thesis, we investigated how electroporation affects changes in transmembrane voltage in Chinese hamster ovary (CHO-K1) cells and how these changes are influenced by the TRPM4 channel inhibitor 9-hydroxyphenanthrene (9-Phenanthrene) and the potassium ion channel inhibitor tetraethylammonium (TEA). We hypothesized that the inhibitor 9-Phenanthrene might have an effect on the changes in TMN, as previous studies have shown that CHO cells express endogenous TRPM4 channels. However, the TEA inhibitor was chosen as a negative control as CHO cells do not express a significant number of potassium channels.
Electroporation was achieved by exposing the cells to a single electrical pulse of 10 µs, 100 µs or 1000 µs. The amplitude of the pulse was determined from measurements of calcium uptake into the cells using the fluorescent dye Fluo4-AM. For each pulse length, we determined the lowest amplitude at which significant calcium uptake into the cells was detected. In addition to this lowest amplitude, which represents the threshold for electroporation, we selected an amplitude approximately 2x higher. Pulses of the selected amplitudes were then used in further measurements of changes in transmembrane voltage. For the latter, we used the FLIPR potentiometric dye, whose fluorescence changes with the change in TMN.
We found that the membrane depolarizes after electroporation, but none of the inhibitors tested had a significant effect on this depolarization, regardless of the pulse length and amplitude. This suggests that the depolarization was largely caused by a non-selective increase in membrane permeability due to electroporation. To facilitate the interpretation of the experimental results, we developed a theoretical model to test the extent to which TRPM4 channels could contribute at all to the changes in TMN after electroporation. The model confirmed that TRPM4 channels do not have a significant effect on depolarization, as under the given conditions the membrane is already fully depolarized due to non-selective ion flux through the pores in the electroporated membrane.
Experiments further revealed that a low-voltage pre-pulse of 45 ms, delivered by a BTX Gemini electroporator to measure the sample resistance before delivering the electroporation pulse, reduces the fluorescence of the FLIPR dye. The mechanisms of this effect are unrelated to the increase in membrane permeability and remain unexplained. In further experiments, it would be advisable to use an electroporator that does not deliver a pre-pulse before the electroporation pulse, to avoid unnecessarily exposing the cell to stress that may affect the electroporation result. At the same time, it would be useful to test other dyes to measure changes in transmembrane voltage that could replace the FLIPR dye.
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