**5. Conclusions**

Theories of pore formation and molecular dynamics simulations suggest recurrent hydrophobic pores or water pores whose occurrence is strongly dependent on the electric field. Experimental information about them is very rare. In our study, planar lipid bilayers of POPC, POPS, and a POPC:POPS (1:1) mixture were exposed to different slopes of linearly increasing voltage or current signal. Such experiments allowed to determine the breakdown voltage of planar lipid bilayers, and taking into account that the membrane can be represented by an RC circuit, the fraction of the planar lipid bilayer occupied by water pores could be calculated. Our results confirm the results of pore formation theories and molecular dynamics models. The fraction of area occupied by water pores at the moment

of planar lipid bilayer rupture is considerably lower when the transmembrane voltage is built up slower than in the case of a fast build-up of transmembrane voltage. In more disordered planar lipid bilayers, water pores are easily formed, and the radii of the water pores are smaller in this case than in more ordered planar lipid bilayers. In the case of lipid mixtures, water pores are formed even more easily, probably due to smaller disordered domains in the planar lipid bilayer or/and weaker regions at the domain boundaries.

**Author Contributions:** Conceptualization, P.K. and A.M.L.; methodology, P.K. and A.M.L.; validation, P.K. and A.M.L.; formal analysis, P.K. and A.M.L.; investigation, P.K. and A.M.L.; data curation, P.K. and A.M.L.; writing—original draft preparation, P.K., A.M.L., M.K., and D.M.; visualization, P.K.; supervision, P.K. and A.M.L.; funding acquisition, D.M. and M.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Slovenian Research Agency (ARRS) research core funding No. P2-0249. The part of experimental work was performed in the scope of Polish-Slovenian Scientific Cooperation: Electroporation of cells in a current-controlled mode funded by Ministry of Science and Higher Education (MNiSW), Poland and Slovenian Research Agency (ARRS), Slovenia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data presented are available in this article.

**Conflicts of Interest:** The authors declare no conflict of interest.
