Interactions of membrane proteins with pulsed electric fields – importance for electroporation-based treatments

High-intensity pulsed electric fields (PEFs) are used increasingly in medicine, as well as in biotechnology and food technology, to achieve a transient increase in cell membrane permeability. The applied electric field triggers a phenomenon called electroporation (also electropermeabilization), which involves creation of nanoscale pores in the cell membrane that allow enhanced exchange of extracellular and intracellular solutes. Most PEF treatments directly or indirectly target muscle and nerve cells. These are excitable cells that can generate and transmit electrical signals called action potentials. Excitability is enabled by specialized membrane proteins, primarily voltage-gated ion channels, which open or close upon changes in the transmembrane voltage, but also other channels and pumps that regulate action potential generation and subsequent restoration of the resting potential. Increasing experimental evidence shows that voltage-gated ion channels can be affected by PEFs. However, it remains poorly understood and poorly explored whether and how these membrane proteins become perturbed by PEFs, under what range of pulse parameters, and what are the downstream consequences of these perturbations. The increasing interest of using PEF to deliver DNA into muscle and neuronal cells, and to ablate cardiac tissue and brain tumors with irreversible electroporation, calls for in-depth investigations of how pulsed electric fields affect voltage-gated ion channels and what role these effects play in the treatment outcome. In this project we will answer this call by developing a mechanistic understanding of how ion channels respond to PEF on the molecular level and how they contribute to increased membrane permeability and other effects associated with electroporation. To do so, we will follow a stepwise approach combining multi-scale computational methods and experiments on engineered biological cells that will allow us to systematically control the complexity of the investigated systems. We will predict and explore electroconformational changes of individual channels as well as changes to protein-protein and lipid-protein interaction that can be elicited by PEFs using atomistic and coarse-grained molecular dynamics simulations. We will investigate the ability of a cell to generate an action potential under different PEF parameters using an engineered cell line, which expresses a minimal complement of sodium and potassium channels required to produce cellular excitability. We will elucidate how the presence or absence of selected types of channels, such as voltage-gated calcium channels, affects the cell’s response to PEF by expressing these channels in host cells using genetic tools. Since different PEF treatments utilize different pulse parameters, we will place special emphasis on exploring the influence of these pulse parameters on our experimental observables. The relevance of the results of this project is far-reaching. The results will be highly relevant to PEF-based applications which target excitable cells, including gene therapy, DNA vaccination, cardiac ablation for treatment of arrhythmias, and nonthermal ablation of brain tumors. The results will also be relevant for treatments, where electroporation of excitable cells is an unwanted side effect, such as electrochemotherapy and irreversible electroporation of various tumors. The relevance will further reach treatments of nonexcitable cells that express voltage-gated ion channels, including cancer cells and stem cells. Finally, this project will be relevant from a fundamental biophysical perspective by developing understanding how external electric fields could modulate membrane protein function.