|Interactions of membrane proteins with pulsed electric fields – importance for electroporation-based treatments (2020-2023)|
|Head:||Assist. Prof. Lea Rems, University of Ljubljana, Faculty of Electrical Engineering|
|Funding:||Slovenian Research Agency (ARRS), Slovenia|
The project is funded by the Slovenian Research Agency (ARRS).
- Member of University of Ljubljana
- Interactions of membrane proteins with pulsed electric fields – importance for electroporation-based treatments
- 01.09.2020 - 31.08.2023
- Amount of financing
- 0,7 FTE
- Research activity
- Tehnika - Sistemi in kibernetika
- Research Organisation
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. 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 PEFbased 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.
- The phases of the project and their realization
The first objective of this project, which will be addressed in work package WP1, is to predict and explore, using atomistic and coarse-grained molecular dynamics simulations, the electroconformational changes of individual channels as well as changes to protein-protein and lipid-protein interaction that can be elicited by PEFs. Molecular dynamics (MD) is a computer simulation that can capture a wide variety of important bio-molecular processes, including conformational change and protein folding at femtosecond temporal resolu-tion. Importantly, such simulations can also predict how biomolecules will respond—at the atomic level—to perturbations such as external electric fields. We will primarily focus our attention to channels that are rele-vant for experiments conducted in WP2, that is, the cardiac sodium channel Nav1.5 and the inward rectifying potassium channel Kir2.1. In addition, we will also investigate membrane proteins, such as the L-type calci-um voltage gated channels, the big potassium (BK) channel, and the TRPM8 channel, for which previous experiments suggested that they are affected by PEF treatment. By gathering results from different ion channels, we aim to determine common biophysical characteristics of the proteins that make them suscepti-ble to electroconformational changes. If we can elucidate the relationship between the protein’s structure and protein’s propensity for electroconformational changes, this will dramatically simplify identification of membrane proteins that are targets of PEF treatment. Atomistic simulations will provide the information on electroconformational changes that can be induced by PEF in individual channels, whereas coarse-grained simulations will enable us to investigate PEF effects on membrane organization, including membrane pro-tein-protein and lipid-protein interaction.
The second objective, which will be addressed in work package WP2, is to use an engineered cell line, which expresses a minimal complement of voltage-gated ion channels required to produce cellular excitabil-ity, to investigate the ability of a cell to generate an action potential under different PEF parameters. To this end we will use OS-HEK cells, which express two subtypes of sodium and potassium channels, Nav1.5 and Kir2.1, which together imbue the cells with the ability to produce action potentials. We will expose OS-HEK cells to electric pulses with different duration (10 ns ─ 10 ms) and strength, and monitor whether the pulses are able to trigger an action potential. We will also monitor membrane permeabilization and prolonged post-pulse depolarization of OS-HEK cells. These experiments will allow us to determine the threshold electric field strengths for excitation and electroporation, depending on the pulse duration and pulse polarity. Fur-thermore, by comparing the response of OS-HEK cells to normal HEK cells, which do not express ion chan-nels, we will investigate the influence of ion channel expression on membrane electroporation. Finally, we will aim to develop and validate a theoretical model of an excitable cell exposed to PEF of different pulse parameters.
- Citations for bibliographic records