Abstract
Nanosecond Pulsed Electric Field (nsPEF or Nano Pulsed Stimulation, NPS) is a technology that delivers a series of pulses of high-voltage electric fields during a short period of time, in the order of nanoseconds. The main consequence of nsPEF upon cells is the formation of nanopores, which is followed by the gating of ionic channels. Literature is conclusive in that the physiological mechanisms governing ion channel gating occur in the order of milliseconds. Hence, understanding how these channels can be activated by a nsPEF would be an important step in order to conciliate fundamental biophysical knowledge with improved nsPEF applications. To get insights on both the kinetics and thermodynamics of ion channel gating induced by nsPEF, in this work, we simulated the Voltage Sensing Domain (VSD) of a voltage-gated Ca channel, inserted in phospholipidic membranes with different concentrations of cholesterol. We studied the conformational changes of the VSD under a nsPEF mimicked by the application of a continuous electric field lasting 50 ns with different intensities as an approach to reveal novel mechanisms leading to ion channel gating in such short timescales. Our results show that using a membrane with high cholesterol content, under an nsPEF of 50 ns and = 0.2 V/nm, the VSD undergoes major conformational changes. As a whole, our work supports the notion that membrane composition may act as an allosteric regulator, specifically cholesterol content, which is fundamental for the response of the VSD to an external electric field. Moreover, changes on the VSD structure suggest that the gating of voltage-gated Ca channels by a nsPEF may be due to major conformational changes elicited in response to the external electric field. Finally, the VSD/cholesterol-bilayer under an nsPEF of 50 ns and = 0.2 V/nm elicits a pore formation across the VSD suggesting a new non-reported effect of nsPEF into cells, which can be called a “protein mediated electroporation”.
Highlights
The application of electricity in humans can be traced back to the 17th century when tissue damage was firstly observed, a phenomenon that is currently explained as an irreversible electroporation (EP) of the cellular membrane [1,2,3,4]
Our results suggest that the activation of voltage-gated Ca2+ channels by a nanosecond Pulsed Electric Field (nsPEF) may be due to major conformational changes elicited by the application of the external electric field
This work was focused on providing insights on the mechanism of activation of a VG Ca2+ channel under the application of an external field applied in the order of nanoseconds, mimicking an nsPEF
Summary
The application of electricity in humans can be traced back to the 17th century when tissue damage was firstly observed, a phenomenon that is currently explained as an irreversible electroporation (EP) of the cellular membrane [1,2,3,4]. The definition of EP has remained intact for over 30 years: EP is the transient loss of semi-permeability of cell membranes subjected to electric pulses, leading to ion leakage, escape of metabolites, and increased cell-uptake of drugs, molecular probes, or DNA [6] This technology is nowadays widely used for other applications than solely DNA transfection, such as electrochemotherapy [11,12], cold EP [13], tissue ablation [1,14], intracellular delivery [15], extraction of various compounds [16,17], and in the food industry [18,19,20,21]. This technology was developed in 1995 with the aim of improving the efficiency of an electric pulse, used in the industry to kill microorganisms responsible for biofouling in cooling systems that used untreated water from lakes, rivers, or the sea [22]
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