Electroporation Of Intracellular Membranes Of Adrenal Chromaffin Cells With High Intensity, 5-ns Electric Pulses: An Experimental And Modeling Study
AuthorEl Zaklit, Josette
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Externally applied nanosecond-duration electric pulses (NEP) can affect cells by permeabilizing the plasma membrane, resulting in the formation of nanopores, as well as by porating membranes of intracellular organelles that can lead to Ca2+ release from internal stores. In this regard, we have previously reported that when excitable adrenal chromaffin cells are exposed to up to ten, 5-ns pulses applied at an electric field amplitude of 5-6 MV/m, the only response observed is Ca2+ influx through voltage-gated Ca2+ channels. The mechanism underlying voltage-gated Ca2+ channel activation is attributed to Na+ influx via nanopores. In contrast, there is no evidence of Ca2+ release from intracellular stores due to intracellular poration of Ca2+-storing organelles, such as the endoplasmic reticulum (ER). Using a combination of experimental and numerical modeling approaches, the goal of this study was to elucidate the basis for the lack of poration of intracellular Ca2+ stores in chromaffin cells exposed to a 5-ns, 5-6 MV/m pulse. Fluorescence imaging of intracellular Ca2+ levels together with whole-cell recordings obtained in patch clamped cells indicated that the E-field amplitude that was required to cause Ca2+ release from intracellular stores, which was identified as the ER, was twice the E-field amplitude required to cause plasma membrane permeabilization (8 MV/m versus 4 MV/m, respectively). A numerical model of a chromaffin cell was constructed to understand the requirement for the higher E-field amplitude to cause intracellular membrane poration. While initial modeling results agreed with the experimental findings with respect to the E-field threshold amplitude required to electroporate the plasma membrane, they did not show that intracellular membrane poration required a higher E-field amplitude. Therefore, the model was refined by using realistic sizes and measured dielectric properties of secretory granules to confirm the source of intracellular Ca2+ released and understand the requirement for the high E-field amplitude to electroporate the membrane of intracellular organelles. Modeling results identified the ER as the primary target of the NEP, and highlighted the importance of knowing accurately the electrical properties of the different structures to understand the basis for intracellular membrane permeabilization. Establishing agreement between the experimental and modeling results is essential for understanding why high E-field thresholds for stimulating chromaffin cells by causing Ca2+ influx that triggers exocytosis and catecholamine release, and those for causing unwanted effects on intracellular structures differ by more than twofold in magnitude. Such knowledge is important for the potential use of NEPs as a novel stimulus for modulating neurosecretion.