Identification of Proteins Involved in Cell Membrane Permeabilization by Nanosecond Electric Pulses (nsEP)

The study was aimed at identifying endogenous proteins which assist or impede the permeabilized state in the cell membrane disrupted by nsEP (20 or 40 pulses, 300 ns width, 7 kV/cm). We employed a LentiArray CRISPR library to generate knockouts (KOs) of 316 genes encoding for membrane proteins in U937 human monocytes stably expressing Cas9 nuclease. The extent of membrane permeabilization by nsEP was measured by the uptake of Yo-Pro-1 (YP) dye and compared to sham-exposed KOs and control cells transduced with a non-targeting (scrambled) gRNA. Only two KOs, for SCNN1A and CLCA1 genes, showed a statistically significant reduction in YP uptake. The respective proteins could be part of electropermeabilization lesions or increase their lifespan. In contrast, as many as 39 genes were identified as likely hits for the increased YP uptake, meaning that the respective proteins contributed to membrane stability or repair after nsEP. The expression level of eight genes in different types of human cells showed strong correlation (R \u3e 0.9, p \u3c 0.02) with their LD50 for lethal nsEP treatments, and could potentially be used as a criterion for the selectivity and efficiency of hyperplasia ablations with nsEP

Research Photo: High-Throughput Screening to Identify Proteins Involved in Nanopores Formation After Nspef

Research photo for “High-Throughput Screening To Identify Proteins Involved In Nanopores Formation After Nspef”https://digitalcommons.odu.edu/bioelectrics-2021retreat-images/1015/thumbnail.jp

The Alpha-1 Subunit of Membrane Na,K-ATPase Is Targeted by Nanosecond Electric Pulses

Electric pulses (EP) can modify the conformation of ion channels, potentially affecting membrane permeability. In our recent high-throughput screening of 328 gene knockouts (KOs) associated with membrane proteins in human monocytes U937, KO of Na,K-ATPase’s alpha 1 (ATP1A1) subunit was found to be less sensitive to nsEP (300ns, 7 kV/cm, at 20 Hz) - YoPro-1 (YP) uptake in ATP1A1 KO cells was up to 20% lower than control after 20 or 40 pulses. The subsequent experiments aimed to validate and further explore these findings. We measured YP uptake within 5 minutes after nsEP in ATP1A1 KO cells using time-lapse imaging on a scanning confocal microscope. YP fluorescence in ATP1A1 KO cells was 15% lower than in control cells, confirming the high-throughput screening results. We also used knock-down (KD) of ATP1A1 by transducing U937 cells with shRNA. In response to nsEP, YP uptake in ATP1A1 KD cells was reduced by 18% and 23% in high-throughput and confocal setups, respectively. Next, we tested whether the inhibition of Na,K-ATPase with ouabain affects YP uptake after nsEP. We found that the presence of ouabain did not reduce YP uptake after nsEP but increased it by 24%. Overall, our findings suggest that the role of ATP1A1 in YP uptake following nsEP exposure may be attributed to a structural feature of the protein rather than its physiological function as an ion pump

High-Throughput Screening to Identify Proteins Involved in Nanopores Formation After nsPEF

It is widely accepted that plasma membrane permeabilization after PEF occurs due to hydrophilic pores formation in the lipid bilayer. The concept of pore formation is confirmed by molecular dynamics (MD) simulations. However, there is increasing evidence that PEF can cause not only hydrophilic pore formation but also modulate membrane proteins and their function, and these changes could be part of increased membrane’s permeability after PEF. Ion channels, especially voltage-gated ion channels, are probable targets of PEF. However, ion channels are an extremely large group, and to identify specific channels responsible for increased conductivity after PEF, high-throughput screening is needed. In this work, we used the lentiviral CRISPR/Cas9 library designed for knockout (KO) of 329 different ion channels or other membrane-associated proteins in the U937/Cas9 cell model. After applying nsPEF (300ns, 7 kV/cm, 20 or 40 pulses at 20 Hz), each KO was screened for YoPro-1 uptake using a custom assembled screening station based on an inverted microscope equipped with automated stage repositioning, IR laser hardware autofocusing (ZDC), and image stitching capabilities. Out of 329 screened KOs, 9 showed lower YO-PRO-1 uptake than control. Such results indicate that proteins coded by these genes either support molecular transport across nsPEF stimulated membrane or proteins could be directly damaged by nsPEF. 20 KOs responded to nsPEF with higher YO-PRO-1 uptake than control, suggesting that these proteins may play the regulatory function of ion channels, or they could be linked to membrane stability and/or repair of the membrane after the electric injury. This is the first high-throughput screening to identify possible nsPEF protein targets in the cell plasma membrane