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1. Lysenin Channels as Sensors for Ions and Molecules

   https://doi.org/10.3390/s20216099  

   Bogard, Andrew ; Abatchev, Gamid ; Hutchinson, Zoe ; Ward, Jason ; Finn, Pangaea W. ; McKinney, Fulton ; Fologea, Daniel ( November 2020 , Sensors)

   null (Ed.)

   Lysenin is a pore-forming protein extracted from the earthworm Eisenia fetida, which inserts large conductance pores in artificial and natural lipid membranes containing sphingomyelin. Its cytolytic and hemolytic activity is rather indicative of a pore-forming toxin; however, lysenin channels present intricate regulatory features manifested as a reduction in conductance upon exposure to multivalent ions. Lysenin pores also present a large unobstructed channel, which enables the translocation of analytes, such as short DNA and peptide molecules, driven by electrochemical gradients. These important features of lysenin channels provide opportunities for using them as sensors for a large variety of applications. In this respect, this literature review is focused on investigations aimed at the potential use of lysenin channels as analytical tools. The described explorations include interactions with multivalent inorganic and organic cations, analyses on the reversibility of such interactions, insights into the regulation mechanisms of lysenin channels, interactions with purines, stochastic sensing of peptides and DNA molecules, and evidence of molecular translocation. Lysenin channels present themselves as versatile sensing platforms that exploit either intrinsic regulatory features or the changes in ionic currents elicited when molecules thread the conducting pathway, which may be further developed into analytical tools of high specificity and sensitivity or exploited for other scientific biotechnological applications. 

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2. Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin

   https://doi.org/10.3390/toxins12050343  

   Shrestha, Nisha ; Thomas, Christopher A. ; Richtsmeier, Devon ; Bogard, Andrew ; Hermann, Rebecca ; Walker, Malyk ; Abatchev, Gamid ; Brown, Raquel J. ; Fologea, Daniel ( May 2020 , Toxins)

   null (Ed.)

   Pore-forming toxins are alluring tools for delivering biologically-active, impermeable cargoes to intracellular environments by introducing large conductance pathways into cell membranes. However, the lack of regulation often leads to the dissipation of electrical and chemical gradients, which might significantly affect the viability of cells under scrutiny. To mitigate these problems, we explored the use of lysenin channels to reversibly control the barrier function of natural and artificial lipid membrane systems by controlling the lysenin’s transport properties. We employed artificial membranes and electrophysiology measurements in order to identify the influence of labels and media on the lysenin channel’s conductance. Two cell culture models: Jurkat cells in suspension and adherent ATDC5 cells were utilized to demonstrate that lysenin channels may provide temporary cytosol access to membrane non-permeant propidium iodide and phalloidin. Permeability and cell viability were assessed by fluorescence spectroscopy and microscopy. Membrane resealing by chitosan or specific media addition proved to be an effective way of maintaining cellular viability. In addition, we loaded non-permeant dyes into liposomes via lysenin channels by controlling their conducting state with multivalent metal cations. The improved control over membrane permeability might prove fruitful for a large variety of biological or biomedical applications that require only temporary, non-destructive access to the inner environment enclosed by natural and artificial membranes. 

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3. Insights into the Voltage Regulation Mechanism of the Pore-Forming Toxin Lysenin

   https://doi.org/10.3390/toxins10080334  

   Bryant, Sheenah ; Clark, Tyler ; Thomas, Christopher ; Ware, Kaitlyn ; Bogard, Andrew ; Calzacorta, Colleen ; Prather, Daniel ; Fologea, Daniel ( August 2018 , Toxins)

   Lysenin, a pore forming toxin (PFT) extracted from Eisenia fetida, inserts voltage-regulated channels into artificial lipid membranes containing sphingomyelin. The voltage-induced gating leads to a strong static hysteresis in conductance, which endows lysenin with molecular memory capabilities. To explain this history-dependent behavior, we hypothesized a gating mechanism that implies the movement of a voltage domain sensor from an aqueous environment into the hydrophobic core of the membrane under the influence of an external electric field. In this work, we employed electrophysiology approaches to investigate the effects of ionic screening elicited by metal cations on the voltage-induced gating and hysteresis in conductance of lysenin channels exposed to oscillatory voltage stimuli. Our experimental data show that screening of the voltage sensor domain strongly affects the voltage regulation only during inactivation (channel closing). In contrast, channel reactivation (reopening) presents a more stable, almost invariant voltage dependency. Additionally, in the presence of anionic Adenosine 5′-triphosphate (ATP), which binds at a different site in the channel’s structure and occludes the conducting pathway, both inactivation and reactivation pathways are significantly affected. Therefore, the movement of the voltage domain sensor into a physically different environment that precludes electrostatically bound ions may be an integral part of the gating mechanism. 

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4. One-step fabrication of robust lithium ion battery separators by polymerization-induced phase separation

   https://doi.org/10.1039/D1TA10730E  

   Manly, Alexander J. ; Tenhaeff, Wyatt E. ( May 2022 , Journal of Materials Chemistry A)

   Conventional lithium ion battery separators are microporous polyolefin membranes that play a passive role in the electrochemical cell. Next generation separators should offer significant performance enhancements, while being fabricated through facile, low cost approaches with the ability to readily tune physicochemical properties. This study presents a single-step manufacturing technique based on UV-initiated polymerization-induced phase separation (PIPS), wherein microporous separators are fabricated from multifunctional monomers and ethylene carbonate (EC), which functions as both the pore-forming agent (porogen) and electrolyte component in the electrochemical cell. By controlling the ratio of the 1,4-butanediol diacrylate (BDDA) monomer to ethylene carbonate, monolithic microporous membranes are readily prepared with 25 μm thickness and pore sizes and porosities ranging from 6.8 to 22 nm and 15.4% to 38.5%, respectively. With 38.5% apparent porosity and an average pore size of 22 nm, the poly(1,4-butanediol diacrylate) (pBDDA) separator takes up 127% liquid electrolyte, resulting in an ionic conductivity of 1.98 mS cm −1 , which is greater than in conventional Celgard 2500. Lithium ion battery half cells consisting of LiNi 0.5 Mn 0.3 Co 0.2 O 2 cathodes and pBDDA separators were shown to undergo reversible charge/discharge cycling with an average discharge capacity of 142 mA h g −1 and a capacity retention of 98.4% over 100 cycles – comparable to cells using state-of-the-art separators. Moreover, similar discharge capacities were achieved in rate performance tests due to the high ionic conductivity and electrolyte uptake of the film. The pBDDA separators were shown to be thermally stable to 374 °C, lack low temperature thermal transitions that can compromise cell safety, and exhibit no thermal shrinkage up to 150 °C. 

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5. Structure and function of the calcium‐selective TRP channel TRPV6

   https://doi.org/10.1113/JP279024  

   Yelshanskaya, Maria V. ; Nadezhdin, Kirill D. ; Kurnikova, Maria G. ; Sobolevsky, Alexander I. ( March 2020 , The Journal of Physiology)

   Epithelial calcium channel TRPV6 is a member of the vanilloid subfamily of TRP channels that is permeable to cations and highly selective to Ca²⁺; it shows constitutive activity regulated negatively by Ca²⁺and positively by phosphoinositol and cholesterol lipids. In this review, we describe the molecular structure of TRPV6 and discuss how its structural elements define its unique functional properties. High Ca²⁺selectivity of TRPV6 originates from the narrow selectivity filter, where Ca²⁺ions are directly coordinated by a ring of anionic aspartate side chains. Divalent cations Ca²⁺and Ba²⁺permeate TRPV6 pore according to the knock‐off mechanism, while tight binding of Gd³⁺to the aspartate ring blocks the channel and prevents Na⁺from permeating the pore. The iris‐like channel opening is accompanied by an α‐to‐π helical transition in the pore‐lining transmembrane helix S6. As a result of this transition, the intracellular halves of the S6 helices bend and rotate by about 100 deg, exposing different residues to the channel pore in the open and closed states. Channel opening is also associated with changes in occupancy of the transmembrane domain lipid binding sites. The inhibitor 2‐aminoethoxydiphenyl borate (2‐APB) binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1‐S4 transmembrane helical bundle and shifts open‐closed channel equilibrium towards the closed state by outcompeting lipids critical for activation. Ca²⁺inhibits TRPV6 via binding to calmodulin (CaM), which mediates Ca²⁺‐dependent inactivation. The TRPV6‐CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head‐to‐tail arrangement. The CaM C‐terminal lobe plugs the channel through a unique cation‐π interaction by inserting the side chain of lysine K115 into a tetra‐tryptophan cage at the ion channel pore intracellular entrance. Recent studies of TRPV6 structure and function described in this review advance our understanding of the role of this channel in physiology and pathophysiology and inform new therapeutic design.image

    

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