Cationic Polymers Inhibit the Conductance of Lysenin Channels

Fologea, Daniel; Krueger, Eric; Rossland, Steve; Bryant, Sheenah; Foss, Wylie; Clark, Tyler

doi:10.1155/2013/316758

Cited by 8 publications

(18 citation statements)

References 46 publications

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“…Several remarkable features make lysenin an excellent candidate for such studies. Lysenin's cytolytic and hemolytic activity has been extensively studied [45,46], and its capability to tamper with the barrier function of artificial lipid bilayers is well-documented [42,43,47,48]. The complete structure of the oligomeric pore inserted into membranes is not yet resolved; however, relatively recent structural data of lysenin interacting with SM in a pre-pore state indicates the existence of a positively charged domain [49] which may promote specific electrostatic interactions with negatively charged adenosine phosphates, similar to the ionotropic P2X receptors [19,50,51].…”

Section: Introductionmentioning

confidence: 99%

Purinergic control of lysenin’s transport and voltage-gating properties

Bryant

Shrestha

Carnig

et al. 2016

Purinergic Signalling

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Lysenin, a pore-forming protein extracted from the coelomic fluid of the earthworm Eisenia foetida, manifests cytolytic activity by inserting large conductance pores in host membranes containing sphingomyelin. In the present study, we found that adenosine phosphates control the biological activity of lysenin channels inserted into planar lipid membranes with respect to their macroscopic conductance and voltage-induced gating. Addition of ATP, ADP, or AMP decreased the macroscopic conductance of lysenin channels in a concentration-dependent manner, with ATP being the most potent inhibitor and AMP the least. ATP removal from the bulk solutions by buffer exchange quickly reinstated the macroscopic conductance and demonstrated reversibility. Singlechannel experiments pointed to an inhibition mechanism that most probably relies on electrostatic binding and partial occlusion of the channel-conducting pathway, rather than ligand gating induced by the highly charged phosphates. The Hill analysis of the changes in macroscopic conduction as a function of the inhibitor concentration suggested cooperative binding as descriptive of the inhibition process. Ionic screening significantly reduced the ATP inhibitory efficacy, in support of the electrostatic binding hypothesis. In addition to conductance modulation, purinergic control over the biological activity of lysenin channels has also been observed to manifest as changes of the voltage-induced gating profile. Our analysis strongly suggests that not only the inhibitor's charge but also its ability to adopt a folded conformation may explain the differences in the observed influence of ATP, ADP, and AMP on lysenin's biological activity.

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Section: Introductionmentioning

confidence: 99%

Purinergic control of lysenin’s transport and voltage-gating properties

Bryant

Shrestha

Carnig

et al. 2016

Purinergic Signalling

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“…EtBr-HD is a relatively long molecule that bears four positive charges that are provided by the included amine groups. These positive charges may induce reversible or irreversible conductance changes by either gating [40] or a gating and trapping mechanism [41].…”

Section: Investigations Of the Effect Of Indicators And Culture Mediamentioning

confidence: 99%

“…This observation was supported by a further increase of the PI concentration in the bulk (i.e., 4.5 µM and 7.5 µM), which also elicited insignificant changes of the macroscopic currents and confirmed PI as a suitable candidate for lysenin-induced permeability experiments. In the same line of preliminary explorations, we asked whether the PI in the bulk solutions might prevent channel blockage that is elicited in the presence of chitosan [41], intended to be added as an irreversible channel blocker. Our results ( Figure 3B) show that chitosan addition (25 nM final concentration) to the HBSS bulk in the presence of 6 µM PI annihilated the macroscopic conductance of lysenin channels inserted into the planar lipid membrane.…”

Section: Investigations Of the Effect Of Indicators And Culture Mediamentioning

confidence: 99%

“…In most cases, multivalent ion removal by chelation or precipitation reinstates the original macroscopic conductance of lysenin channels reconstituted in artificial membrane systems [39,40]. In contrast, cationic polymers, such as polyethylenimine or chitosan, irreversibly block the channels and obliterate the membrane's macroscopic conductance through a mechanism that might employ induced-gating and polymer trapping into the channel's lumen [41].…”

Section: Introductionmentioning

confidence: 99%

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Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin

Shrestha

Thomas

Richtsmeier

et al. 2020

Toxins

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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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“…However, many of those may prove toxic for living cells, thus restricting their applicability [11]. To avoid such limitations, we used another unique and remarkable feature of lysenin channels, namely irreversible blockage by chitosan [120]. Chitosan is a highly charged linear polysaccharide consisting of N-acetylglucosamine and glucosamine with β-1, 4-linkages.…”

Section: Lysenin As a Prototype Channel For Sensing And Controlled Pementioning

confidence: 99%

Lysenin Channels as Single Molecule Nano-Sensors and Nano-Switches for Controlled Membrane Permeability

Shrestha¹

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The completion of this dissertation would not have been possible without support from many individuals. Foremost, I would like to express immense gratitude to my amazing advisor Dr. Daniel Fologea, who not only encouraged and supported me, but also provided me with all the freedom and flexibility to carry out my research projects. His enthusiasm, intelligence, passion, contagious energy, and cheerful personality have always been my motivational force and have made all of these years a pleasant journey. My ambition of getting a doctoral degree would not have been possible if he hadn't welcomed me in his lab, believed in me and my ability to succeed. I am indebted to him forever for all the tremendous support he bestowed on me during my Ph.D. endeavor. I would also like to extend my heartfelt gratitude to my other advisor, Dr. Juliette Tinker, for her invaluable advice, dedication, and friendly nature. Her support and motivation have always provided me with confidence to move forward with my graduate degree. I would also like to thank Dr. Denise Wingett for her expert advice, support, encouragement, and her warm welcoming personality. I would like to deeply acknowledge Dr. Xinzhu Pu for his meticulous training with mass spectroscopy techniques and all his guidance and support during my Ph.D. training. My graduate journey would have been a turbulent voyage without the financial support from the Biomolecular Sciences Graduate Program and the Biomolecular Research Center (BRC) at Boise State University. My sincere thanks to the BRC for two years of fellowships and all of the generous grants, training, and support, without which vi the completion of my research would not have been possible. I would like to thank Raquel Brown for training me in confocal microscopy, and for all of her warmness and supportive nature. I am also grateful to Dr. Julie Oxford for supporting me in my graduate endeavor. My deepest and sincere thanks to Dr. Rebecca Hermann for her help with my Jurkat cells experiments and for training me in flow cytometry. My time at Boise State University would not have been enjoyable without my extremely amusing and incredibly smart colleagues Sheenah Bryant,

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Cationic Polymers Inhibit the Conductance of Lysenin Channels

Cited by 8 publications

References 46 publications

Purinergic control of lysenin’s transport and voltage-gating properties

Purinergic control of lysenin’s transport and voltage-gating properties

Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin

Lysenin Channels as Single Molecule Nano-Sensors and Nano-Switches for Controlled Membrane Permeability

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