Physical Sciences and Mathematics Commons^™

Modulation Of Voltage-Gating And Hysteresis Of Lysenin Channels By Cu2+ Ions, Andrew Bogard, Pangaea W. Finn, Aviana R. Smith, Ilinca M. Flacau, Rose Whiting, Daniel Fologea

Physics Faculty Publications and Presentations

The intricate voltage regulation presented by lysenin channels reconstituted in artificial lipid membranes leads to a strong hysteresis in conductance, bistability, and memory. Prior investigations on lysenin channels indicate that the hysteresis is modulated by multivalent cations which are also capable of eliciting single-step conformational changes and transitions to stable closed or sub-conducting states. However, the influence on voltage regulation of Cu²⁺ ions, capable of completely closing the lysenin channels in a two-step process, was not sufficiently addressed. In this respect, we employed electrophysiology approaches to investigate the response of lysenin channels to variable voltage stimuli in the presence …

The Ionic Selectivity Of Lysenin Channels In Open And Sub-Conducting States, Andrew Bogard, Pangaea W. Finn, Fulton Mckinney, Ilinca M. Flacau, Aviana R. Smith, Rosey Whiting, Daniel Fologea

Physics Faculty Publications and Presentations

The electrochemical gradients established across cell membranes are paramount for the execution of biological functions. Besides ion channels, other transporters, such as exogenous pore-forming toxins, may present ionic selectivity upon reconstitution in natural and artificial lipid membranes and contribute to the electrochemical gradients. In this context, we utilized electrophysiology approaches to assess the ionic selectivity of the pore-forming toxin lysenin reconstituted in planar bilayer lipid membranes. The membrane voltages were determined from the reversal potentials recorded upon channel exposure to asymmetrical ionic conditions, and the permeability ratios were calculated from the fit with the Goldman–Hodgkin–Katz equation. Our work shows that …

Rapid Production And Purification Of Dye-Loaded Liposomes By Electrodialysis-Driven Depletion, Gamid Abatchev, Andrew Bogard, Zoe Hutchinson, Jason Ward, Daniel Fologea

Physics Faculty Publications and Presentations

Liposomes are spherical-shaped vesicles that enclose an aqueous milieu surrounded by bilayer or multilayer membranes formed by self-assembly of lipid molecules. They are intensively exploited as either model membranes for fundamental studies or as vehicles for delivery of active substances in vivo and in vitro. Irrespective of the method adopted for production of loaded liposomes, obtaining the final purified product is often achieved by employing multiple, time consuming steps. To alleviate this problem, we propose a simplified approach for concomitant production and purification of loaded liposomes by exploiting the Electrodialysis-Driven Depletion of charged molecules from solutions. Our investigations show that …

Liposomes Prevent In Vitro Hemolysis Induced By Streptolysin O And Lysenin, Marcelo Ayllon, Gamid Abatchev, Andrew Bogard, Rosey Whiting, Sarah E. Hobdey, Daniel Fologea

Physics Faculty Publications and Presentations

The need for alternatives to antibiotics in the fight against infectious diseases has inspired scientists to focus on antivirulence factors instead of the microorganisms themselves. In this respect, prior work indicates that tiny, enclosed bilayer lipid membranes (liposomes) have the potential to compete with cellular targets for toxin binding, hence preventing their biological attack and aiding with their clearance. The effectiveness of liposomes as decoy targets depends on their availability in the host and how rapidly they are cleared from the circulation. Although liposome PEGylation may improve their circulation time, little is known about how such a modification influences their …

Lysenin Channels As Sensors For Ions And Molecules, Andrew Bogard, Gamid Abatchev, Zoe Hutchinson, Jason Ward, Pangaea W. Finn, Fulton Mckinney, Daniel Fologea

Physics Faculty Publications and Presentations

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 …

Temporary Membrane Permeabilization Via The Pore-Forming Toxin Lysenin, Nisha Shrestha, Christopher A. Thomas, Devon Richtsmeier, Andrew Bogard, Rebecca Hermann, Malyk Walker, Gamid Abatchev, Raquel J. Brown, Daniel Fologea

Physics Faculty Publications and Presentations

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 …

Insights Into The Voltage Regulation Mechanism Of The Pore-Forming Toxin Lysenin, Sheenah Lynn Bryant, Tyler Clark, Christopher Alex Thomas, Kaitlyn Summer Ware, Andrew Bogard, Colleen Calzacorta, Daniel Prather, Daniel Fologea

Physics Faculty Publications and Presentations

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 …

Zno Nanoparticles Modulate The Ionic Transport And Voltage Regulation Of Lysenin Nanochannels, Sheenah L. Bryant, Josh E. Eixenberger, Steven Rossland, Holly Apsley, Connor Hoffman, Nisha Shrestha, Michael Mchugh, Alex Punnoose, Daniel Fologea

Physics Faculty Publications and Presentations

Background: The insufficient understanding of unintended biological impacts from nanomaterials (NMs) represents a serious impediment to their use for scientific, technological, and medical applications. While previous studies have focused on understanding nanotoxicity effects mostly resulting from cellular internalization, recent work indicates that NMs may interfere with transmembrane transport mechanisms, hence enabling contributions to nanotoxicity by affecting key biological activities dependent on transmembrane transport. In this line of inquiry, we investigated the effects of charged nanoparticles (NPs) on the transport properties of lysenin, a pore-forming toxin that shares fundamental features with ion channels such as regulation and high transport rate.

Results: …

Intramembrane Congestion Effects On Lysenin Channel Voltage-Induced Gating, Eric Krueger, Sheenah Bryant, Nisha Shrestha, Tyler Clark, Charles Hanna, David Pink, Daniel Fologea

Physics Faculty Publications and Presentations

All cell membranes are packed with proteins. The ability to investigate the regulatory mechanisms of protein channels in experimental conditions mimicking their congested native environment is crucial for understanding the environmental physicochemical cues that may fundamentally contribute to their functionality in natural membranes. Here we report on investigations of the voltage-induced gating of lysenin channels in congested conditions experimentally achieved by increasing the number of channels inserted into planar lipid membranes. Typical electrophysiology measurements reveal congestion-induced changes to the voltage-induced gating, manifested as a significant reduction of the response to external voltage stimuli. Furthermore, we demonstrate a similar diminished voltage …

A Model For The Hysteresis Observed In Gating Of Lysenin Channels, Eric Krueger, Radwan Al Faouri, Daniel Fologea, Ralph Henry, David Straub, Greg J. Salamo

Physics Faculty Publications and Presentations

The pore-forming toxin lysenin self-inserts to form conductance channels in natural and artificial lipid membranes containing sphingomyelin. The inserted channels exhibit voltage regulation and hysteresis of the macroscopic current during the application of positive periodic voltage stimuli. We explored the bi-stable behavior of lysenin channels and present a theoretical approach for the mechanism of the hysteresis to explain its static and dynamic components. This investigation develops a model to incorporate the role of charge accumulation on the bilayer lipid membrane in influencing the channel conduction state. Our model is supported by experimental results and also provides insight into the temperature …

Bi-Stability, Hysteresis, And Memory Of Voltage-Gated Lysenin Channels, Daniel Fologea, Eric Krueger, Yuriy I. Mazur, Christine Stith, Yui Okuyama, Ralph Henry, Greg J. Salamo

Physics Faculty Publications and Presentations

Lysenin, a 297 amino acid pore-forming protein extracted from the coelomic fluid of the earthworm E. foetida, inserts constitutively open large conductance channels in natural and artificial lipid membranes containing sphingomyelin. The inserted channels show voltage regulation and slowly close at positive applied voltages. We report on the consequences of slow voltage-induced gating of lysenin channels inserted into a planar Bilayer Lipid Membrane (BLM), and demonstrate that these pore-forming proteins constitute memory elements that manifest gating bi-stability in response to variable external voltages. The hysteresis in macroscopic currents dynamically changes when the time scale of the voltage variation is …