Abstract
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.
Highlights
Lysenin, a pore forming toxin (PFT) found in the coelomic fluid of the red earthworm E. fetida, induces cytolysis and hemolysis of cells that contain sphingomyelin in their plasmalemma [1,2,3,4,5].Electrophysiology [2,6,7] and atomic force microscopy [8,9,10] investigations of lysenin inserted into artificial membrane systems have shown that this lytic activity stems from self-insertion of large conducting pores in the target membrane
Since both ionic strength and pH strongly modulate the voltage-induced gating of lysenin channels [6], it is natural to assume that the charged voltage domain sensor is exposed to the bulk ionic solution at rest, and screened
The macroscopic ionic currents through a large population of lysenin channels, which were inserted into the bilayer membrane and exposed to increasing monovalent ion concentrations, underwent visible changes with regards to their magnitude and voltage required to initiate the open-close transitions during ascending voltage ramps, as our group reported in a previous study [6]
Summary
A pore forming toxin (PFT) found in the coelomic fluid of the red earthworm E. fetida, induces cytolysis and hemolysis of cells that contain sphingomyelin in their plasmalemma [1,2,3,4,5]. Temperature has negligible influence on channel reactivation (descending voltage ramps) and the open probability (Popen ) is invariant This stable reactivation pathway leads to a static hysteresis [17], which could be better understood by gaining more insights into the gating mechanism. Structural data reveals the presence of hinge-like, flexible structures essential for pore formation [19,20,21] which may allow the elusive voltage domain sensor to move Since both ionic strength and pH strongly modulate the voltage-induced gating of lysenin channels [6], it is natural to assume that the charged voltage domain sensor is exposed to the bulk ionic solution at rest (the state in which all the channels are open at zero transmembrane potential), and screened. Investigations conducted by employing ATP, which modulate the macroscopic conductance by binding to the channel lumen and partially occluding the conducting pathway [13], show a quantitatively and qualitatively different influence on voltage-induced gating, in support of the hypothesized gating mechanism
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