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

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.

Modulation of Voltage-Gating and Hysteresis of Lysenin Channels by Cu2+ Ions

“…The intrinsic regulatory mechanisms manifest by adjustments of the channel’s conformation and conductance in response to stimuli of physical and chemical origin [ 13 , 14 , 15 ]. Lysenin channels reconstituted in artificial membrane systems comprising anionic lipids present voltage-induced gating at low positive voltages [ 2 , 16 , 17 ], but this feature vanishes when the channels are reconstituted in neutral membranes [ 2 , 11 ]. When exposed to multivalent cations, lysenin channels present ligand-induced gating in both charged and neutral lipid membranes [ 11 , 13 , 15 ].…”

“…A salient feature of lysenin channels is the prominent hysteresis in conductance and bistability, indicative of molecular memory [ 16 , 17 , 18 , 19 ]. Hysteresis manifests when the response to a particular stimulation is not fixed and depends on the history of the system [ 20 ].…”

“…In the case of lysenin, the observed hysteresis is not dynamic, and seemingly originates in an invariant reopening pathway of the channels previously closed by applied positive voltages [ 16 , 18 ]. Notably, the reactivation pathway is also temperature-independent, while the inactivation pathway is strongly modulated by temperature variations [ 18 ].…”

“…K + ions and higher pH lead to a right shift in the voltage-induced gating, suggesting modulation of voltage regulation by electrostatic interactions [ 14 ]. The same monovalent cations adjust the hysteresis in conductance by shifting the voltage needed to close the channels to higher values, yet the reactivation pathway is rather invariant [ 16 ]. Multivalent ions present a more intricate interaction with lysenin channels, dominated by the resulting ligand-induced gating [ 11 , 13 , 15 , 31 ].…”

“…In the same line of intricacy, voluminous organic cations (spermidine, spermine) present an inhibition profile resembling divalent metal cations [ 13 , 15 ], suggesting that charge density as opposed to charge alone influences the pathway adopted for conformational changes (i.e., full closing, as opposed to sub-conducting). La 3+ ions, at concentrations sufficiently small to render ligand-induced gating negligible, influence hysteresis similarly to monovalent cations: a right shift of the voltage gating and open probability during ascending voltage ramps, and a rather invariant reopening pathway during descending voltage ramps [ 16 ]. The interaction with anionic ATP adds another level of intricacy: the conductance is inhibited in a concentration-dependent manner, yet voltage-gating and open probability show a strong right shift for both ascending and descending voltage ramps [ 16 , 31 ].…”