Modulation of Voltage-Gating and Hysteresis of Lysenin Channels by Cu\u3csup\u3e2+\u3c/sup\u3e Ions

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 of small concentrations of Cu2+ ions. Our experimental results showed that the hysteretic behavior, recorded in response to variable voltage ramps, is accentuated in the presence of Cu2+ ions. Using simultaneous AC/DC stimulation, we were able to determine that Cu2+ prevents the reopening of channels previously closed by depolarizing potentials and the channels remain in the closed state even in the absence of a transmembrane voltage. In addition, we showed that Cu2+ addition reinstates the voltage gating and hysteretic behavior of lysenin channels reconstituted in neutral lipid membranes in which lysenin channels lose their voltage-regulating properties. In the presence of Cu2+ ions, lysenin not only regained the voltage gating but also behaved like a long-term molecular memory controlled by electrical potentials

Lysenin Channels as Sensors for Ions and Molecules

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

Modulation of Lysenin’s Memory by Cu\u3csup\u3e2+\u3c/sup\u3e Ions

Lysenin is a pore-forming protein extracted from the red earthworm E. fetida, which forms voltage-gated channels in artificial and natural lipid membranes. A prominent feature of the channels is their memory, originating in the conductance hysteresis that occurs during the application of slow oscillatory voltages. In this work, we showed this innate memory was strongly influenced by the addition of small amounts of Cu2+ ions. After Cu2+ addition, the lysenin channels previously closed by an applied voltage showed a stronger preference for the closed state, indicative of major changes in kinetics and equilibrium. However, the physiology behind this shift is still obscure. To fill this gap in our knowledge, we employed electrophysiology measurements to identify the changes in the closing and opening rates of lysenin channels exposed to Cu2+ ions and step voltages. We found Cu2+ simultaneously reduced the closing rates and increased the reopening rates, leading to a more prominent hysteretic behavior and improved memory. These findings may constitute the starting point on investigations of the memory of brainless microorganisms, and potential applications to bioelectronics and development of smart biological switches and nano-valves

Mechanical Stress Modulates the Ionic Conductance of Bilayer Lipid Membranes

The modulation of the transmembrane voltage of receptor cells using mechanical stimuli is an essential component of touch and hearing senses. Such stimuli influence the conducting state of mechano-sensitive channels, which in turn adjusts the ionic permeation and consequently the transmembrane voltage. The necessity of ion channels in these transduction processes seems obvious due to the non-conductive nature of a lipid membrane. However, our electrophysiology experiments show that a bare, artificial lipid membrane exposed to mechanical stress allows the passage of inorganic ions. We concluded that lipid membranes may constitute an important component of the transduction mechanism under mechanical stimuli

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

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 of small concentrations of Cu2+ ions. Our experimental results showed that the hysteretic behavior, recorded in response to variable voltage ramps, is accentuated in the presence of Cu2+ ions. Using simultaneous AC/DC stimulation, we were able to determine that Cu2+ prevents the reopening of channels previously closed by depolarizing potentials and the channels remain in the closed state even in the absence of a transmembrane voltage. In addition, we showed that Cu2+ addition reinstates the voltage gating and hysteretic behavior of lysenin channels reconstituted in neutral lipid membranes in which lysenin channels lose their voltage-regulating properties. In the presence of Cu2+ ions, lysenin not only regained the voltage gating but also behaved like a long-term molecular memory controlled by electrical potentials

The Ionic Selectivity of Lysenin Channels in Open and Sub-Conducting States

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 lysenin channels are ion-selective and the determined permeability coefficients are cation and anion-species dependent. We also exploited the unique property of lysenin channels to transition to a stable sub-conducting state upon exposure to calcium ions and assessed their subsequent change in ionic selectivity. The observed loss of selectivity was implemented in an electrical model describing the dependency of reversal potentials on calcium concentration. In conclusion, our work demonstrates that this pore-forming toxin presents ionic selectivity but this is adjusted by the particular conduction state of the channels

Experimental Investigations on the Conductance of Lipid Membranes Under Differential Hydrostatic Pressure

The unassisted transport of inorganic ions through lipid membranes has become increasingly relevant to an expansive range of biological phenomena. Recent simulations indicate a strong influence of a lipid membrane’s curvature on its permeability, which may be part of the overall cell sensitivity to mechanical stimulation. However, most ionic permeability experiments employ a flat, uncurved lipid membrane, which disregards the physiological relevance of curvature on such investigations. To fill this gap in our knowledge, we adapted a traditional experimental system consisting of a planar lipid membrane, which we exposed to a controlled, differential hydrostatic pressure. Our electrophysiology experiments indicate a strong correlation between the changes in membrane geometry elicited by the application of pressure, as inferred from capacitance measurements, and the resulting conductance. Our experiments also confirmed the well-established influence of cholesterol addition to lipid membranes in adjusting their mechanical properties and overall permeability. Therefore, the proposed experimental system may prove useful for a better understanding of the intricate connections between membrane mechanics and adjustments of cellular functionalities upon mechanical stimulation, as well as for confirmation of predictions made by simulations and theoretical modeling