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

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

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