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.