Potential analytical applications of lysenin channels for detection of multivalent ions

Abstract

Transmembrane protein transporters possessing binding sites for ions, toxins, pharmaceutical drugs, and other molecules constitute excellent candidates for developing sensitive and selective biosensing devices. Their attractiveness for analytical purposes is enhanced by the intrinsic amplification capabilities shown when the binding event leads to major changes in the transportation of ions or molecules other than the analyte itself. The large-scale implementation of such transmembrane proteins in biosensing devices is limited by the difficulties encountered in inserting functional transporters into artificial bilayer lipid membranes and by the limitations in understanding and exploiting the changes induced by the interaction with the analyte for sensing purposes. Here, we show that lysenin, a pore-forming toxin extracted from earthworm Eisenia foetida, which inserts stable and large conductance channels into artificial bilayer lipid membranes, functions as a multivalent ion-sensing device. The analytical response consists of concentration and ionic-species-dependent macroscopic conductance inhibition most probably linked to a ligand-induced gating mechanism. Multivalent ion removal by chelation or precipitation restores, in most cases, the initial conductance and demonstrates reversibility. Changes in lipid bilayer membrane compositions leading to the absence of voltage-induced gating do not affect the analytical response to multivalent ions. Microscopic current analysis performed on individual lysenin channels in the presence of Cu(2+) revealed complex open-closed transitions characterized by unstable intermediate sub-conducting states. Lysenin channels provide an analytical tool with a built-in sensing mechanism for inorganic and organic multivalent ions, and the excellent stability in an artificial environment recommend lysenin as a potential candidate for single-molecule detection and analysis.

Intramembrane congestion effects on lysenin channel voltage-induced gating

“…S1) indicative of individual channel insertions (Ide et al 2006). The stepwise open current distribution for four inserted channels yielded a unitary conductance of 0.391 ± 0.025 nS/channel, consistent with previous reports (Fologea et al 2011a; Ide et al 2006; Kwiatkowska et al 2007). The I–V curves recorded in response to voltage ramps ranging from −100 to 100 mV at 0.2 mV/s demonstrated the linear behavior in the negative voltage region (hence absence of conformational changes and a constant conductance in the negative voltage range), followed by voltage induced gating at positive potentials (Fologea et al 2011b; Ide et al 2006) (Electronic Supplementary Material Fig.…”

Purinergic control of lysenin’s transport and voltage-gating properties

“…3). Unlike what has been observed for multivalent cations, i.e., gating indicated by a fast step-wise decrease of the ionic current [52,53], ATP addition yielded a slow and monotonic variation. This suggests that ligand-induced gating is not the mechanism responsible for the observed conductance inhibition.…”

“…Lysenin channels have been shown to reversibly gate in a voltage-independent manner by specifically interacting with multivalent metal and organic cations [52,53]. This ligand-induced gating is identified in single-channel experiments as sudden single-step variations of the ionic current, indicative of fast conformational changes in response to multivalent cations [52,53]. We therefore asked whether or not highly charged anions such as ATP might trigger a similar gating mechanism after interacting with lysenin channels.…”

“…The complete structure of the oligomeric pore inserted into membranes is not yet resolved; however, relatively recent structural data of lysenin interacting with SM in a pre-pore state indicates the existence of a positively charged domain [49] which may promote specific electrostatic interactions with negatively charged adenosine phosphates, similar to the ionotropic P2X receptors [19,50,51]. In addition, unlike many other PFTs, lysenin is endowed with regulatory mechanisms by voltage and ligands [41][42][43][52][53][54], which are in fact remarkable features of ion channels. Lysenin channels present voltage regulation manifested as voltage-induced gating at positive transmembrane voltages greater than ∼20 mV [41,54].…”

Cationic Polymers Inhibit the Conductance of Lysenin Channels

“…The biological function of lysenin has not yet been uncovered, but its cytolytic and hemolytic activities are indicative of a PFT [33]. The channel's properties and behaviors have been recently characterized [31, 34–37], and the investigations revealed considerable similarities with ion channels and functionalities uncommon among PFTs: asymmetrical voltage-induced gating [31, 35], reversible conductance inhibition induced by interactions with multivalent cations [34, 36], a strong dependence of the voltage-induced gating on temperature [37], and an unusual hysteresis of the open-close transition [37]. The exact mechanism of this particular type of conductance modulation by multivalent ions is unknown.…”