ZnO nanoparticles modulate the ionic transport and voltage regulation of lysenin nanochannels.

BACKGROUND: The insufficient understanding of unintended biological impacts from nanomaterials (NMs) represents a serious impediment to their use for scientific, technological, and medical applications. While previous studies have focused on understanding nanotoxicity effects mostly resulting from cellular internalization, recent work indicates that NMs may interfere with transmembrane transport mechanisms, hence enabling contributions to nanotoxicity by affecting key biological activities dependent on transmembrane transport. In this line of inquiry, we investigated the effects of charged nanoparticles (NPs) on the transport properties of lysenin, a pore-forming toxin that shares fundamental features with ion channels such as regulation and high transport rate. RESULTS: The macroscopic conductance of lysenin channels greatly diminished in the presence of cationic ZnO NPs. The inhibitory effects were asymmetrical relative to the direction of the electric field and addition site, suggesting electrostatic interactions between ZnO NPs and a binding site. Similar changes in the macroscopic conductance were observed when lysenin channels were reconstituted in neutral lipid membranes, implicating protein-NP interactions as the major contributor to the reduced transport capabilities. In contrast, no inhibitory effects were observed in the presence of anionic SnO2 NPs. Additionally, we demonstrate that inhibition of ion transport is not due to the dissolution of ZnO NPs and subsequent interactions of zinc ions with lysenin channels. CONCLUSION: We conclude that electrostatic interactions between positively charged ZnO NPs and negative charges within the lysenin channels are responsible for the inhibitory effects on the transport of ions. These interactions point to a potential mechanism of cytotoxicity, which may not require NP internalization.

Stochastic sensing of Angiotensin II with lysenin channels.

The ability of pore-forming proteins to interact with various analytes has found vast applicability in single molecule sensing and characterization. In spite of their abundance in organisms from all kingdoms of life, only a few pore-forming proteins have been successfully reconstituted in artificial membrane systems for sensing purposes. Lysenin, a pore-forming toxin extracted from the earthworm E. fetida, inserts large conductance nanopores in lipid membranes containing sphingomyelin. Here we show that single lysenin channels may function as stochastic nanosensors by allowing the short cationic peptide angiotensin II to be electrophoretically driven through the conducting pathway. Long-term translocation experiments performed using large populations of lysenin channels allowed unequivocal identification of the unmodified analyte by Liquid Chromatography-Mass Spectrometry. However, application of reverse voltages or irreversible blockage of the macroscopic conductance of lysenin channels by chitosan addition prevented analyte translocation. This investigation demonstrates that lysenin channels have the potential to function as nano-sensing devices capable of single peptide molecule identification and characterization, which may be further extended to other macromolecular analytes.