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.

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.

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.