Том 11, № 3 (2017)

Extracellular vesicles (EVs) are bilayer membrane fragments that are released by different cell types upon activation or death. The most well studied EVs are those of blood plasma. Two types of EVs are usually distinguished: exosomes (formed by the membranes of intracellular compartments, 50–100 nm in diameter) and ectosomes (also called microparticles or microvesicles, formed from plasma membrane, 100–1000 nm in diameter). The real picture is much more complicated and is still poorly understood. EVs are enriched by various proteins, mRNA and miRNA, and the EV lipid and protein composition can substantially differ from that of the parental cells, from which EV originate. The blood concentration of EVs greatly increases in many diseases and conditions. EVs have a wide spectrum of biological activities, from pro-coagulant to immunomodulating ones. This activity can be physiologically important and is believed to be absolutely important pathophysiologically. In recent studies, EVs are considered to be important not only as objects of basic research, but also as potential biomarkers, drug candidates, drug carriers, or therapeutic targets.

Theoretical model of a through pore formation in lipid bilayer membrane under applied lateral tension was developed. In the framework of elastic theory of liquid crystals adapted to lipid membranes, we calculated a continuous trajectory from intact bilayer through a hydrophobic defect to a through pore. It was shown that the major energetic characteristic of membrane stability with respect to the pore formation, i. e., line tension, depends both on the pore radius and on the value of the applied lateral tension. This leads to a non-monotonous dependence of the average waiting time of the pore formation on the lateral tension: at low tensions the waiting time was large, then there was a local minimum, after which the average waiting time was increasing again. For membranes formed from stearoyl oleoyl phosphatidylcholine, the local minimum corresponded to the lateral tension of 7 mN/m; the calculated value of the edge line tension of a large pore was 16.5 pN. These results are consistent with available experimental data.

Investigation of the transport phenomena in the nanoscopic channels/pores with the diameter smaller than 100 nm is of utmost importance for various biological, medical, and technical applications. Presently, the main line of development of nanofluidics is creation of biosensors capable of detecting single molecules and manipulating them. Detection of molecules is based on the measurement of electric current through a channel of appropriate size: when the molecule enters the channel, which diameter is comparable with the molecule size, the ion current reduces. In order to improve transport properties of such channels, their walls are often coated with a lipid bilayer, which behaves as two-dimensional liquid and thus is capable of supporting transport phenomena. In the present work, we utilized this property of lipid membranes for the development of a method for detecting and controlling transport of single-stranded DNA through channels formed by membrane cylinders with the luminal radii of 5–7 nm. We have demonstrated that in the conditions of small ion strength, the appearance of a DNA molecule inside such channel is accompanied by an increase of its ion conductivity and can be controlled by the polarity of the applied voltage. The amplitude of the ion current increase allows evaluating the amount of DNA molecules inside the channels. It was also demonstrated that upon adsorption of DNA molecules on the lipid bilayer surface, the membrane cylinder behaves as a voltage-sensitive selective ion channel.

The effect of the most hydrophobic bile acid–lithocholic–as an inducer of two different Ca²⁺-dependent inner membrane permeability systems was studied on isolated rat liver mitochondria. It is shown that the addition of lithocholic acid at a concentration of 20 μM to the Ca²⁺-loaded mitochondria leads to swelling of the organelles, rapid release of Ca²⁺ from the matrix and almost complete collapse of Δψ. Mitochondrial pore blocker cyclosporin A (CsA) eliminates mitochondrial swelling but has no effect on the process of Ca²⁺ release and Δψ collapse. In the absence of Ca²⁺ lithocholic acid causes only a transient decrease of Δψ with subsequent complete recovery. Ruthenium red, inhibitor of mitochondrial Ca²⁺ uniporter, which blocks Ca²⁺ influx into the matrix, prevents mitochondrial swelling induced by lithocholic acid. At the same time, ruthenium red, which is added before lithocholic acid to the Ca²⁺-preloaded mitochondria, does not affect the swelling of the organelles but reduces the CsA-insensitive drop in Δψ. It is concluded that lithocholic acid is able to induce two Ca²⁺-dependent energy dissipation systems in the inner membrane of liver mitochondria: CsA-sensitive mitochondrial pore and CsA-insensitive permeability, which exhibits sensitivity to ruthenium red. It is found that the effect of this bile acid as an inductor of CsA-sensitive mitochondrial pore is not associated with the modulation of Pi effects. It is assumed that CsA-insensitive action of lithocholic acid is associated with the induction of Ca²⁺ efflux from the matrix in exchange for protons. In this case, the energy-dependent Ca²⁺ transport in the opposite direction with the participation of mitochondrial calcium uniporter sensitive to ruthenium red leads to the formation of calcium cycle and thereby to energy dissipation.

Here we show that positive modulators (CyPPA and NS309) of Ca²⁺-activated K⁺ channels of small (SK) and intermediate (IK) conductances in cerebellar neurons decrease glutamate-evoked Ca²⁺ entry into neurons independently on the presence of Mg²⁺ in extracellular media. An analysis of neuronal viability after long-term (240 min) glutamate treatments demonstrated neuroprotective action of CyPPA and NS309. Extracellular Mg²⁺ did not protect neurons from apoptosis during prolonged treatment with glutamate. Activation of SK and IK channels results in local membrane hyperpolarization, which enhances Mg²⁺ block of NMDA receptors and reduces activation of voltage-dependent Ca²⁺ channels, which can explain neuroprotection caused by CyPPA or NS309. The obtained results reveal an important role Ca²⁺-activated K⁺ channels of small and intermediate conductance in the regulation of Ca²⁺ entry into cerebellar neurons via NMDA receptors and voltage-gated Ca²⁺ channels.