Molecular mechanism of olesoxime-mediated neuroprotection through targeting α-synuclein interaction with mitochondrial VDAC.

An intrinsically disordered neuronal protein α-synuclein (αSyn) is known to cause mitochondrial dysfunction, contributing to loss of dopaminergic neurons in Parkinson's disease. Through yet poorly defined mechanisms, αSyn crosses mitochondrial outer membrane and targets respiratory complexes leading to bioenergetics defects. Here, using neuronally differentiated human cells overexpressing wild-type αSyn, we show that the major metabolite channel of the outer membrane, the voltage-dependent anion channel (VDAC), is a pathway for αSyn translocation into the mitochondria. Importantly, the neuroprotective cholesterol-like synthetic compound olesoxime inhibits this translocation. By applying complementary electrophysiological and biophysical approaches, we provide mechanistic insights into the interplay between αSyn, VDAC, and olesoxime. Our data suggest that olesoxime interacts with VDAC ß-barrel at the lipid-protein interface thus hindering αSyn translocation through the VDAC pore and affecting VDAC voltage gating. We propose that targeting αSyn translocation through VDAC could represent a key mechanism for the development of new neuroprotective strategies.

Lipid-mediated regulation of pore-forming activity of syringomycin E by thyroid hormones and xanthene dyes.

The effects of dipole modifiers, thyroid hormones (thyroxine and triiodothyronine) and xanthene dyes (Rose Bengal, phloxineB, erythrosin, eosinY and fluorescein) on the pore-forming activity of the lipopeptide syringomycin E (SRE) produced by Pseudomonas syringae were studied in a model bilayer. Thyroxine does not noticeably influence the steady-state number of open SRE channels (Nop), whereas triiodothyronine decreases it 10-fold at -50mV. Rose Bengal, phloxine B and erythrosin significantly increase Nop by 350, 100 and 70 times, respectively. Eosin Y and fluorescein do not practically affect the pore-forming activity of SRE. Recently, we showed that hormones decrease the dipole potential of lipid bilayers by approximately 60mV at 50µM, while Rose Bengal, phloxine B and erythrosin at 2.5µM reduce the membrane dipole potential by 120, 80 and 50mV, respectively. In the present study using differential scanning microcalorimetry, confocal fluorescence microscopy, the calcein release technique and measurements of membrane curvature elasticity, we show that triiodothyronine strongly affects the fluidity of model membranes: its addition leads to a significant decrease in the temperature and cooperativity of the main phase transition of DPPC, calcein leakage from DOPC vesicles, fluidization of solid domains in DOPC/DPPC liposomes, and promotion of lipid curvature stress. Thyroxine exerts a weaker effect. Xanthene dyes do not influence the phase transition of DPPC. Despite the decrease in the dipole potential, thyroid hormones modulate SRE channels predominantly via the elastic properties of the membrane, whereas the xanthene dyes Rose Bengal, phloxine B and erythrosine affect SRE channels via bilayer electrostatics.

Interaction of Quercetin, Cyanidin, and Their O-Glucosides with Planar Lipid Models: Implications for Their Biological Effects.

Flavonoids are specialized metabolites produced by plants, as free aglycones or as glycosylated derivatives, which are particularly endowed with a variety of beneficial health properties. The antioxidant, anti-inflammatory, antimicrobial, anticancer, antifungal, antiviral, anti-Alzheimer's, anti-obesity, antidiabetic, and antihypertensive effects of flavonoids are now known. These bioactive phytochemicals have been shown to act on different molecular targets in cells including the plasma membrane. Due to their polyhydroxylated structure, lipophilicity, and planar conformation, they can either bind at the bilayer interface or interact with the hydrophobic fatty acid tails of the membrane. The interaction of quercetin, cyanidin, and their O-glucosides with planar lipid membranes (PLMs) similar in composition to those of the intestine was monitored using an electrophysiological approach. The obtained results show that the tested flavonoids interact with PLM and form conductive units. The modality of interaction with the lipids of the bilayer and the alteration of the biophysical parameters of PLMs induced by the tested substances provided information on their location in the membrane, helping to elucidate the mechanism of action which underlies some pharmacological properties of flavonoids. To our knowledge, the interaction of quercetin, cyanidin, and their O-glucosides with PLM surrogates of the intestinal membrane has never been previously monitored.

Electrophysiological Characterization of Microbial Rhodopsin Transport Properties: Electrometric and ΔpH Measurements Using Planar Lipid Bilayer, Collodion Film, and Fluorescent Probe Approaches.

Electrophysiological approaches to the study of the activity of retinal-containing protein bacteriorhodopsin (bR) or other proteins of this family are based usually on measurements of electrical current through a planar bilayer lipid membrane (BLM) with proteoliposomes attached to the BLM surface at one side of the membrane. Here, we describe the measurements of the pumping activity of bR and channelrhodopsin 2 (ChR2) with special attention to the study of voltage dependence of the light-induced currents. Strong voltage dependence of ChR2 suggests light-triggered ion channel activity of ChR2. We also describe electrophysiological measurements with the help of collodion film instead of BLM for the measurements of fast stages of a rhodopsin photocycle as well as the estimation of the activity of proteoliposomes without a macro membrane using fluorescent probes such as oxonol VI or 9-aminoacridine.

The Cardiac Ryanodine Receptor Provides a Suitable Pathway for the Rapid Transport of Zinc (Zn2+).

The sarcoplasmic reticulum (SR) in cardiac muscle is suggested to act as a dynamic storage for Zn2+ release and reuptake, albeit it is primarily implicated in the Ca2+ signaling required for the cardiac cycle. A large Ca2+ release from the SR is mediated by the cardiac ryanodine receptor (RYR2), and while this has a prominent conductance for Ca2+ in vivo, it also conducts other divalent cations in vitro. Since Zn2+ and permeant Mg2+ have similar physical properties, we tested if the RYR2 channel also conducts Zn2+. Using the method of planar lipid membranes, we evidenced that the RYR2 channel is permeable to Zn2+ with a considerable conductance of 81.1 ± 2.4 pS, which was significantly lower than the values for Ca2+ (127.5 ± 1.8 pS) and Mg2+ (95.3 ± 1.4 pS), obtained under the same asymmetric conditions. Despite similar physical properties, the intrinsic Zn2+ permeability (PCa/PZn = 2.65 ± 0.19) was found to be ~2.3-fold lower than that of Mg2+ (PCa/PMg = 1.146 ± 0.071). Further, we assessed whether the channel itself could be a direct target of the Zn2+ current, having the Zn2+ finger extended into the cytosolic vestibular portion of the permeation pathway. We attempted to displace Zn2+ from the RYR2 Zn2+ finger to induce its structural defects, which are associated with RYR2 dysfunction. Zn2+ chelators were added to the channel cytosolic side or strongly competing cadmium cations (Cd2+) were allowed to permeate the RYR2 channel. Only the Cd2+ current was able to cause the decay of channel activity, presumably as a result of Zn2+ to Cd2+ replacement. Our findings suggest that the RYR2 channel can provide a suitable pathway for rapid Zn2+ escape from the cardiac SR; thus, the channel may play a role in local and/or global Zn2+ signaling in cardiomyocytes.

Study of Resveratrol's Interaction with Planar Lipid Models: Insights into Its Location in Lipid Bilayers.

Resveratrol, a polyphenolic molecule found in edible fruits and vegetables, shows a wide range of beneficial effects on human health, including anti-microbial, anti-inflammatory, anti-cancer, and anti-aging properties. Due to its poor water solubility and high liposome-water partition coefficient, the biomembrane seems to be the main target of resveratrol, although the mode of interaction with membrane lipids and its location within the cell membrane are still unclear. In this study, using electrophysiological measurements, we study the interaction of resveratrol with planar lipid membranes (PLMs) of different composition. We found that resveratrol incorporates into palmitoyl-oleoyl-phosphatidylcholine (POPC) and POPC:Ch PLMs and forms conductive units unlike those found in dioleoyl-phosphatidylserine (DOPS):dioleoyl-phosphatidylethanolamine (DOPE) PLMs. The variation of the biophysical parameters of PLMs in the presence of resveratrol provides information on its location within a lipid double layer, thus contributing to an understanding of its mechanism of action.

Luminal addition of non-permeant Eu3+ interferes with luminal Ca2+ regulation of the cardiac ryanodine receptor.

Dysregulation of the cardiac ryanodine receptor (RYR2) by luminal Ca2+ has been implicated in a life-threatening, stress-induced arrhythmogenic disease. The mechanism of luminal Ca2+-mediated RYR2 regulation is under debate, and it has been attributed to Ca2+ binding on the cytosolic face (the Ca2+ feedthrough mechanism) and/or the luminal face of the RYR2 channel (the true luminal mechanism). The molecular nature and location of the luminal Ca2+ site is unclear. At the single-channel level, we directly probed the RYR2 luminal face by Eu3+, considering the non-permeant nature of trivalent cations and their high binding affinities for Ca2+ sites. Without affecting essential determinants of the Ca2+ feedthrough mechanism, we found that luminal Eu3+ competitively antagonized the activation effect of luminal Ca2+ on RYR2 responsiveness to cytosolic caffeine, and no appreciable effect was observed for luminal Ba2+ (mimicking the absence of luminal Ca2+). Importantly, luminal Eu3+ caused no changes in RYR2 gating. Our results indicate that two distinct Ca2+ sites (available for luminal Ca2+ even when the channel is closed) are likely involved in the true luminal mechanism. One site facing the lumen regulates channel responsiveness to caffeine, while the other site, presumably positioned in the channel pore, governs the gating behavior.

What planar lipid membranes tell us about the pore-forming activity of cholesterol-dependent cytolysins.

Pore-forming toxins are an important group of natural molecules that damage cellular membranes by forming transmembrane pores. They are used by many organisms for attack or defense and similar proteins are employed in the immune system of vertebrates. Various biophysical approaches have been used to understand how these proteins act at the molecular level. One of the most useful, in terms of monitoring pore formation in real time, is a method that employs planar lipid membranes and involves ionic current measurements. Here we highlight the advantages and possibilities that this approach offers and show how it can advance understanding of the pore-forming mechanism and pore properties for one of the most important families of natural toxins, the cholesterol-dependent cytolysins.