Publications by authors named "Tatyana I Rokitskaya"

44 Publications

Rhodopsin Channel Activity Can Be Evaluated by Measuring the Photocurrent Voltage Dependence in Planar Bilayer Lipid Membranes.

Biochemistry (Mosc) 2021 Apr;86(4):409-419

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia.

The studies of the functional properties of retinal-containing proteins often include experiments in model membrane systems, e.g., measurements of electric current through planar bilayer lipid membranes (BLMs) with proteoliposomes adsorbed on one of the membrane surfaces. However, the possibilities of this method have not been fully explored yet. We demonstrated that the voltage dependence of stationary photocurrents for two light-sensitive proteins, bacteriorhodopsin (bR) and channelrhodopsin 2 (ChR2), in the presence of protonophore had very different characteristics. In the case of the bR (proton pump), the photocurrent through the BLM did not change direction when the polarity of the applied voltage was switched. In the case of the photosensitive channel protein ChR2, the photocurrent increased with the increase in voltage and the current polarity changed with the change in the voltage polarity. The protonophore 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole (TTFB) was more efficient in the maximizing stationary photocurrents. In the presence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP), the amplitude of the measured photocurrents for bR significantly decreased, while in the case of ChR2, the photocurrents virtually disappeared. The difference between the effects of TTFB and CCCP was apparently due to the fact that, in contrast to TTFB, CCCP transfers protons across the liposome membranes with a higher rate than through the decane-containing BLM used as a surface for the proteoliposome adsorption.
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http://dx.doi.org/10.1134/S0006297921040039DOI Listing
April 2021

Lipophilic ion aromaticity is not important for permeability across lipid membranes.

Biochim Biophys Acta Biomembr 2021 01 28;1863(1):183483. Epub 2020 Sep 28.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.

To clarify the contribution of charge delocalization in a lipophilic ion to the efficacy of its permeation through a lipid membrane, we compared the behavior of alkyl derivatives of triphenylphosphonium, tricyclohexylphosphonium and trihexylphosphonium both in natural and artificial membranes. Exploring accumulation of the lipophilic cations in response to inside-negative membrane potential generation in mitochondria by using an ion-selective electrode revealed similar mitochondrial uptake of butyltricyclohexylphosphonium (CTCHP) and butyltriphenylphosphonium (CTPP). Fluorescence correlation spectroscopy also demonstrated similar membrane potential-dependent accumulation of fluorescein derivatives of tricyclohexyldecylphosphonium and decyltriphenylphosphonium in mitochondria. The rate constant of lipophilic cation translocation across the bilayer lipid membrane (BLM), measured by the current relaxation method, moderately increased in the following sequence: trihexyltetradecylphosphonium ([P]) < triphenyltetradecylphosphonium (CTPP) < tricyclohexyldodecylphosphonium (CTCHP). In line with these results, measurements of the BLM stationary conductance indicated that membrane permeability for CTCHP is 2.5 times higher than that for CTPP. Values of the difference in the free energy of ion solvation in water and octane calculated using the density functional theory and the polarizable continuum solvent model were similar for methyltriphenylphosphonium, tricyclohexylmethylphosphonium and trihexylmethylphosphonium. Our results prove that both cyclic and aromatic moieties are not necessary for lipophilic ions to effectively permeate through lipid membranes.
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http://dx.doi.org/10.1016/j.bbamem.2020.183483DOI Listing
January 2021

Usnic acid as calcium ionophore and mast cells stimulator.

Biochim Biophys Acta Biomembr 2020 09 3;1862(9):183303. Epub 2020 Apr 3.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia. Electronic address:

Usnic acid (UA), a secondary lichen metabolite, has long been popular as one of natural fat-burning dietary supplements. Similar to 2,4-dinitrophenol, the weight-loss effect of UA is assumed to be associated with its protonophoric uncoupling activity. Recently, we have shown that the ability of UA to shuttle protons across both mitochondrial and artificial membranes is strongly modulated by the presence of calcium ions in the medium. Here, by using fluorescent probes, we studied the calcium-transporting capacity of usnic acid in a variety of membrane systems comprising liposomes, isolated rat liver mitochondria, erythrocytes and rat basophilic leukemia cell culture (RBL-2H3). At concentrations of tens of micromoles, UA appeared to be able to carry calcium ions across membranes in all the systems studied. Similar to the calcium ionophore A23187, UA caused degranulation of RBL-2H3 cells. Therefore, UA, being a protonophoric uncoupler of oxidative phosphorylation, at higher concentrations manifests itself as a calcium ionophore, which could be relevant to its overdose toxicity in humans and also its phytotoxicity.
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http://dx.doi.org/10.1016/j.bbamem.2020.183303DOI Listing
September 2020

Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins.

Biophys J 2019 11 24;117(10):1845-1857. Epub 2019 Oct 24.

Institute of Physiology, Pathophysiology and Biophysics, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria. Electronic address:

Genipin, a natural compound from Gardenia jasminoides, is a well-known compound in Chinese medicine that is used for the treatment of cancer, inflammation, and diabetes. The use of genipin in classical medicine is hindered because of its unknown molecular mechanisms of action apart from its strong cross-linking ability. Genipin is increasingly applied as a specific inhibitor of proton transport mediated by mitochondrial uncoupling protein 2 (UCP2). However, its specificity for UCP2 is questionable, and the underlying mechanism behind its action is unknown. Here, we investigated the effect of genipin in different systems, including neuroblastoma cells, isolated mitochondria, isolated mitochondrial proteins, and planar lipid bilayer membranes reconstituted with recombinant proteins. We revealed that genipin activated dicarboxylate carrier and decreased the activity of UCP1, UCP3, and complex III of the respiratory chain alongside with UCP2 inhibition. Based on competitive inhibition experiments, the use of amino acid blockers, and site-directed mutagenesis of UCP1, we propose a mechanism of genipin's action on UCPs. At low concentrations, genipin binds to arginine residues located in the UCP funnel, which leads to a decrease in UCP's proton transporting function in the presence of long chain fatty acids. At concentrations above 200 μM, the inhibitory action of genipin on UCPs is overlaid by increased nonspecific membrane conductance due to the formation of protein-genipin aggregates. Understanding the concentration-dependent mechanism of genipin action in cells will allow its targeted application as a drug in the above-mentioned diseases.
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http://dx.doi.org/10.1016/j.bpj.2019.10.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031773PMC
November 2019

Effect of methyl and halogen substituents on the transmembrane movement of lipophilic ions.

Phys Chem Chem Phys 2019 Nov 17;21(42):23355-23363. Epub 2019 Oct 17.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.

Penetrating cations are widely used for the design of bioactive mitochondria-targeted compounds. The introduction of various substituents into the phenyl rings of dodecyltriphenylphosphonium and the measurement of the flip-flop of the synthesized cations by the current relaxation method revealed that methyl groups accelerated significantly the cation penetration through the lipid membrane, depending on the number of groups introduced. However, halogenation slowed down the penetration of the analogues. This result is strictly opposite to the flip-flop acceleration observed for halogenated tetraphenylborate anions. Density functional theory and the polarizable continuum solvent model were used to calculate the solvation energies of methyltriphenylphosphonium and methyltriphenylborate analogues. A good agreement was demonstrated between the difference in the free energy of ion solvation in water and octane and the absolute value of the central free energy barrier estimated from experimental data. Our results reveal that increasing the size of the lipophilic ion can lead to both acceleration and deceleration of the transmembrane flip-flop rate depending on the substituent and sign of the ion. This finding also emphasizes the different nature of ion-water interactions for structurally similar substituted hydrophobic anions and cations.
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http://dx.doi.org/10.1039/c9cp03460aDOI Listing
November 2019

Zwitterionic Protonophore Derived from 2-(2-Hydroxyaryl)alkenylphosphonium as an Uncoupler of Oxidative Phosphorylation.

Bioconjug Chem 2019 09 16;30(9):2435-2443. Epub 2019 Aug 16.

Belozersky Institute of Physico-Chemical Biology , Lomonosov Moscow State University , Leninskie Gory 1 , Moscow 119991 , Russian Federation.

2-(2-Hydroxyaryl)alkenylphosphonium salts (here coined as PPR) representing derivatives of quaternary phosphonium with two phenyl (P) and one alkyl (R) substituents linked through alkenyl bridge to substituted phenol were applied here to planar bilayer lipid membranes (BLM), isolated mitochondria, and cell culture. PPR with six carbon atoms in R (PP6) induced proton-selective currents across BLM and caused mitochondrial uncoupling. In particular, PP6 at submicromolar concentrations accelerated respiration, decreased membrane potential, and reduced ATP synthesis in isolated rat liver mitochondria (RLM). Methylation of a hydroxyl group substantially suppressed the protonophoric activity of PP6 on BLM and its uncoupling potency in RLM. Of note, the methylated derivative PP6-OMe was synthesized here via a new synthetic route including cyclization of PP6 with subsequent ring opening. PPR were considered as protonophoric uncouplers of a zwitterionic type, capable of penetrating membranes both as a zwitterion composed of a deprotonated phenol and a cationic quaternary phosphonium, and as a protonated cation. The protonophoric and uncoupling properties of PPR found here were speculated to account for their strong antibacterial activity described previously.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00516DOI Listing
September 2019

Mechanism of action of an old antibiotic revisited: Role of calcium ions in protonophoric activity of usnic acid.

Biochim Biophys Acta Bioenerg 2019 04 30;1860(4):310-316. Epub 2019 Jan 30.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia.

Usnic acid (UA), an old antibiotic and one of the first described mitochondrial uncouplers, has demonstrated many beneficial activities, such as antimicrobial, antiviral, antitumour and anti-inflammatory properties. Here, we performed a thorough investigation of effects of usnic acid and its analogues on artificial planar bilayer lipid membrane (BLM), rat liver mitochondria and bacteria. Surprisingly enough, all of the three hydroxyl groups of UA appeared to be involved in its proton-shuttling activity on BLM. We ascribed this fact to an ability of UA to form complexes with calcium ions, aiding it in cycling protons across the membrane. Actually, the addition of calcium ions markedly stimulated the UA-induced electrical current across BLM. By using the calcium ionophore A23187, we proved the involvement of calcium ions in the UA uncoupling action on isolated rat liver mitochondria. The calcium-chelating property of UA was demonstrated here by the method of extracting metal ions into a hydrophobic phase. Modification of any of the hydroxyl groups in UA dramatically reduced not only the UA-induced current across BLM and the UA-mediated calcium extraction, but also the uncoupling activity of UA in mitochondria and the inhibiting effect of UA on the growth of Bacillus subtilis. The ability of UA to cause dissipation of membrane potential in isolated liver mitochondria and bacterial cells was shown here for the first time. In view of the data obtained, the protonophoric activity of UA is considered to make a significant contribution to its antibacterial action.
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http://dx.doi.org/10.1016/j.bbabio.2019.01.005DOI Listing
April 2019

Carborane derivatives of 1,2,3-triazole depolarize mitochondria by transferring protons through the lipid part of membranes.

Biochim Biophys Acta Biomembr 2019 03 15;1861(3):573-583. Epub 2018 Dec 15.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation. Electronic address:

Boron containing polyhedra (carboranes) are three-dimensional delocalized aromatic systems. These structures have been shown to transport protons through lipid membranes and mitochondria. Conjugation of carboranes to various organic moieties is aimed at obtaining biologically active compounds with novel properties. Taking advantage of 1,2,3-triazoles as the scaffolds valuable in medicinal chemistry, we synthesized 1-(o-carboranylmethyl)-4-pentyl-1,2,3-triazole (c-triazole) and 1-(o-carboranylmethyl)-4-pentyl-1,2,3-triazolium iodide (c-triazolium). Both compounds interacted with model lipid membranes and exhibited a proton carrying activity in planar bilayers and liposomes in a concentration- and pH-dependent manner. Importantly, mechanisms of the protonophoric activity differed; namely, protonation-deprotonation reactions of the triazole and the o-carborane moieties were involved in the transport cycles of c-triazole and c-triazolium, respectively. At micromolar concentrations, c-triazole and c-triazolium stimulated respiration of isolated rat liver mitochondria and depolarized their membrane potential, with c-triazole being more potent. In living K562 (human chronic myelogenous leukemia) cells, both c-triazolium and c-triazole altered the mitochondrial membrane potential as determined by a decreased intracellular accumulation of the potential-dependent dye tetramethylrhodamine ethyl ester. Finally, cell viability testing demonstrated a cytotoxic potency of c-triazolium and, to a lesser extent, of c-triazole against K562 cells, whereas non-malignant fibroblasts were much less sensitive. In all tests, the reference boron-free benzyl-4-pentyl-1,2,3-triazole showed little-to-no effects. These results demonstrated that carboranyltriazoles carry protons across biological membranes, a property potentially important in anticancer drug design.
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http://dx.doi.org/10.1016/j.bbamem.2018.12.009DOI Listing
March 2019

Effect of Alkyl Chain Length on Translocation of Rhodamine B n-Alkyl Esters across Lipid Membranes.

Biophys J 2018 08 9;115(3):514-521. Epub 2018 Jul 9.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

Voltage-dependent translocation of a series of cationic rhodamine B derivatives differing in n-alkyl chain length (ethyl, butyl, octyl, dodecyl, octadecyl) from one lipid monolayer to another was studied by measuring electrical current relaxation after a voltage jump on a planar bilayer phosphatidylcholine (PC) membrane. The rate of the translocation decreased in the following series of lipids: diphytanyl-PC > dioleyl-PC > diphytanoyl-PC > dierucoyl-PC. For all the lipids studied, the rate increased with lengthening of the hydrocarbon chain of the rhodamine derivatives, with the increase being most pronounced for the compounds having a short alkyl chain. The results could be well explained by involvement of molecule reorientations in the process of transmembrane flip-flop of the hydrophobic membrane-bound compounds. However, an impact of membrane dipole potential on the translocation rate could not be excluded, because the dipole potential could contribute to the energy barrier for translocation of the compounds located at different depths in the water-membrane interface. Based on the data obtained, a difference in the dipole potential of ester diphytanoyl-PC membranes with respect to ether diphytanyl-PC was estimated to be 108 mV, highlighting the contribution of a layer of oriented carbonyl groups of the lipids to the membrane dipole potential.
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http://dx.doi.org/10.1016/j.bpj.2018.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6084523PMC
August 2018

Role of mitochondrial outer membrane in the uncoupling activity of N-terminally glutamate-substituted gramicidin A.

Biochim Biophys Acta Biomembr 2019 01 22;1861(1):281-287. Epub 2018 Jun 22.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia. Electronic address:

Of a series of gramicidin A (gA) derivatives, we have earlier found the peptide [Glu1]gA exhibiting very low toxicity toward mammalian cells, although dissipating mitochondrial membrane potential with almost the same efficiency as gA. Substitution of glutamate for valine at position 1 of the gA amino acid sequence, which is supposed to interfere with the formation of ion-conducting gA channels via head-to-head dimerization, reduces both channel-forming potency of the peptide in planar lipid bilayer membranes and its photonophoric activity in unilamellar liposomes. Here, we compared [Glu1]gA and gA abilities to cause depolarization of the inner mitochondrial membrane in mitochondria and mitoplasts, the latter lacking the outer mitochondrial membrane. Importantly, much less gA was needed to decrease the membrane potential in mitoplasts than in mitochondria, whereas the depolarizing potency of [Glu1]gA was nearly the same in these systems. Moreover, in multilamellar liposomes, [Glu1]gA exhibited more pronounced protonophoric activity than gA, in contrast to the data for unilamellar liposomes. These results allowed us to conclude that [Glu1]gA has a much higher permeability between adjacent lipid membranes than gA. Therefore, the fraction of peptide molecules, reaching the inner mitochondrial membrane upon the addition to cells, is much higher for [Glu1]gA compared to gА. Under these conditions, the decreased cytotoxicity of [Glu1]gA could be associated with its low efficiency as a channel-former dissipating potassium and sodium ion gradients across plasma membrane. The present study highlighted the role of the ability to permeate among various biological membranes for intracellular efficiency of ionophores.
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http://dx.doi.org/10.1016/j.bbamem.2018.06.013DOI Listing
January 2019

A conjugate of decyltriphenylphosphonium with plastoquinone can carry cyclic adenosine monophosphate, but not cyclic guanosine monophosphate, across artificial and natural membranes.

Biochim Biophys Acta Biomembr 2018 Feb 14;1860(2):329-334. Epub 2017 Oct 14.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia. Electronic address:

The present study demonstrated for the first time the interaction between adenosine 3',5'-cyclic monophosphate (cAMP), one of the most important signaling compounds in living organisms, and the mitochondria-targeted antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1). The data obtained on model liquid membranes and human platelets revealed the ability of SkQ1 to selectively transport cAMP, but not guanosine 3',5'-cyclic monophosphate (cGMP), across both artificial and natural membranes. In particular, SkQ1 elicited translocation of cAMP from the source to the receiving phase of a Pressman-type cell, while showing low activity with cGMP. Importantly, only conjugate with plastoquinone, but not dodecyl-triphenylphosphonium, was effective in carrying cAMP. In human platelets, SkQ1 also appeared to serve as a carrier of cAMP, but not cGMP, from outside to inside the cell, as measured by phosphorylation of the vasodilator stimulated phosphoprotein. The SkQ1-induced transfer of cAMP across the plasma membrane found here can be tentatively suggested to interfere with cAMP signaling pathways in living cells.
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http://dx.doi.org/10.1016/j.bbamem.2017.10.013DOI Listing
February 2018

Fast flip-flop of halogenated cobalt bis(dicarbollide) anion in a lipid bilayer membrane.

Phys Chem Chem Phys 2017 Sep;19(36):25122-25128

A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov Str., 119991, Moscow, Russian Federation.

Transmembrane translocation (flip-flop) of cobalt bis(dicarbollide) (COSAN) anions, elicited by application of a voltage-jump across the lipid bilayer membrane, manifested itself in monoexponential electrical current transients in the microsecond time scale. Halogenation of COSAN led to multi-fold acceleration of the flip-flop, the effect increasing with the molecular weight of the halogens. The exception was a fluorinated analog which exhibited slowing of the translocation kinetics. Measurements of the fluorescence ratio of the dye di-4-ANEPPS in lipid vesicles showed significant differences in the adsorption of studied hydrophobic anions. Based on these data, it can be concluded that COSAN and COSAN-F were located on the surface of the lipid membrane in the cisoid conformation increasing the dipole potential of the lipid membrane, while other halogenated COSAN analogs were adsorbed in the transoid conformation. Differences in the flip-flop kinetics of COSAN analogs were attributed to variation in the molecular volume of the anions and their orientation on the membrane surface.
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http://dx.doi.org/10.1039/c7cp04207hDOI Listing
September 2017

Blocking of Single α-Hemolysin Pore by Rhodamine Derivatives.

Biophys J 2017 Jun;112(11):2327-2335

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

Measurements of ion conductance through α-hemolysin pore in a bilayer lipid membrane revealed blocking of the ion channel by a series of rhodamine 19 and rhodamine B esters. The longest dwell closed time of the blocking was observed with rhodamine 19 butyl ester (C4R1), whereas the octyl ester (C8R1) was of poor effect. Voltage asymmetry in the binding kinetics indicated that rhodamine derivatives bound to the stem part of the aqueous pore lumen. The binding frequency was proportional to a quadratic function of rhodamine concentrations, thereby showing that the dominant binding species were rhodamine dimers. Two levels of the pore conductance and two dwell closed times of the pore were found. The dwell closed times lengthened as the voltage increased, suggesting impermeability of the channel for the ligands. Molecular docking analysis revealed two distinct binding sites within the lumen of the stem of the α-hemolysin pore for the C4R1 dimer, but only one binding site for the C8R1 dimer. The blocking of the α-hemolysin nanopore by rhodamines could be utilized in DNA sequencing as additional optical sensing owing to bright fluorescence of rhodamines if used for DNA labeling.
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http://dx.doi.org/10.1016/j.bpj.2017.04.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474841PMC
June 2017

pH-Dependent properties of ion channels formed by N-terminally glutamate substituted gramicidin A in planar lipid bilayers.

Biochim Biophys Acta Biomembr 2017 May 7;1859(5):896-902. Epub 2017 Feb 7.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia. Electronic address:

The N-terminally glutamate substituted analogue of the pentadecapeptide gramicidin A [Glu1]gA has been previously described as a low-toxic uncoupler of mitochondrial oxidative phosphorylation and neuroprotector. Here, we studied ion channel-forming activity of this peptide in planar bilayer lipid membranes (BLMs). [Glu1]gA exhibited an ability to induce both macroscopic current and single channels in a broad pH range, albeit with a lower potency than the parent gramicidin A (gA). Single-channel recordings in 1M KCl at pH about 4 showed channel openings of one type with the conductance (about 26pS), similar to that of gA, and the lifetime (40ms), much shorter than that of gA. By contrast, two populations of channels were found at pH9, one of which had much longer duration (several seconds) and lower conductance (3.5-10pS). Autocorrelation function of the current noise of [Glu1]gA revealed a marked shift towards longer correlation times upon alkalinization. The sensitized photoinactivation technique also revealed substantial differences in [Glu1]gA conducting properties at alkaline and acidic pH, in particular deceleration of the photoinactivation kinetics and a sharp decrease in its amplitude upon alkalinization. A double-logarithmic plot of the concentration dependence of [Glu1]gA-induced BLM conductance had the slope of about 3, which pointed to peptide aggregation in the membrane. The data were discussed in relation to pH-dependent aggregation of [Glu1]gA, resulting from deprotonation of the glutamate side chain at alkaline pH.
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http://dx.doi.org/10.1016/j.bbamem.2017.02.004DOI Listing
May 2017

A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted uncoupler and fluorescent neuro- and nephroprotector.

Biochim Biophys Acta 2016 11 20;1860(11 Pt A):2463-2473. Epub 2016 Jul 20.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.

Background: Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. Linking a triphenyl-phosphonium cation to fluorescein through a decyl (C10) spacer yields a fluorescent uncoupler, coined mitoFluo, that selectively accumulates in energized mitochondria (Denisov et al., Chem.Commun. 2014).

Methods: Proton-transport activity of mitoFluo was tested in liposomes reconstituted with bacteriorhodopsin. To examine the uncoupling action on mitochondria, we monitored mitochondrial membrane potential in parallel with oxygen consumption. Neuro- and nephroprotecting activity was detected by a limb-placing test and a kidney ischemia/reperfusion protocol, respectively.

Results: We compared mitoFluo properties with those of its newly synthesized analog having a short (butyl) spacer (C4-mitoFluo). MitoFluo, but not C4-mitoFluo, caused collapse of mitochondrial membrane potential resulting in stimulation of mitochondrial respiration. The dramatic difference in the uncoupling activity of mitoFluo and C4-mitoFluo was in line with the difference in their protonophoric activity on a lipid membrane. The accumulation of mitoFluo in mitochondria was more pronounced than that of C4-mitoFluo. MitoFluo decreased the rate of ROS production in mitochondria. MitoFluo was effective in preventing consequences of brain trauma in rats: it suppressed trauma-induced brain swelling and reduced a neurological deficit. Besides, mitoFluo attenuated acute kidney injury after ischemia/reperfusion in rats.

Conclusions: A long alkyl linker was proved mandatory for mitoFluo to be a mitochondria- targeted uncoupler. MitoFluo showed high protective efficacy in certain models of oxidative stress-related diseases.

General Significance: MitoFluo is a candidate for developing therapeutic and fluorescence imaging agents to treat brain and kidney pathologies.
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http://dx.doi.org/10.1016/j.bbagen.2016.07.014DOI Listing
November 2016

Weak C-H acids as protonophores can carry hydrogen ions through lipid membranes and mitochondria: a case of o-carborane.

Phys Chem Chem Phys 2016 Jun 6;18(24):16476-82. Epub 2016 Jun 6.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119899, Russian Federation.

ortho-Carborane (1,2-C2B10H12) was found to be a carrier of protons in both mitochondrial and artificial lipid membranes, suggesting that this dicarborane can reversibly release hydrogen ions and diffuse through the membranes in neutral and anionic forms. Similar to conventional uncouplers (e.g. 2,4-dinitrophenol), o-carborane stimulated mitochondrial respiration and decreased the membrane potential at concentrations of tens of micromoles. Protonophoric activity of o-carborane was observed both by a fluorometric assay using pyranine-loaded liposomes and electrical current measurements across planar lipid bilayers. Substantial contribution of the proton flux to the o-carborane-mediated current was proved by a shift of the zero current voltage upon imposing a pH gradient across the membrane. Meta-carborane (1,7-C2B10H12) lacked the protonophoric activity in line with its reduced C-H acidity. The results suggest that weak C-H acids can exhibit protonophoric activity in the biological environment. The finding of a new class of protonophoric compounds is of substantial interest due to promising anti-obesity and anti-diabetic properties of uncouplers.
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http://dx.doi.org/10.1039/c6cp02581aDOI Listing
June 2016

Ubiquinol and plastoquinol triphenylphosphonium conjugates can carry electrons through phospholipid membranes.

Bioelectrochemistry 2016 Oct 28;111:23-30. Epub 2016 Apr 28.

Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology, Vorobyevy Gory 1, Moscow 119991, Russia; Lomonosov Moscow State University, Institute of Mitoengineering, Vorobyevy Gory 1, Moscow 119991, Russia. Electronic address:

Many mitochondria-targeted antioxidants (MTAs) that comprise a quinol moiety covalently attached through an aliphatic carbon chain to the lipophilic triphenylphosphonium cation are widely used for evaluating the role of mitochondria in pathological processes involving oxidative stress. The potency of MTAs to carry electrons across biological membranes and thereby mediate transmembrane redox processes was unknown. To assess this, we measured the rate of ferricyanide reduction inside liposomes by external ascorbate. Here, we show that MTAs containing ubiquinone (MitoQ series) or plastoquinone (SkQ series) can carry electrons through lipid membranes, with the rate being inversely proportional to the length of the hydrocarbon linker group. Furthermore, this process was stimulated by the hydrophobic anion tetraphenylborate suggesting that permeation of the cationic MTA through the membrane was the rate-limiting step of the process. This conclusion was supported by the observation that the rate of MTA-induced electron transfer was insensitive to nigericin, in contrast to electron transfer mediated by neutral quinone derivatives. These findings indicate that MTAs can be utilized to transfer electrons across lipid membranes and this may be applicable to the study of the electron-transport chain in mitochondria and other natural membranes exhibiting redox processes.
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http://dx.doi.org/10.1016/j.bioelechem.2016.04.009DOI Listing
October 2016

Fullerenol C60(OH)24 increases ion permeability of lipid membranes in a pH-dependent manner.

Biochim Biophys Acta 2016 Jun 10;1858(6):1165-74. Epub 2016 Feb 10.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russian Federation. Electronic address:

Fullerenols are water-soluble analogs of fullerene exhibiting both antioxidant and prooxidant activities in vitro and in vivo. Here we report, for the first time, that fullerenol C60(OH)24 can induce ion permeability of a planar lipid bilayer membrane via the formation of ion pores or conductive defects with a preference for cations over anions. The fullerenol-mediated electrical current displayed non-linear concentration dependence and was reversibly enhanced by alkalinization. Calcium and magnesium ions decreased the fullerenol-induced potassium ion permeability. Voltage dependence of the current was sensitive to membrane composition, with the conductance being well pronounced in fully saturated diphytanoylphosphatidylcholine. Fullerenol did not induce carboxyfluorescein leakage from liposomes, suggesting a small size of fullerenol-induced pores. In contrast to ion permeability, the binding of C60(OH)24 to liposomes increased at acidic pH, as measured by fluorescence quenching of pyrene-labeled lipid. In line with this, the photodynamic action of fullerenol on the peptide gramicidin A also increased at low pH. It is hypothesized that aggregates of fullerenol may stabilize transient conductive lipid defects or pores formed under a variety of stress conditions.
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http://dx.doi.org/10.1016/j.bbamem.2016.02.009DOI Listing
June 2016

Single channel activity of OmpF-like porin from Yersinia pseudotuberculosis.

Biochim Biophys Acta 2016 Apr 17;1858(4):883-91. Epub 2016 Feb 17.

Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia. Electronic address:

To gain a mechanistic insight in the functioning of the OmpF-like porin from Yersinia pseudotuberculosis (YOmpF), we compared the effect of pH variation on the ion channel activity of the protein in planar lipid bilayers and its binding to lipid membranes. The behavior of YOmpF channels upon acidification was similar to that previously described for Escherichia coli OmpF. In particular, a decrease in pH of the bathing solution resulted in a substantial reduction of YOmpF single channel conductance, accompanied by the emergence of subconductance states. Similar subconductance substates were elicited by the addition of lysophosphatidylcholine. This observation, made with porin channels for the first time, pointed to the relevance of lipid-protein interactions, in particular, the lipid curvature stress, to the appearance of subconductance states at acidic pH. Binding of YOmpF to membranes displayed rather modest dependence on pH, whereas the channel-forming potency of the protein tremendously decreased upon acidification.
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http://dx.doi.org/10.1016/j.bbamem.2016.02.005DOI Listing
April 2016

Intramitochondrial accumulation of cationic Atto520-biotin proceeds via voltage-dependent slow permeation through lipid membrane.

Biochim Biophys Acta 2015 Jun 6;1848(6):1277-84. Epub 2015 Mar 6.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation.

Conjugation to penetrating cations is a general approach for intramitochondrial delivery of physiologically active compounds, supported by a high membrane potential of mitochondria having negative sign on the matrix side. By using fluorescence correlation spectroscopy, we found here that Atto520-biotin, a conjugate of a fluorescent cationic rhodamine-based dye with the membrane-impermeable vitamin biotin, accumulated in energized mitochondria in contrast to biotin-rhodamine 110. The energy-dependent uptake of Atto520-biotin by mitochondria, being slower than that of the conventional mitochondrial dye tetramethyl-rhodamine ethyl ester, was enhanced by the hydrophobic anion tetraphenylborate (TPB). Atto520-biotin also exhibited accumulation in liposomes driven by membrane potential resulting from potassium ion gradient in the presence valinomycin. The induction of electrical current across planar bilayer lipid membrane by Atto520-biotin proved the ability of the compound to permeate through lipid membrane in a cationic form. Atto520-biotin stained mitochondria in a culture of L929 cells, and the staining was enhanced in the presence of TPB. Therefore, the fluorescent Atto520 moiety can serve as a vehicle for intramitochondrial delivery of hydrophilic drugs. Of importance for biotin-streptavidin technology, binding of Atto520-biotin to streptavidin was found to cause quenching of its fluorescence similar to the case of fluorescein-4-biotin.
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http://dx.doi.org/10.1016/j.bbamem.2015.02.028DOI Listing
June 2015

Methodology for use of mitochondria-targeted cations in the field of oxidative stress-related research.

Methods Mol Biol 2015 ;1265:149-59

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Bldg. 40, Leninskie Gory 1, Moscow, 119992, Russia.

For many pathological conditions, reactive oxygen species (ROS) generated in mitochondria are considered to have a role as a trigger. When mitochondrial ROS (mROS) are formed in the inner mitochondrial membrane, they initiate free radical-mediated chain reactions of lipid peroxidation and are thus especially damaging. The consequences of membrane damage are decreased electrical resistance of the membrane, oxidative damage to cardiolipin (a mitochondria specific lipid essential for functioning of respiratory chain proteins and H(+)-ATP synthase), and damage to mitochondrial DNA localized in close vicinity to the inner membrane, with consequent mitochondrial dysfunction and induction of apoptotic cascade and cell death. To target the starting point of such undesirable events, antioxidants conjugated with mitochondria-targeted, membrane-penetrating cations can be used to scavenge ROS inside mitochondria. The most demonstrative indications favoring this conclusion originate from recent discoveries of the in vivo effects of such cations belonging to the MitoQ and SkQ groups. Here we describe some essential methodological aspects of the application of mitochondria-targeted cations promising in treating oxidative stress-related pathologies.
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http://dx.doi.org/10.1007/978-1-4939-2288-8_12DOI Listing
October 2015

Electrogenic and nonelectrogenic ion fluxes across lipid and mitochondrial membranes mediated by monensin and monensin ethyl ester.

Biochim Biophys Acta 2015 Apr 17;1848(4):995-1004. Epub 2015 Jan 17.

Department of Bioorganic Chemistry, Faculty of Chemistry, A. Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland.

Monensin is a carrier of cations through lipid membranes capable of exchanging sodium (potassium) cations for protons by an electroneutral mechanism, whereas its ethyl ester derivative ethyl-monensin is supposed to transport sodium (potassium) cations in an electrogenic manner. To elucidate mechanistic details of the ionophoric activity, ion fluxes mediated by monensin and ethyl-monensin were measured on planar bilayer lipid membranes, liposomes, and mitochondria. In particular, generation of membrane potential on liposomes was studied via the measurements of rhodamine 6G uptake by fluorescence correlation spectroscopy. In mitochondria, swelling experiments were expounded by the additional measurements of respiration, membrane potential, and matrix pH. It can be concluded that both monensin and ethyl-monensin can perform nonelectrogenic exchange of potassium (sodium) ions for protons and serve as electrogenic potassium ion carriers similar to valinomycin. The results obtained are in line with the predictions based on the crystal structures of the monensin complexes with sodium ions and protons (Huczyński et al., Biochim. Biophys. Acta, 1818 (2012) pp. 2108-2119). The functional activity observed for artificial membranes and mitochondria can be applied to explain the activity of ionophores in living systems. It can also be important for studying the antitumor activity of monensin.
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http://dx.doi.org/10.1016/j.bbamem.2015.01.005DOI Listing
April 2015

A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector.

Biochim Biophys Acta 2014 Oct 16;1837(10):1739-47. Epub 2014 Jul 16.

Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology, Vorobyevy Gory 1, Moscow 119991, Russia; Lomonosov Moscow State University, Institute of Mitoengineering, Vorobyevy Gory 1, Moscow 119991, Russia; Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, Vorobyevy Gory 1, Moscow 119991, Russia. Electronic address:

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C4R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C12R1). Surprisingly, C12R1 increased H(+) conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C4R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C4R1. In experiments with HeLa cells, C4R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C4R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C12R1. Thus, C4R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.
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http://dx.doi.org/10.1016/j.bbabio.2014.07.006DOI Listing
October 2014

Unsaturated lipids protect the integral membrane peptide gramicidin A from singlet oxygen.

FEBS Lett 2014 May 5;588(9):1590-5. Epub 2014 Mar 5.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation.

In contrast to expectations that unsaturated fatty acids contribute to oxidative stress by providing a source of lipid peroxides, we demonstrated the protective effect of double bonds in lipids on oxidative damage to membrane proteins. Photodynamic inactivation of gramicidin channels was decreased in unsaturated lipid compared to saturated lipid bilayers. By estimating photosensitizer (boronated chlorine e6 amide) binding to the membrane with the current relaxation technique, the decrease in gramicidin photoinactivation was attributed to singlet oxygen scavenging by double bonds in lipids rather than to the reduction in photosensitizer binding. Gramicidin protection by unsaturated lipids was also observed upon induction of oxidative stress with tert-butyl hydroperoxide.
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http://dx.doi.org/10.1016/j.febslet.2014.02.046DOI Listing
May 2014

Photodynamic activity of the boronated chlorin e6 amide in artificial and cellular membranes.

Biochim Biophys Acta 2014 Mar 25;1838(3):793-801. Epub 2013 Nov 25.

Biological Department, Lomonosov Moscow State University, Moscow, Russian Federation.

Photodynamic tumor-destroying activity of the boronated chlorin e6 derivative BACE (chlorin e6 13(1)-N-{2-[N-(1-carba-closo-dodecaboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester), previously described in Moisenovich et al. (2010) PLoS ONE 5(9) e12717, was shown here to be enormously higher than that of unsubstituted chlorin e6, being supported by the data on much higher photocytotoxicity of BACE in M-1 sarcoma cell culture. To validate membrane damaging effect as the basis of the enhanced tumoricidal activity, BACE was compared with unsubstituted chlorin e6 in the potency to photosensitize dye leakage from liposomes, transbilayer lipid flip-flop, inactivation of gramicidin A ionic channels in planar lipid membranes and erythrocyte hemolysis. In all the models comprising artificial and cellular membranes, the photodynamic effect of BACE exceeded that of chlorin e6. BACE substantially differed from chlorin e6 in the affinity to liposomes and erythrocytes, as monitored by fluorescence spectroscopy, flow cytometry and centrifugation. The results support the key role of membrane binding in the photodynamic effect of the boronated chlorin e6 amide.
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http://dx.doi.org/10.1016/j.bbamem.2013.11.012DOI Listing
March 2014

Penetrating cations enhance uncoupling activity of anionic protonophores in mitochondria.

PLoS One 2013 23;8(4):e61902. Epub 2013 Apr 23.

M.V. Lomonosov Moscow State University, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.

Protonophorous uncouplers causing a partial decrease in mitochondrial membrane potential are promising candidates for therapeutic applications. Here we showed that hydrophobic penetrating cations specifically targeted to mitochondria in a membrane potential-driven fashion increased proton-translocating activity of the anionic uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluorophenylhydrazone (FCCP). In planar bilayer lipid membranes (BLM) separating two compartments with different pH values, DNP-mediated diffusion potential of H(+) ions was enhanced in the presence of dodecyltriphenylphosphonium cation (C12TPP). The mitochondria-targeted penetrating cations strongly increased DNP- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-mediated steady-state current through BLM when a transmembrane electrical potential difference was applied. Carboxyfluorescein efflux from liposomes initiated by the plastoquinone-containing penetrating cation SkQ1 was inhibited by both DNP and FCCP. Formation of complexes between the cation and CCCP was observed spectophotometrically. In contrast to the less hydrophobic tetraphenylphosphonium cation (TPP), SkQ1 and C12TPP promoted the uncoupling action of DNP and FCCP on isolated mitochondria. C12TPP and FCCP exhibited a synergistic effect decreasing the membrane potential of mitochondria in yeast cells. The stimulating action of penetrating cations on the protonophore-mediated uncoupling is assumed to be useful for medical applications of low (non-toxic) concentrations of protonophores.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061902PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633956PMC
November 2013

Electrogenic proton transport across lipid bilayer membranes mediated by cationic derivatives of rhodamine 19: comparison with anionic protonophores.

Eur Biophys J 2013 Jun 5;42(6):477-85. Epub 2013 Apr 5.

A. N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.

Protonophores can be considered as candidates for anti-obesity drugs and tools to prevent excessive reactive oxygen species production in mitochondria by means of a limited decrease in the mitochondrial potential. Experimentally used protonophores are weak acids that can carry protons across a membrane in a neutral (protonated) form, and they come back in an anionic (deprotonated) form. A cationic derivative of rhodamine 19 and plastoquinone (SkQR1) was recently shown to possess uncoupling activity in mitochondria and in intact cells. In this article, we studied the mechanism of action of SkQR1 and its plastoquinone-lacking analog (C12R1) on a planar bilayer lipid membrane by applying voltage jumps. The steady-state current was proportional to the C12R1 concentration in a manner as if the monomeric form of the carrier were operative. As predicted by the carrier model, at high pH, when rhodamines were mainly deprotonated, the current changed immediately following a jump in the applied potential and then remained constant. By contrast, at low pH, the current relaxed from an initially high value to a lower value since the protonated carrier cations were redistributed in the membrane. An inverse pH dependence was revealed with the anionic protonophore CCCP. The dependence of the SkQR1 protonophorous activity on voltage exhibited an increase at high voltages, an effect that might facilitate mild (self-limited) uncoupling of mitochondria.
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http://dx.doi.org/10.1007/s00249-013-0898-9DOI Listing
June 2013

N-terminally glutamate-substituted analogue of gramicidin A as protonophore and selective mitochondrial uncoupler.

PLoS One 2012 24;7(7):e41919. Epub 2012 Jul 24.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

Limited uncoupling of oxidative phosphorylation could be beneficial for cells by preventing excessive generation of reactive oxygen species. Typical uncouplers are weak organic acids capable of permeating across membranes with a narrow gap between efficacy and toxicity. Aimed at designing a nontoxic uncoupler, the protonatable amino acid residue Glu was substituted for Val at the N-terminus of the pentadecapeptide gramicidin A (gA). The modified peptide [Glu1]gA exhibited high uncoupling activity in isolated mitochondria, in particular, abolishing membrane potential at the inner mitochondrial membrane with the same or even larger efficacy as gA. With mitochondria in cell culture, the depolarizing activity of [Glu1]gA was observed at concentrations by an order of magnitude lower than those of gA. On the contrary, [Glu1]gA was much less potent in forming proton channels in planar lipid bilayers than gA. Remarkably, at uncoupling concentrations, [Glu1]gA did not alter cell morphology and was nontoxic in MTT test, in contrast to gA showing high toxicity. The difference in the behavior of [Glu1]gA and gA in natural and artificial membranes could be ascribed to increased capability of [Glu1]gA to permeate through membranes and/or redistribute between different membranes. Based on the protective role of mild uncoupling, [Glu1]gA and some other proton-conducting gA analogues may be considered as prototypes of prospective therapeutic agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041919PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404012PMC
February 2013

Effect of liposomes on energy-dependent uptake of the antioxidant SkQR1 by isolated mitochondria.

J Bioenerg Biomembr 2012 Aug 22;44(4):453-60. Epub 2012 Jun 22.

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Vorobyevy Gory 1, Moscow, 119991, Russia.

The mitochondria-targeted antioxidant SkQR1 composed of a plastoquinone part covalently bound to a cationic rhodamine 19 moiety via a decane linker was previously shown to effectively protect brain and kidney from ischemia injury accompanying generation of reactive oxygen species. In the present paper the energy-dependent SkQR1 uptake by isolated rat liver mitochondria was studied by fluorescence correlation spectroscopy peak intensity analysis (FCS PIA). This approach can be used to measure the number of fluorescent molecules per single mitochondrion. A large portion of SkQR1 appeared to be taken up by mitochondria in an energy-independent fashion because of its high affinity to membranes. Liposomes were found to compete effectively with mitochondria for the energy-independent SkQR1 binding, thereby facilitating, as an "SkQR1-buffer", observation of energy-dependent SkQR1 accumulation in mitochondria. The rate of energy-dependent SkQR1 uptake by mitochondria observed in the presence of liposomes was rather low (minutes) which was apparently due to slow redistribution of SkQR1 between liposomal and mitochondrial membranes. This can explain the low rate of staining of mitochondria by SkQR1 in living cells containing, besides mitochondria, other membrane components (endoplasmic reticulum, Golgi membranes, endosomes, lysosomes, etc.) which can compete with mitochondria for the energy-independent SkQR1 binding.
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http://dx.doi.org/10.1007/s10863-012-9449-9DOI Listing
August 2012

The pH-dependent induction of lipid membrane ionic permeability by N-terminally lysine-substituted analogs of gramicidin A.

Eur Biophys J 2012 Feb 1;41(2):129-38. Epub 2011 Nov 1.

A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119991, Russia.

Insertion of charged groups at the N-terminus of the gramicidin A (gA) amino acid sequence is considered to be fatal for peptide channel-forming activity because of hindrance to the head-to-head dimer formation. Here the induction of ionic conductivity in planar bilayer lipid membranes (BLM) was studied with gA analogs having lysine either in the first ([Lys1]gA) or the third ([Lys3]gA) position. If added to the bathing solution at neutral or acidic pH, these analogs, being protonated and thus positively charged, were unable to induce ionic current across BLM. By contrast, at pH 11 the induction of BLM conductivity was observed with both lysine-substituted analogs. Based on the dependence of the macroscopic current on the side of the peptide addition, sensitivity to calcium ions and susceptibility to sensitized photoinactivation, as well as on the single-channel properties of the analogs, we surmise that at alkaline pH [Lys1]gA formed channels with predominantly single-stranded structure of head-to-head helical dimers, whereas [Lys3]gA open channels had the double-stranded helical structure. CD spectra of the lysine-substituted analogs in liposomes were shown to be pH-dependent.
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http://dx.doi.org/10.1007/s00249-011-0764-6DOI Listing
February 2012