Neoteny, Prolongation of Youth: From Naked Mole Rats to "Naked Apes" (Humans).

It has been suggested that highly social mammals, such as naked mole rats and humans, are long-lived due to neoteny (the prolongation of youth). In both species, aging cannot operate as a mechanism facilitating natural selection because the pressure of this selection is strongly reduced due to 1) a specific social structure where only the "queen" and her "husband(s)" are involved in reproduction (naked mole rats) or 2) substituting fast technological progress for slow biological evolution (humans). Lists of numerous traits of youth that do not disappear with age in naked mole rats and humans are presented and discussed. A high resistance of naked mole rats to cancer, diabetes, cardiovascular and brain diseases, and many infections explains why their mortality rate is very low and almost age-independent and why their lifespan is more than 30 years, versus 3 years in mice. In young humans, curves of mortality versus age start at extremely low values. However, in the elderly, human mortality strongly increases. High mortality rates in other primates are observed at much younger ages than in humans. The inhibition of the aging process in humans by specific drugs seems to be a promising approach to prolong our healthspan. This might be a way to retard aging, which is already partially accomplished via the natural physiological phenomenon neoteny.

Age-Dependent Changes in the Production of Mitochondrial Reactive Oxygen Species in Human Skeletal Muscle.

A decrease in muscle mass and its functionality (strength, endurance, and insulin sensitivity) is one of the integral signs of aging. One of the triggers of aging is an increase in the production of mitochondrial reactive oxygen species. Our study was the first to examine age-dependent changes in the production of mitochondrial reactive oxygen species related to a decrease in the proportion of mitochondria-associated hexokinase-2 in human skeletal muscle. For this purpose, a biopsy was taken from m. vastus lateralis in 10 young healthy volunteers and 70 patients (26-85 years old) with long-term primary arthrosis of the knee/hip joint. It turned out that aging (comparing different groups of patients), in contrast to inactivity/chronic inflammation (comparing young healthy people and young patients), causes a pronounced increase in peroxide production by isolated mitochondria. This correlated with the age-dependent distribution of hexokinase-2 between mitochondrial and cytosolic fractions, a decrease in the rate of coupled respiration of isolated mitochondria and respiration when stimulated with glucose (a hexokinase substrate). It is discussed that these changes may be caused by an age-dependent decrease in the content of cardiolipin, a potential regulator of the mitochondrial microcompartment containing hexokinase. The results obtained contribute to a deeper understanding of age-related pathogenetic processes in skeletal muscles and open prospects for the search for pharmacological/physiological approaches to the correction of these pathologies.

Mild depolarization of the inner mitochondrial membrane is a crucial component of an anti-aging program.

The mitochondria of various tissues from mice, naked mole rats (NMRs), and bats possess two mechanistically similar systems to prevent the generation of mitochondrial reactive oxygen species (mROS): hexokinases I and II and creatine kinase bound to mitochondrial membranes. Both systems operate in a manner such that one of the kinase substrates (mitochondrial ATP) is electrophoretically transported by the ATP/ADP antiporter to the catalytic site of bound hexokinase or bound creatine kinase without ATP dilution in the cytosol. One of the kinase reaction products, ADP, is transported back to the mitochondrial matrix via the antiporter, again through an electrophoretic process without cytosol dilution. The system in question continuously supports H+-ATP synthase with ADP until glucose or creatine is available. Under these conditions, the membrane potential, ∆ψ, is maintained at a lower than maximal level (i.e., mild depolarization of mitochondria). This ∆ψ decrease is sufficient to completely inhibit mROS generation. In 2.5-y-old mice, mild depolarization disappears in the skeletal muscles, diaphragm, heart, spleen, and brain and partially in the lung and kidney. This age-dependent decrease in the levels of bound kinases is not observed in NMRs and bats for many years. As a result, ROS-mediated protein damage, which is substantial during the aging of short-lived mice, is stabilized at low levels during the aging of long-lived NMRs and bats. It is suggested that this mitochondrial mild depolarization is a crucial component of the mitochondrial anti-aging system.

Six Functions of Respiration: Isn't It Time to Take Control over ROS Production in Mitochondria, and Aging Along with It?

Cellular respiration is associated with at least six distinct but intertwined biological functions. (1) biosynthesis of ATP from ADP and inorganic phosphate, (2) consumption of respiratory substrates, (3) support of membrane transport, (4) conversion of respiratory energy to heat, (5) removal of oxygen to prevent oxidative damage, and (6) generation of reactive oxygen species (ROS) as signaling molecules. Here we focus on function #6, which helps the organism control its mitochondria. The ROS bursts typically occur when the mitochondrial membrane potential (MMP) becomes too high, e.g., due to mitochondrial malfunction, leading to cardiolipin (CL) oxidation. Depending on the intensity of CL damage, specific programs for the elimination of damaged mitochondria (mitophagy), whole cells (apoptosis), or organisms (phenoptosis) can be activated. In particular, we consider those mechanisms that suppress ROS generation by enabling ATP synthesis at low MMP levels. We discuss evidence that the mild depolarization mechanism of direct ATP/ADP exchange across mammalian inner and outer mitochondrial membranes weakens with age. We review recent data showing that by protecting CL from oxidation, mitochondria-targeted antioxidants decrease lethality in response to many potentially deadly shock insults. Thus, targeting ROS- and CL-dependent pathways may prevent acute mortality and, hopefully, slow aging.

Sodium Energetic Cycle in the Natronophilic Bacterium Thioalkalivibrio versutus.

As inhabitants of soda lakes, Thioalkalivibrio versutus are halo- and alkaliphilic bacteria that have previously been shown to respire with the first demonstrated Na+-translocating cytochrome-c oxidase (CO). The enzyme generates a sodium-motive force (Δs) as high as -270 mV across the bacterial plasma membrane. However, in these bacteria, operation of the possible Δs consumers has not been proven. We obtained motile cells and used them to study the supposed Na+ energetic cycle in these bacteria. The resulting motility was activated in the presence of the protonophore 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), in line with the same effect on cell respiration, and was fully blocked by amiloride-an inhibitor of Na+-motive flagella. In immotile starving bacteria, ascorbate triggered CO-mediated respiration and motility, both showing the same dependence on sodium concentration. We concluded that, in T. versutus, Na+-translocating CO and Na+-motive flagella operate in the Na+ energetic cycle mode. Our research may shed light on the energetic reason for how these bacteria are confined to a narrow chemocline zone and thrive in the extreme conditions of soda lakes.

A Crosstalk between the Biorhythms and Gatekeepers of Longevity: Dual Role of Glycogen Synthase Kinase-3.

This review discusses genetic and molecular pathways that link circadian timing with metabolism, resulting in the emergence of positive and negative regulatory feedback loops. The Nrf2 pathway is believed to be a component of the anti-aging program responsible for the healthspan and longevity. Nrf2 enables stress adaptation by activating cell antioxidant defense and other metabolic processes via control of expression of over 200 target genes in response to various types of stress. The GSK3 system represents a "regulating valve" that controls fine oscillations in the Nrf2 level, unlike Keap1, which prevents significant changes in the Nrf2 content in the absence of oxidative stress and which is inactivated by the oxidative stress. Furthermore, GSK3 modifies core circadian clock proteins (Bmal1, Clock, Per, Cry, and Rev-erbα). Phosphorylation by GSK3 leads to the inactivation and degradation of circadian rhythm-activating proteins (Bmal1 and Clock) and vice versa to the activation and nuclear translocation of proteins suppressing circadian rhythms (Per and Rev-erbα) with the exception of Cry protein, which is likely to be implicated in the fine tuning of biological clock. Functionally, GSK3 appears to be one of the hubs in the cross-regulation of circadian rhythms and antioxidant defense. Here, we present the data on the crosstalk between the most powerful cell antioxidant mechanism, the Nrf2 system, and the biorhythm-regulating system in mammals, including the impact of GSK3 overexpression and knockout on the Nrf2 signaling. Understanding the interactions between the regulatory cascades linking homeostasis maintenance and cell response to oxidative stress will help in elucidating molecular mechanisms that underlie aging and longevity.

Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase.

Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which are widespread among pathogenic bacteria, are the least understood. In particular, the proton-pumping machinery of these Coxs has not yet been elucidated despite the availability of X-ray structure information. Here, we report the discovery of the first (to our knowledge) sodium-pumping Cox (Scox), a cbb3 cytochrome from the extremely alkaliphilic bacterium Thioalkalivibrio versutus. This finding offers clues to the previously unknown structure of the ion-pumping channel in the C-type Coxs and provides insight into the functional properties of this enzyme.

Neuroprotective Effects of Mitochondria-Targeted Plastoquinone in a Rat Model of Neonatal Hypoxic⁻Ischemic Brain Injury.

Neonatal hypoxia⁻ischemia is one of the main causes of mortality and disability of newborns. To study the mechanisms of neonatal brain cell damage, we used a model of neonatal hypoxia⁻ischemia in seven-day-old rats, by annealing of the common carotid artery with subsequent hypoxia of 8% oxygen. We demonstrate that neonatal hypoxia⁻ischemia causes mitochondrial dysfunction associated with high production of reactive oxygen species, which leads to oxidative stress. Targeted delivery of antioxidants to the mitochondria can be an effective therapeutic approach to treat the deleterious effects of brain hypoxia⁻ischemia. We explored the neuroprotective properties of the mitochondria-targeted antioxidant SkQR1, which is the conjugate of a plant plastoquinone and a penetrating cation, rhodamine 19. Being introduced before or immediately after hypoxia⁻ischemia, SkQR1 affords neuroprotection as judged by the diminished brain damage and recovery of long-term neurological functions. Using vital sections of the brain, SkQR1 has been shown to reduce the development of oxidative stress. Thus, the mitochondrial-targeted antioxidant derived from plant plastoquinone can effectively protect the brain of newborns both in pre-ischemic and post-stroke conditions, making it a promising candidate for further clinical studies.

Low concentration of uncouplers of oxidative phosphorylation decreases the TNF-induced endothelial permeability and lethality in mice.

Mitochondrial dysfunctions occur in many diseases linked to the systemic inflammatory response syndrome (SIRS). Mild uncoupling of oxidative phosphorylation is known to rescue model animals from pathologies related to mitochondrial dysfunctions and overproduction of reactive oxygen species (ROS). To study the potential of SIRS therapy by uncoupling, we tested protonophore dinitrophenol (DNP) and a free fatty acid (FFA) anion carrier, lipophilic cation dodecyltriphenylphosphonium (C12TPP) in mice and in vitro models of SIRS. DNP and C12TPP prevented the body temperature drop and lethality in mice injected with high doses of a SIRS inducer, tumor necrosis factor (TNF). The mitochondria-targeted antioxidant plastoquinonyl decyltriphenylphosphonium (SkQ1) which also catalyzes FFA-dependent uncoupling revealed similar protective effects and downregulated expression of the NFκB-regulated genes (VCAM1, ICAM1, MCP1, and IL-6) involved in the inflammatory response of endothelium in aortas of the TNF-treated mice. In vitro mild uncoupling rescued from TNF-induced endothelial permeability, disassembly of cell contacts and VE-cadherin cleavage by the matrix metalloprotease 9 (ММР9). The uncouplers prevented TNF-induced expression of MMP9 via inhibition of NFκB signaling. Water-soluble antioxidant Trolox also prevented TNF-induced activation and permeability of endothelium in vitro via inhibition of NFκB signaling, suggesting that the protective action of the uncouplers is linked to their antioxidant potential.

Protective effect of mitochondria-targeted antioxidants in an acute bacterial infection.

Acute pyelonephritis is a potentially life-threatening infection of the upper urinary tract. Inflammatory response and the accompanying oxidative stress can contribute to kidney tissue damage, resulting in infection-induced intoxication that can become fatal in the absence of antibiotic therapy. Here, we show that pyelonephritis was associated with oxidative stress and renal cell death. Oxidative stress observed in pyelonephritic kidney was accompanied by a reduced level of mitochondrial B-cell lymphoma 2 (Bcl-2). Importantly, renal cell death and animal mortality were both alleviated by mitochondria-targeted antioxidant 10(6'-plastoquinonyl) decylrhodamine 19 (SkQR1). These findings suggest that pyelonephritis can be treated by reducing mitochondrial reactive oxygen species and thus by protecting mitochondrial integrity and lowering kidney damage.

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

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.

Neuroprotective Effects of Mitochondria-Targeted Plastoquinone and Thymoquinone in a Rat Model of Brain Ischemia/Reperfusion Injury.

We explored the neuroprotective properties of natural plant-derived antioxidants plastoquinone and thymoquinone (2-demethylplastoquinone derivative) modified to be specifically accumulated in mitochondria. The modification was performed through chemical conjugation of the quinones with penetrating cations: Rhodamine 19 or tetraphenylphosphonium. Neuroprotective properties were evaluated in a model of middle cerebral artery occlusion. We demonstrate that the mitochondria-targeted compounds, introduced immediately after reperfusion, possess various neuroprotective potencies as judged by the lower brain damage and higher neurological status. Plastoquinone derivatives conjugated with rhodamine were the most efficient, and the least efficiency was shown by antioxidants conjugated with tetraphenylphosphonium. Antioxidants were administered intraperitoneally or intranasally with the latter demonstrating a high level of penetration into the brain tissue. The therapeutic effects of both ways of administration were similar. Long-term administration of antioxidants in low doses reduced the neurological deficit, but had no effect on the volume of brain damage. At present, cationic decylrhodamine derivatives of plastoquinone appear to be the most promising anti-ischemic mitochondria-targeted drugs of the quinone family. We suggest these antioxidants could be potentially used for a stroke treatment.

Evolution of cytochrome bc complexes: from membrane-anchored dehydrogenases of ancient bacteria to triggers of apoptosis in vertebrates.

This review traces the evolution of the cytochrome bc complexes from their early spread among prokaryotic lineages and up to the mitochondrial cytochrome bc1 complex (complex III) and its role in apoptosis. The results of phylogenomic analysis suggest that the bacterial cytochrome b6f-type complexes with short cytochromes b were the ancient form that preceded in evolution the cytochrome bc1-type complexes with long cytochromes b. The common ancestor of the b6f-type and the bc1-type complexes probably resembled the b6f-type complexes found in Heliobacteriaceae and in some Planctomycetes. Lateral transfers of cytochrome bc operons could account for the several instances of acquisition of different types of bacterial cytochrome bc complexes by archaea. The gradual oxygenation of the atmosphere could be the key evolutionary factor that has driven further divergence and spread of the cytochrome bc complexes. On the one hand, oxygen could be used as a very efficient terminal electron acceptor. On the other hand, auto-oxidation of the components of the bc complex results in the generation of reactive oxygen species (ROS), which necessitated diverse adaptations of the b6f-type and bc1-type complexes, as well as other, functionally coupled proteins. A detailed scenario of the gradual involvement of the cardiolipin-containing mitochondrial cytochrome bc1 complex into the intrinsic apoptotic pathway is proposed, where the functioning of the complex as an apoptotic trigger is viewed as a way to accelerate the elimination of the cells with irreparably damaged, ROS-producing mitochondria. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Cationic antioxidants as a powerful tool against mitochondrial oxidative stress.

This review describes evidence that mitochondrial reactive oxygen species (mROS) are of great importance under many physiological and pathological conditions. The most demonstrative indications favoring this conclusion originate from recent discoveries of the in vivo effects of mitochondria-targeted antioxidants (MitoQ and SkQs). The latter compounds look promising in treating several incurable pathologies as well as aging.

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

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.

Penetrating cation/fatty acid anion pair as a mitochondria-targeted protonophore.

A unique phenomenon of mitochondria-targeted protonophores is described. It consists in a transmembrane H(+)-conducting fatty acid cycling mediated by penetrating cations such as 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) or dodecyltriphenylphosphonium (C(12)TPP). The phenomenon has been modeled by molecular dynamics and directly proved by experiments on bilayer planar phospholipid membrane, liposomes, isolated mitochondria, and yeast cells. In bilayer planar phospholipid membrane, the concerted action of penetrating cations and fatty acids is found to result in conversion of a pH gradient (DeltapH) to a membrane potential (Deltapsi) of the Nernstian value (about 60 mV Deltapsi at DeltapH = 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C(12)TPP, but not cetyltrimethylammonium, potentiated fatty acid-induced (i) uncoupling of respiration and phosphorylation, and (ii) inhibition of H(2)O(2) formation. In intact yeast cells, C(12)TPP stimulated respiration regardless of the extracellular pH value, whereas a nontargeted protonophorous uncoupler (trifluoromethoxycarbonylcyanide phenylhydrazone) stimulated respiration at pH 5 but not at pH 3. Hydrophobic penetrating cations might be promising to treat obesity, senescence, and some kinds of cancer that require mitochondrial hyperpolarization.

Derivatives of rhodamine 19 as mild mitochondria-targeted cationic uncouplers.

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.

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

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.

Efim Liberman toward the comprehension of the phenomenon of life.

Efim Liberman was a proud scholar of biophysics-a genuine scientific discipline that strives for high accuracy in contrast to descriptive biology. He played a major role at the Pushchino Institute of Biophysics and participated in Gelfand's famous seminar. He developed a groundbreaking model of neural impulses and discovered the decisive role of cell membranes and membrane channels in neural cell excitation. Efim Liberman studied artificial and biological membranes, and conducted seminal experiments to proof Mitchell's hypothesis of oxidative phosphorylation. He discovered penetrating ions in pioneering studies of mitochondrial electrical energetics. He put forward a trailblazing idea of an intracell analog-digital molecular computer capable of using genetically coded algorithms for processing information. His ideas and research gave impetus to academic studies in various areas, such as a role of small RNАs as universal regulators of life. Efim Liberman was an outstanding, versatile and bright personality whose lifelong journey was to pursue the comprehension of the Universe and to understand the phenomenon of life.

Mitochondria-targeted penetrating cations as carriers of hydrophobic anions through lipid membranes.

High negative electric potential inside mitochondria provides a driving force for mitochondria-targeted delivery of cargo molecules linked to hydrophobic penetrating cations. This principle is utilized in construction of mitochondria-targeted antioxidants (MTA) carrying quinone moieties which produce a number of health benefitting effects by protecting cells and organisms from oxidative stress. Here, a series of penetrating cations including MTA were shown to induce the release of the liposome-entrapped carboxyfluorescein anion (CF), but not of glucose or ATP. The ability to induce the leakage of CF from liposomes strongly depended on the number of carbon atoms in alkyl chain (n) of alkyltriphenylphosphonium and alkylrhodamine derivatives. In particular, the leakage of CF was maximal at n about 10-12 and substantially decreased at n=16. Organic anions (palmitate, oleate, laurylsulfate) competed with CF for the penetrating cation-induced efflux. The reduced activity of alkylrhodamines with n=16 or n=18 as compared to that with n=12 was ascribed to a lower rate of partitioning of the former into liposomal membranes, because electrical current relaxation studies on planar bilayer lipid membranes showed rather close translocation rate constants for alkylrhodamines with n=18 and n=12. Changes in the alkylrhodamine absorption spectra upon anion addition confirmed direct interaction between alkylrhodamines and the anion. Thus, mitochondria-targeted penetrating cations can serve as carriers of hydrophobic anions across bilayer lipid membranes.