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2.
Dokl Biochem Biophys ; 495(1): 342-346, 2020 Nov.
Article in English | MEDLINE | ID: mdl-33368048

ABSTRACT

This work provides the first characteristics of the rhodopsin SpaR from Sphingomonas paucimobilis, aerobic bacteria associated with opportunistic infections. The sequence analysis of SpaR has shown that this protein has unusual DTS motif which has never reported in rhodopsins from Proteobacteria. We report that SpaR operates as an outward proton pump at low pH; however, proton pumping is almost absent at neutral and alkaline pH. The photocycle of this rhodopsin in detergent micelles slows down with an increase in pH because of longer Schiff base reprotonation. Our results show that the novel microbial ion transporter SpaR of interest both as an object for basic research of membrane proteins and as a promising optogenetic tool.


Subject(s)
Proton Pumps/metabolism , Rhodopsin/metabolism , Rhodopsins, Microbial/metabolism , Sphingomonas/metabolism , Hydrogen-Ion Concentration , Light , Optogenetics/methods , Proton Pumps/genetics , Rhodopsin/genetics , Rhodopsins, Microbial/genetics , Sphingomonas/genetics
3.
Biochemistry (Mosc) ; 85(12): 1578-1590, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33705296

ABSTRACT

Appending lipophilic cations to small molecules has been widely used to produce mitochondria-targeted compounds with specific activities. In this work, we obtained a series of derivatives of the well-known fluorescent dye 7-nitrobenzo-2-oxa-1,3-diazole (NBD). According to the previous data [Denisov et al. (2014) Bioelectrochemistry, 98, 30-38], alkyl derivatives of NBD can uncouple isolated mitochondria at concentration of tens of micromoles despite a high pKa value (~11) of the dissociating group. Here, a number of triphenylphosphonium (TPP) derivatives linked to NBD via hydrocarbon spacers of varying length (C5, C8, C10, and C12) were synthesized (mitoNBD analogues), which accumulated in the mitochondria in an energy-dependent manner. NBD-C10-TPP (C10-mitoNBD) acted as a protonophore in artificial lipid membranes (liposomes) and uncoupled isolated mitochondria at micromolar concentrations, while the derivative with a shorter linker (NBD-C5-TPP, or C5-mitoNBD) exhibited no such activities. In accordance with this data, C10-mitoNBD was significantly more efficient than C5-mitoNBD in suppressing the growth of Bacillus subtilis. C10-mitoNBD and C12-mitoNBD demonstrated the highest antibacterial activity among the investigated analogues. C10-mitoNBD also exhibited the neuroprotective effect in the rat model of traumatic brain injury.


Subject(s)
Anti-Bacterial Agents/pharmacology , Brain Injuries/prevention & control , Mitochondria, Liver/drug effects , Neuroprotective Agents/pharmacology , Nitrobenzenes/pharmacology , Organophosphorus Compounds/pharmacology , Oxadiazoles/pharmacology , Animals , Bacillus subtilis/drug effects , Disease Models, Animal , Energy Metabolism , Mitochondria, Liver/metabolism , Nitrobenzenes/chemistry , Organophosphorus Compounds/chemistry , Oxadiazoles/chemistry , Rats , Thermogenesis
4.
Biochemistry (Mosc) ; 84(10): 1151-1165, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31694511

ABSTRACT

Uncouplers of oxidative phosphorylation in mitochondria, which have been essential in elucidating the basic principles of cell bioenergetics, have recently attracted a considerable interest as compounds with therapeutic, e.g., neuroprotective, properties. Here, we report the effect of mitofluorescein (mitoFluo), a new protonophoric uncoupler representing a conjugate of fluorescein with decyl(triphenyl)phosphonium, on the electrical activity of neurons from Lymnaea stagnalis. Incubation with mitoFluo in the dark led to a decrease in the absolute value of the resting membrane potential of the neurons and alterations in their spike activity, such as spike broadening, spike amplitude reduction, and increase in the spike frequency. Prolonged incubation at high (tens micromoles) mitoFluo concentrations resulted in complete suppression of neuronal electrical activity. The effect of mitoFluo on the neurons was qualitatively similar to that of the classical mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) but manifested itself after much longer incubation and at higher concentrations. The distinctive feature of mitoFluo is its light-induced effect on the electrical activity of neurons. Changes in the parameters of the neuronal activity upon illumination in the presence of mitoFluo were similar to the light-induced effects of the well-known photosensitizer Rose Bengal, although less pronounced. It was suggested that the effects of mitoFluo on the electrical activity of neurons, both as a mitochondrial uncoupler and a photosensitizer, are mediated by the changes in the cytoplasmic calcium concentration.


Subject(s)
Electric Stimulation , Fluorescein/pharmacology , Fluorescent Dyes/pharmacology , Neurons/drug effects , Photochemotherapy , Protons , Animals , Fluorescein/chemistry , Fluorescent Dyes/chemistry , Neurons/metabolism , Ponds , Snails
5.
Biochemistry (Mosc) ; 82(10): 1140-1146, 2017 Oct.
Article in English | MEDLINE | ID: mdl-29037134

ABSTRACT

In this work, it was found that the ability of common uncouplers - carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol (DNP) - to reduce membrane potential of isolated rat liver mitochondria was diminished in the presence of millimolar concentrations of the known cytochrome c oxidase inhibitor - cyanide. In the experiments, mitochondria were energized by addition of ATP in the presence of rotenone, inhibiting oxidation of endogenous substrates via respiratory complex I. Cyanide also reduced the uncoupling effect of FCCP and DNP on mitochondria energized by succinate in the presence of ferricyanide. Importantly, cyanide did not alter the protonophoric activity of FCCP and DNP in artificial bilayer lipid membranes. The causes of the effect of cyanide on the efficiency of protonophoric uncouplers in mitochondria are considered in the framework of the suggestion that conformational changes of membrane proteins could affect the state of lipids in their vicinity. In particular, changes in local microviscosity and vacuum permittivity could change the efficiency of protonophore-mediated translocation.


Subject(s)
Carbonyl Cyanide m-Chlorophenyl Hydrazone/analogs & derivatives , Membrane Potential, Mitochondrial/drug effects , Mitochondria, Liver/drug effects , Uncoupling Agents/pharmacology , 2,4-Dinitrophenol/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Electron Transport Complex I/antagonists & inhibitors , Electron Transport Complex I/metabolism , Mitochondria, Liver/metabolism , Mitochondrial Membranes/drug effects , Potassium Cyanide/pharmacology , Rats , Rotenone/pharmacology
6.
Biochemistry (Mosc) ; 80(12): 1589-97, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26638684

ABSTRACT

A series of permeating cations based on alkyl derivatives of triphenylphosphonium (C(n)-TPP(+)) containing linear hydrocarbon chains (butyl, octyl, decyl, and dodecyl) was investigated in systems of isolated mitochondria, bacteria, and liposomes. In contrast to some derivatives (esters) of rhodamine-19, wherein butyl rhodamine possessed the maximum activity, in the case of C(n)-TPP a stimulatory effect on mitochondrial respiration steadily increased with growing length of the alkyl radical. Tetraphenylphosphonium and butyl-TPP(+) at a dose of several hundred micromoles exhibited an uncoupling effect, which might be related to interaction between C(n)-TPP(+) and endogenous fatty acids and induction of their own cyclic transfer, resulting in transport of protons across the mitochondrial membrane. Such a mechanism was investigated by measuring efflux of carboxyfluorescein from liposomes influenced by C(n)-TPP(+). Experiments with bacteria demonstrated that dodecyl-TPP(+), decyl-TPP(+), and octyl-TPP(+) similarly to quinone-containing analog (SkQ1) inhibited growth of the Gram-positive bacterium Bacillus subtilis, wherein the inhibitory effect was upregulated with growing lipophilicity. These cations did not display toxic effect on growth of the Gram-negative bacterium Escherichia coli. It is assumed that the difference in toxic action on various bacterial species might be related to different permeability of bacterial coats for the examined triphenylphosphonium cations.


Subject(s)
Bacillus subtilis/drug effects , Mitochondria, Liver/drug effects , Onium Compounds/toxicity , Organophosphorus Compounds/chemistry , Animals , Bacillus subtilis/metabolism , Fatty Acids/metabolism , Liposomes , Membrane Potential, Mitochondrial/drug effects , Mitochondria, Liver/metabolism , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Onium Compounds/chemistry , Organophosphorus Compounds/toxicity , Plastoquinone/analogs & derivatives , Plastoquinone/chemistry , Plastoquinone/toxicity , Protons , Rats , Structure-Activity Relationship
7.
Biochemistry (Mosc) ; 80(6): 745-51, 2015 Jun.
Article in English | MEDLINE | ID: mdl-26531019

ABSTRACT

The impact of double bonds in fatty acyl tails of unsaturated lipids on the photodynamic inactivation of ion channels formed by the pentadecapeptide gramicidin A in a planar bilayer lipid membrane was studied. The presence of unsaturated acyl tails protected gramicidin A against photodynamic inactivation, with efficacy depending on the depth of a photosensitizer in the membrane. The protective effect of double bonds was maximal with membrane-embedded chlorin e6-monoethylenediamine monoamide dimethyl ester, and minimal - in the case of water-soluble tri-sulfonated aluminum phthalocyanine (AlPcS3) known to reside at the membrane surface. By contrast, the protective effect of the hydrophilic singlet oxygen scavenger ascorbate was maximal for AlPcS3 and minimal for amide of chlorin e6 dimethyl ester. The depth of photosensitizer position in the lipid bilayer was estimated from the quenching of photosensitizer fluorescence by iodide. Thus, the protective effect of a singlet oxygen scavenger against photodynamic inactivation of the membrane-inserted peptide is enhanced upon location of the photosensitizer and scavenger molecules in close vicinity to each other.


Subject(s)
Gramicidin/chemistry , Ion Channels/chemistry , Lipid Bilayers/chemistry , Photosensitizing Agents/pharmacology , Ascorbic Acid/pharmacology , Gramicidin/metabolism , Hydrophobic and Hydrophilic Interactions , Indoles/chemistry , Ion Channels/metabolism , Lipid Bilayers/metabolism , Organometallic Compounds/chemistry , Photochemistry , Porphyrins/chemistry , Porphyrins/metabolism , Singlet Oxygen/chemistry , Singlet Oxygen/metabolism
8.
Biochemistry (Mosc) ; 77(9): 975-82, 2012 Sep.
Article in English | MEDLINE | ID: mdl-23157256

ABSTRACT

Boronated derivatives of porphyrins are studied extensively as promising compounds for boron-neutron capture therapy and photodynamic therapy. Understanding of the mechanism of their permeation across cell membranes is a key step in screening for the most efficient compounds. In the present work, we studied the ability of boronated derivatives of chlorin e(6) and porphyrins, which are mono-, di-, and tetra-anions, to permeate through planar bilayer lipid membranes (BLM). The translocation rate constants through the hydrophobic part of the lipid bilayer were estimated for monocarborane and its conjugate with chlorin e(6) by the method of electrical current relaxation. They were similar, 6.6 and 6.8 sec(-1), respectively. Conjugates of porphyrins carrying two and four carborane groups were shown to permeate efficiently through a BLM although they carry two charges and four charges, respectively. The rate of permeation of the tetraanion estimated by the BLM current had superlinear dependence on the BLM voltage. Because the resting potential of most mammalian cells is negative inside, it can be concluded that the presence of negatively-charged boronated groups in compounds should hinder the accumulation of the porphyrins in cells.


Subject(s)
Boron Compounds/metabolism , Fluorides/metabolism , Lipid Bilayers/metabolism , Porphyrins/metabolism , Anions/chemistry , Anions/metabolism , Boron Compounds/chemistry , Chlorophyllides , Fluorides/chemistry , Lipid Bilayers/chemistry , Molecular Structure , Porphyrins/chemistry
9.
Biochemistry (Mosc) ; 77(9): 983-95, 2012 Sep.
Article in English | MEDLINE | ID: mdl-23157257

ABSTRACT

Novel mitochondria-targeted compounds composed entirely of natural constituents have been synthesized and tested in model lipid membranes, in isolated mitochondria, and in living human cells in culture. Berberine and palmatine, penetrating cations of plant origin, were conjugated by nonyloxycarbonylmethyl residue with the plant electron carrier and antioxidant plastoquinone. These conjugates (SkQBerb, SkQPalm) and their analogs lacking the plastoquinol moiety (C10Berb and C10Palm) penetrated across planar bilayer phospholipid membrane in their cationic forms and accumulated in isolated mitochondria or in mitochondria in living human cells in culture. Reduced forms of SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations. In isolated mitochondria and in living cells, the berberine and palmatine moieties were not reduced, so antioxidant activity belonged exclusively to the plastoquinol moiety. In human fibroblasts, nanomolar SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by exogenous hydrogen peroxide. At higher concentrations, conjugates of berberine and palmatine induced proton transport mediated by free fatty acids both in model and in mitochondrial membrane. In mitochondria this process was facilitated by the adenine nucleotide carrier. As an example of application of the novel mitochondria-targeted antioxidants SkQBerb and SkQPalm to studies of signal transduction, we discuss induction of cell cycle arrest, differentiation, and morphological normalization of some tumor cells. We suggest that production of oxygen radicals in mitochondria is necessary for growth factors-MAP-kinase signaling, which supports proliferation and transformed phenotype.


Subject(s)
Berberine Alkaloids/chemistry , Berberine Alkaloids/metabolism , Berberine/chemistry , Berberine/metabolism , Mitochondria/metabolism , Plastoquinone/chemistry , Plastoquinone/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Antioxidants/chemistry , Antioxidants/metabolism , Antioxidants/pharmacology , Berberine/pharmacology , Berberine Alkaloids/pharmacology , Humans , Mitochondria/drug effects , Plastoquinone/pharmacology
10.
Biochemistry (Mosc) ; 77(9): 1029-37, 2012 Sep.
Article in English | MEDLINE | ID: mdl-23157263

ABSTRACT

It is generally accepted that mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150 mV. Due to this, high values of the membrane potential are highly dangerous, specifically under pathological conditions associated with oxidative stress. Mild uncoupling of oxidative phosphorylation is an approach to preventing hyperpolarization of the mitochondrial membrane. We confirmed data obtained earlier in our group that dodecylrhodamine 19 (C(12)R1) (a penetrating cation from SkQ family not possessing a plastoquinone group) has uncoupling properties, this fact making it highly potent for use in prevention of pathologies associated with oxidative stress induced by mitochondrial hyperpolarization. Further experiments showed that C(12)R1 provided nephroprotection under ischemia/reperfusion of the kidney as well as under rhabdomyolysis through diminishing of renal dysfunction manifested by elevated level of blood creatinine and urea. Similar nephroprotective properties were observed for low doses (275 nmol/kg) of the conventional uncoupler 2,4-dinitrophenol. Another penetrating cation that did not demonstrate protonophorous activity (SkQR4) had no effect on renal dysfunction. In experiments with induced ischemic stroke, C(12)R1 did not have any effect on the area of ischemic damage, but it significantly lowered neurological deficit. We conclude that beneficial effects of penetrating cation derivatives of rhodamine 19 in renal pathologies and brain ischemia may be at least partially explained by uncoupling of oxidation and phosphorylation.


Subject(s)
Brain Ischemia/drug therapy , Kidney/drug effects , Neuroprotective Agents/pharmacology , Rhabdomyolysis/drug therapy , Rhodamines/chemistry , Rhodamines/pharmacology , Uncoupling Agents/pharmacology , Animals , Brain Ischemia/metabolism , Brain Ischemia/pathology , Cations/chemistry , Cations/pharmacology , Cell Respiration/drug effects , Disease Models, Animal , Kidney/metabolism , Kidney/pathology , Neuroprotective Agents/chemistry , Oxidative Phosphorylation/drug effects , Rats , Reperfusion Injury/drug therapy , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Rhabdomyolysis/metabolism , Rhabdomyolysis/pathology , Uncoupling Agents/chemistry
11.
Biochemistry (Mosc) ; 77(9): 1038-43, 2012 Sep.
Article in English | MEDLINE | ID: mdl-23157264

ABSTRACT

Using dialkylphospholipid (diphytanyl phosphatidylcholine) instead of the conventional diacylphospholipid (diphytanoyl phosphatidylcholine) in planar lipid bilayer membranes (BLM) led to an increase in the diffusion potential of the penetrating cation plastoquinonyl-decyl-triphenylphosphonium (SkQ1), making it close to the Nernst value, and accelerated translocation of SkQ1 across the BLM as monitored by the kinetics of a decrease in the transmembrane electric current after applying a voltage (current relaxation). The consequences of changing from an ester to an ether linkage between the head groups and the hydrocarbon chains are associated with a substantial reduction in the membrane dipole potential known to originate from dipoles of tightly bound water molecules and carbonyl groups in ester bonds. The difference in the dipole potential between BLM formed of the ester phospholipid and that of the ether phospholipid was estimated to be 100 mV. In the latter case, suppression of SkQ1-mediated proton conductivity of the BLM was also observed.


Subject(s)
Cell Membrane Permeability/drug effects , Esters/chemistry , Ethers/chemistry , Lipid Bilayers/chemistry , Phospholipids/chemistry , Plastoquinone/analogs & derivatives , Cations/chemistry , Cations/pharmacology , Molecular Structure , Plastoquinone/chemistry , Plastoquinone/pharmacology
12.
J Membr Biol ; 222(3): 141-9, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18493812

ABSTRACT

The antioxidant activity of mitochondria-targeted small molecules, SkQ1 and MitoQ (conjugates of a lipophilic decyltriphenylphosphonium cation with an antioxidant moiety of a plastoquinone and ubiquinone, respectively), was studied in aqueous solution and in a lipid environment, i.e., micelles, liposomes and planar bilayer lipid membranes. Reactive oxygen species (ROS) were generated by azo initiators or ferrous ions with or without tert-butyl-hydroperoxide (t-BOOH). Chemiluminescence, fluorescence, oxygen consumption and inactivation of gramicidin peptide channels were measured to detect antioxidant activity. In all of the systems studied, SkQ1 was shown to effectively scavenge ROS. The scavenging was inherent to the reduced form of the quinone (SkQ1H(2)). In the majority of the above model systems, SkQ1 exhibited higher antioxidant activity than MitoQ. It is concluded that SkQ1H(2) operates as a ROS scavenger in both aqueous and lipid environments, being effective at preventing ROS-induced damage to membrane lipids as well as membrane-embedded peptides.


Subject(s)
Antioxidants/pharmacology , Free Radical Scavengers/pharmacology , Mitochondrial Membranes/drug effects , Onium Compounds/pharmacology , Plastoquinone/analogs & derivatives , Water/metabolism , Antioxidants/chemistry , Cations , Drug Delivery Systems , Free Radical Scavengers/chemistry , Gramicidin/antagonists & inhibitors , Gramicidin/pharmacology , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Liposomes , Micelles , Mitochondrial Membranes/chemistry , Models, Biological , Onium Compounds/chemistry , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/pharmacology , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/metabolism , Plastoquinone/chemistry , Plastoquinone/pharmacology , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Reactive Oxygen Species/toxicity , Ubiquinone/analogs & derivatives , Ubiquinone/chemistry , Ubiquinone/pharmacology , Water/chemistry
13.
Biochemistry (Mosc) ; 73(12): 1273-87, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19120014

ABSTRACT

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the "window" between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH*. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C(1/2) values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10(-11) and 8x10(-13) M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Deltapsi values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000 : 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3x10(8) times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.


Subject(s)
Aging , Antioxidants/metabolism , Mitochondria/metabolism , Plastoquinone/metabolism , Antioxidants/chemical synthesis , Antioxidants/chemistry , Apoptosis , Biological Transport , Cells, Cultured , Fibroblasts/chemistry , Fibroblasts/cytology , Fibroblasts/metabolism , HeLa Cells , Humans , Mitochondria/chemistry , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/metabolism , Necrosis , Oxidation-Reduction , Plastoquinone/analogs & derivatives , Plastoquinone/chemical synthesis
14.
J Membr Biol ; 199(1): 51-62, 2004 May 01.
Article in English | MEDLINE | ID: mdl-15366423

ABSTRACT

Chemical modification and photodynamic treatment of the colicin E1 channel-forming domain (P178) in vesicular and planar bilayer lipid membranes (BLMs) was used to elucidate the role of tryptophan residues in colicin E1 channel activity. Modification of colicin tryptophan residues by N-bromosuccinimide (NBS), as judged by the loss of tryptophan fluorescence, resulted in complete suppression of wild-type P178 channel activity in BLMs formed from fully saturated (diphytanoyl) phospholipids, both at the macroscopic-current and single-channel levels. The similar effect on both the tryptophan fluorescence and the electric current across BLM was observed also after NBS treatment of gramicidin channels. Of the single-tryptophan P178 mutants studied, W460 showed the highest sensitivity to NBS treatment, pointing to the importance of the water-exposed Trp460 in colicin channel activity. In line with previous work, the photodynamic treatment (illumination with visible light in the presence of a photosensitizer) led to suppression of P178 channel activity in diphytanoyl-phospholipid membranes concomitant with the damage to tryptophan residues detected here by a decrease in tryptophan fluorescence. The present work revealed novel effects: activation of P178 channels as a result of both NBS and photodynamic treatments was observed with BLMs formed from unsaturated (dioleoyl) phospholipids. These phenomena are ascribed to the effect of oxidative modification of double-bond-containing lipids on P178 channel formation. The pronounced stimulation of the colicin-mediated ionic current observed after both pretreatment with NBS and sensitized photomodification of the BLMs support the idea that distortion of membrane structure can facilitate channel formation.


Subject(s)
Colicins/metabolism , Gramicidin/metabolism , Ion Channels/physiology , Lipid Bilayers/metabolism , Membrane Lipids/metabolism , Bromosuccinimide/pharmacology , Ion Channels/drug effects , Mutation/genetics , Oxidation-Reduction/drug effects , Tryptophan/metabolism
15.
Biochemistry ; 43(15): 4575-82, 2004 Apr 20.
Article in English | MEDLINE | ID: mdl-15078104

ABSTRACT

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.


Subject(s)
Biotin/chemistry , Biotin/metabolism , Gramicidin/chemistry , Gramicidin/metabolism , Ion Channels/chemistry , Ion Channels/metabolism , Streptavidin/chemistry , Streptavidin/metabolism , Binding Sites , Biotinylation , Electric Conductivity , Gramicidin/antagonists & inhibitors , Ion Channels/antagonists & inhibitors , Kinetics , Ligands , Lipid Bilayers/chemistry , Models, Chemical , Mutation , Patch-Clamp Techniques , Photolysis , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/chemistry , Streptavidin/genetics , Surface Properties
16.
Biochemistry (Mosc) ; 69(2): 220-7, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15000691

ABSTRACT

Biotin-avidin (or streptavidin) high affinity binding has been widely applied as a universal tool for basic research as well as diagnostic and therapeutic purposes. Here we studied the interaction of streptavidin with ionic channels formed by biotinylated gramicidin in planar bilayer lipid membranes (BLM) using the method of sensitized photoinactivation. As shown previously, the addition of streptavidin leads to a profound increase in the lifetime (tau) of gA5XB, a biotinylated analog of gramicidin A with a linker arm of five aminocaproyl groups (Rokitskaya et al. (2000) Biochemistry, 39, 13053-13058). The present study has revealed that the increase in tau is related to multivalent interaction of streptavidin with biotinylated gramicidin, i.e., to formation of a complex of streptavidin with several gramicidin channels, whereas binding of streptavidin to a single channel does not change the value of tau. A rather long linker arm attaching biotin to the C-terminus of gramicidin appeared to be required for the multivalent interaction of streptavidin with gramicidin channels, as the increase in tau was not observed with channels formed by gA2XB, the biotinylated gramicidin analog with a linker arm comprising only two aminocaproyl groups. However, the formation of a stoichiometric (1 : 1) complex of streptavidin with gA2XB apparently occurred. The multivalent interaction of streptavidin with gA5XB disappeared if biotinylated lipids were included into the diphytanoylphosphatidylcholine membrane. It is suggested that the slowing of gramicidin channel kinetics provoked by streptavidin binding is due to membrane-mediated elastic interactions between two neighboring channels.


Subject(s)
Anti-Bacterial Agents/chemistry , Biotin/chemistry , Gramicidin/chemistry , Ion Channels/chemistry , Lipid Bilayers/chemistry , Streptavidin/chemistry , Anti-Bacterial Agents/metabolism , Avidin/chemistry , Avidin/metabolism , Biotin/metabolism , Gramicidin/metabolism , Ion Channels/metabolism , Kinetics , Lipid Bilayers/metabolism , Lipids/chemistry , Models, Chemical , Protein Binding
17.
J Membr Biol ; 189(2): 119-30, 2002 Sep 15.
Article in English | MEDLINE | ID: mdl-12235487

ABSTRACT

Clustering of membrane proteins, in particular of ion channels, plays an important role in their functioning. To further elucidate the mechanism of such ion channel activity regulation, we performed experiments with a model system comprising the negatively-charged gramicidin analog, O-pyromellitylgramicidin (OPg) that forms ion channels in bilayer lipid membrane (BLM), and polycations. The effect of polylysines on the kinetics of OPg channels in BLM was studied by the method of sensitized photoinactivation. As found in our previous work, the interaction of polylysine with OPg led to the deceleration of the OPg photoinactivation kinetics, i.e., to the increase in the characteristic time of OPg photoinactivation. It was shown here that in a certain range of polylysine concentrations the photoinactivation kinetics displayed systematic deviations from a monoexponential curve and was well described by a sum of two exponentials. The deviations from the monoexponential approximation were more pronounced with polylysines having a lower degree of polymerization. These deviations increased also upon the elevation of the ionic strength of the bathing solution and the addition of calcium ions. A theoretical model is presented that relates the OPg photoinactivation kinetics at different concentration ratios of OPg and polylysine to the distribution of OPg molecules among OPg-polylysine clusters of different stoichiometry. This model is shown to explain qualitatively the experimental results, although the quantitative description of the whole body of evidence requires further development, assuming that the interaction of polylysine with OPg causes segregation of membrane domains enriched in OPg channels. The single-channel data, which revealed the insensitivity of the single-channel lifetime of OPg to the addition of polylysine, are in good agreement with the theoretical model.


Subject(s)
Gramicidin/chemistry , Ion Channels/chemistry , Lipid Bilayers/chemistry , Models, Chemical , Polylysine/chemistry , Cations/chemistry , Gramicidin/analogs & derivatives , Gramicidin/radiation effects , Ion Channels/classification , Ion Channels/radiation effects , Kinetics , Light , Lipid Bilayers/radiation effects , Membrane Microdomains/chemistry , Membrane Microdomains/radiation effects , Phosphatidylcholines/chemistry , Phosphatidylcholines/radiation effects , Photochemistry , Sensitivity and Specificity , Static Electricity
18.
FEBS Lett ; 505(1): 147-50, 2001 Sep 07.
Article in English | MEDLINE | ID: mdl-11557058

ABSTRACT

The bacterial toxin colicin E1 is known to induce voltage-gated currents across a planar bilayer lipid membrane. In the present study, it is shown that the colicin-induced current decreased substantially upon illumination of the membrane in the presence of the photosensitizer, aluminum phthalocyanine. This effect was almost completely abolished by the singlet oxygen quencher, sodium azide. Using single tryptophan mutants of colicin E1, Trp495 was identified as the amino acid residue responsible for the sensitized photodamage of the colicin channel activity. Thus, the distinct participation of a specific amino acid residue in the sensitized photoinactivation of a defined protein function was demonstrated. It is suggested that Trp495 is critical for the translocation and/or anchoring of the colicin channel domain in the membrane.


Subject(s)
Colicins/chemistry , Lipid Bilayers/chemistry , Photochemistry , Tryptophan/chemistry , Amino Acid Substitution , Colicins/genetics , Indoles , Mutation , Organometallic Compounds , Osmolar Concentration
19.
Z Med Phys ; 11(1): 39-43, 2001.
Article in German | MEDLINE | ID: mdl-11487858

ABSTRACT

The electrostatic equilibrium on the surface of an ion-impermeable membrane was not influenced by ultrasound fields. Only after incorporation of an ion transporter did the ultrasound induce changes of the membrane surface potential. Because the ultrasound effect was completely reversible, measurements of the surface potential of a flat lipid bilayer membrane containing the calcium transporter calcimycin were performed, simultaneously to the ultrasound exposure. The ultrasound-induced volume flow, also called quartz wind, favored the mass transfer through the diffusion boundary close to the membrane, thereby leading to increased calcium concentrations in the immediate vicinity of the membrane. This, in turn, became manifest as a reduction of the negative surface charge density.


Subject(s)
Calcimycin , Lipid Bilayers/radiation effects , Membrane Potentials/radiation effects , Ultrasonics , Calcium , Kinetics , Static Electricity , Surface Properties
20.
Photochem Photobiol ; 74(1): 1-7, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11460529

ABSTRACT

Interaction of potent photodynamic agents, sulfonated aluminum phthalocyanines (AlPcSn where n is a number of sulfonic groups), with biological membranes was studied here using model systems: sensitized photoinactivation of gramicidin channels in planar lipid bilayers and adsorption on lipid monolayers. Fluoride anions known to form complexes with aluminum were found to inhibit both the adsorption of aluminum phthalocyanines on lipid monolayers, as measured with a Langmuir trough by surface pressure and surface potential changes, and photodynamic efficacy of the dyes, as studied by gramicidin channel photoinactivation. The similar effects were caused by the alkalinization of the medium. Fluoride anions appeared to be much more effective in the case of AlPcS4 as compared to AlPcS3. The suppression of the photodynamic potency of aluminum phthalocyanines was attributed to desorption of the dyes from lipid bilayers induced by fluoride or hydroxyl ions. With AlPcS4 an enhancement of the dye aggregation leading to a decrease in the sensitizing activity was probably involved in the fluoride effect as revealed by absorption and fluorescence spectral measurements. Capillary electrophoresis was employed to understand the mechanism of the dye desorption. The results of these experiments indicated that the reduction in the membrane affinity was associated with an increase in the negative charge of the dye molecules due to the binding of fluoride or hydroxyl ions.


Subject(s)
Fluorides/pharmacology , Gramicidin/radiation effects , Gramicidin/chemistry , Hydrogen-Ion Concentration , In Vitro Techniques , Indoles/pharmacology , Isoindoles , Lipid Bilayers/chemistry , Photochemistry , Photochemotherapy , Photosensitizing Agents/pharmacology
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