Characterization of a novel Ca2+-Activated potassium channel in rat brain rough endoplasmic reticulum.

OBJECTIVES: Potassium channels in the endoplasmic reticulum (ER) are crucial for maintaining calcium balance during calcium fluxes. Disruption in ER calcium balance leads to ER stress, implicated in diseases like diabetes and Alzheimer's disease (AD). However, limited data exists on ER potassium channels in excitable tissues such as the brain. To fill this gap, we aimed to evaluate potassium currents in rat brain rough endoplasmic reticulum (RER). METHODS: Rats were euthanized under deep anesthesia and their brains were immediately removed. The brains were then homogenized in ice-cold sucrose buffer, followed by the extraction of RER microsomes through a series of centrifugation processes. Purity of sample was evaluated using western blotting technique. Single channel recordings were done in voltage steps from +50 to -60 mV following incorporation of rat brain RER vesicles into planar bilayers. RESULTS: We observed a voltage-dependent potassium channel with an approximate conductance of 188 pS. Channel open probability was low at negative voltages, increasing at positive voltages. The channel was blocked by Charybdotoxin but not by Iberiotoxin. Additionally, TRAM-34, a specific KCa3.1 channel blocker, suppressed channel current amplitude and open probability. Western blot analysis revealed specific bands for anti-KCa3.1 antibody, approximately 50 kDa in brain homogenate and RER fraction. CONCLUSION: Our study provides strong evidence for the presence of an KCa3.1 channel on the RER membrane in rat brain, exhibiting distinct electro-pharmacological profile compared to plasma membrane and other organelles.

Membrane-Disruptive Effects of Fatty Acid and Monoglyceride Mitigants on E. coli Bacteria-Derived Tethered Lipid Bilayers.

We report electrochemical impedance spectroscopy measurements to characterize the membrane-disruptive properties of medium-chain fatty acid and monoglyceride mitigants interacting with tethered bilayer lipid membrane (tBLM) platforms composed of E. coli bacterial lipid extracts. The tested mitigants included capric acid (CA) and monocaprin (MC) with 10-carbon long hydrocarbon chains, and lauric acid (LA) and glycerol monolaurate (GML) with 12-carbon long hydrocarbon chains. All four mitigants disrupted E. coli tBLM platforms above their respective critical micelle concentration (CMC) values; however, there were marked differences in the extent of membrane disruption. In general, CA and MC caused larger changes in ionic permeability and structural damage, whereas the membrane-disruptive effects of LA and GML were appreciably smaller. Importantly, the distinct magnitudes of permeability changes agreed well with the known antibacterial activity levels of the different mitigants against E. coli, whereby CA and MC are inhibitory and LA and GML are non-inhibitory. Mechanistic insights obtained from the EIS data help to rationalize why CA and MC are more effective than LA and GML at disrupting E. coli membranes, and these measurement capabilities support the potential of utilizing bacterial lipid-derived tethered lipid bilayers for predictive assessment of antibacterial drug candidates and mitigants.

Retinal-Based Anion Pump from the Cyanobacterium Tolypothrix campylonemoides.

In this work, TcaR rhodopsin from the cyanobacterium Tolypothrix campylonemoides was characterized. Analysis of the amino acid sequence of TcaR revealed that this protein possesses a TSD motif that differs by only one amino acid from the TSA motif of the known halorhodopsin chloride pump. The TcaR protein was expressed in E. coli, purified, and incorporated into proteoliposomes and nanodiscs. Functional activity was measured by electric current generation through the planar bilayer lipid membranes (BLMs) with proteoliposomes adsorbed on one side of the membrane surface, as well as by fluorescence using the voltage-dependent dye oxonol VI. We have shown that TcaR rhodopsin functions as a powerful anion pump. Our results show that the novel microbial anion transporter, TcaR, deserves deeper investigation and may be of interest both for fundamental studies of membrane proteins and as a tool for optogenetics.

MXene supported biomimetic bilayer lipid membrane biosensor for zeptomole detection of BRCA1 gene.

A biomimetic bilayer lipid membrane supported MXene based biosensor is reported for electrochemical hybridization detection of the most prevalent and potential BC biomarker BRCA1. 2D MXene nanosheet-anchored gold nanoparticle-decorated biomimetic bilayer lipid membrane (AuNP@BLM) biosensor is used for the attachment of thiolated single-stranded DNA (HS-ssDNA) targeting hybridization detection. The interaction of biomimetic bilayer lipid membrane with 2D MXene nanosheets is explored in this work for the first time. The synergistic combination of MXene and AuNP@BLM has proven to efficiently improve the detection signal to several folds. The sensor provides hybridization signals only to the complementary DNA (cDNA) sequence with a linearity range 10 zM to 1 µM and LOD of 1 zM without the need of any further amplification. The specificity of the biosensor is validated using non-complementary (ncDNA) and double base mis-match oligonucleotide DNA (dmmDNA) sequences. The sensor successfully distinguishes the signal for different target DNAs with good reproducibility indicated by the RSD value of 4.9%. Hence, we envision that the reported biosensor can be used to construct efficient diagnostic point-of-care tools based on molecular affinity interactions.

Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes.

Membrane proteins play an important role in key cellular functions, such as signal transduction, apoptosis, and metabolism. Therefore, structural and functional studies of these proteins are essential in fields such as fundamental biology, medical science, pharmacology, biotechnology, and bioengineering. However, observing the precise elemental reactions and structures of membrane proteins is difficult, despite their functioning through interactions with various biomolecules in living cells. To investigate these properties, methodologies have been developed to study the functions of membrane proteins that have been purified from biological cells. In this paper, we introduce various methods for creating liposomes or lipid vesicles, from conventional to recent approaches, as well as techniques for reconstituting membrane proteins into artificial membranes. We also cover the different types of artificial membranes that can be used to observe the functions of reconstituted membrane proteins, including their structure, number of transmembrane domains, and functional type. Finally, we discuss the reconstitution of membrane proteins using a cell-free synthesis system and the reconstitution and function of multiple membrane proteins.

In Situ Spectroscopic Detection of Large-Scale Reorientations of Transmembrane Helices During Influenza A M2 Channel Opening.

Viroporins are small ion channels in membranes of enveloped viruses that play key roles during viral life cycles. To use viroporins as drug targets against viral infection requires in-depth mechanistic understanding and, with that, methods that enable investigations under in situ conditions. Here, we apply surface-enhanced infrared absorption (SEIRA) spectroscopy to Influenza A M2 reconstituted within a solid-supported membrane, to shed light on the mechanics of its viroporin function. M2 is a paradigm of pH-activated proton channels and controls the proton flux into the viral interior during viral infection. We use SEIRA to track the large-scale reorientation of M2's transmembrane α-helices in situ during pH-activated channel opening. We quantify this event as a helical tilt from 26° to 40° by correlating the experimental results with solid-state nuclear magnetic resonance-informed computational spectroscopy. This mechanical motion is impeded upon addition of the inhibitor rimantadine, giving a direct spectroscopic marker to test antiviral activity. The presented approach provides a spectroscopic tool to quantify large-scale structural changes and to track the function and inhibition of the growing number of viroporins from pathogenic viruses in future studies.

Mechanistic Evaluation of Antimicrobial Lipid Interactions with Tethered Lipid Bilayers by Electrochemical Impedance Spectroscopy.

There is extensive interest in developing real-time biosensing strategies to characterize the membrane-disruptive properties of antimicrobial lipids and surfactants. Currently used biosensing strategies mainly focus on tracking membrane morphological changes such as budding and tubule formation, while there is an outstanding need to develop a label-free biosensing strategy to directly evaluate the molecular-level mechanistic details by which antimicrobial lipids and surfactants disrupt lipid membranes. Herein, using electrochemical impedance spectroscopy (EIS), we conducted label-free biosensing measurements to track the real-time interactions between three representative compounds-glycerol monolaurate (GML), lauric acid (LA), and sodium dodecyl sulfate (SDS)-and a tethered bilayer lipid membrane (tBLM) platform. The EIS measurements verified that all three compounds are mainly active above their respective critical micelle concentration (CMC) values, while also revealing that GML induces irreversible membrane damage whereas the membrane-disruptive effects of LA are largely reversible. In addition, SDS micelles caused membrane solubilization, while SDS monomers still caused membrane defect formation, shedding light on how antimicrobial lipids and surfactants can be active in, not only micellar form, but also as monomers in some cases. These findings expand our mechanistic knowledge of how antimicrobial lipids and surfactants disrupt lipid membranes and demonstrate the analytical merits of utilizing the EIS sensing approach to comparatively evaluate membrane-disruptive antimicrobial compounds.

Usnic Acid-Mediated Exchange of Protons for Divalent Metal Cations across Lipid Membranes: Relevance to Mitochondrial Uncoupling.

Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic proton exchange for calcium or magnesium cations. Here, we evaluated the ability of various divalent metal cations to stimulate a proton transport through both planar and vesicular bilayer lipid membranes by measuring the transmembrane electrical current and fluorescence-detected pH gradient dissipation in pyranine-loaded liposomes, respectively. Thus, we obtained the following selectivity series of calcium, magnesium, zinc, manganese and copper cations: Zn2+ > Mn2+ > Mg2+ > Ca2+ >> Cu2+. Remarkably, Cu2+ appeared to suppress the UA-mediated proton transport in both lipid membrane systems. The data on the divalent metal cation/proton exchange were supported by circular dichroism spectroscopy of UA in the presence of the corresponding cations.

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

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.

Lipoic acid nanoforms based on phosphatidylcholine: production and characteristics.

We describe the dynamics of lipoic acid (LA) alone, incorporated in liposomes and as a part of nanoemulsions. Mass spectrometry shows that LA in water forms aggregates of two or three molecules in the form of a negatively charged ion and a neutral molecule. Phosphatidylcholine (PC)-based nanoforms of LA as liposomes and nanoemulsions with a particle size equal to 145 nm are characterized by a high degree of incorporation of LA into the nanoparticles and long-term stability during storage at room temperature. Dynamic light scattering (DLS) gives the polydispersity index of the nanoforms (> 0.3), characterizing the homogeneity of the obtained nanodispersions. We found that such emulsions can significantly (5 ×) increase the concentration of LA in the aqueous phase (5-7 mg/mL) when compared with an aqueous solution of LA (1 mg/mL) and by 40% when compared with PC liposomes (4 mg/mL). Moreover, the inclusion of LA in liposomes and nanoemulsions from PC did not change the neutral ζ-potential characteristic of PC nanoforms. CryoTEM established that the structural organization of the liposomes practically did not differ from nanoemulsions and both nanoforms contained both multilayer and single-layer vesicles. When studying the release kinetics of LA from phosphatidylcholine nanoforms, we found that at 22 h, 45-55% of LA was released from nanoparticles, but that at the initial stage of the process LA was slowly released from the nanoemulsions and rapidly from the liposomes. Conductance measurements indicate that LA delivered in all the three forms increase membrane permeability, though this result is most marked with the LA in PC liposomes.

Porin from Marine Bacterium Marinomonas primoryensis KMM 3633T: Isolation, Physico-Chemical Properties, and Functional Activity.

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin.

Integrated microfluidic biosensing platform for simultaneous confocal microscopy and electrophysiological measurements on bilayer lipid membranes and ion channels.

Combining high-resolution imaging and electrophysiological recordings is key for various types of experimentation on lipid bilayers and ion channels. Here, we propose an integrated biosensing platform consisting of a microfluidic cartridge and a dedicated chip-holder to conduct such dual measurements on suspended lipid bilayers, in a user-friendly manner. To illustrate the potential of the integrated platform, we characterize lipid bilayers in terms of thickness and fluidity while simultaneously monitoring single ion channel currents. For that purpose, POPC lipid bilayers are supplemented with a fluorescently-tagged phospholipid (NBD-PE, 1% mol) for Fluorescence Recovery After Photobleaching (FRAP) measurements and a model ion channel (gramicidin, 1 nM). These combined measurements reveal that NBD-PE has no effect on the lipid bilayer thickness while gramicidin induces thinning of the membrane. Furthermore, the presence of gramicidin does not alter the lipid bilayer fluidity. Surprisingly, in lipid bilayers supplemented with both probes, a reduction in gramicidin open probability and lifetime is observed compared to lipid bilayers with gramicidin only, suggesting an influence of NBD-PE on the gramicidin ion function. Altogether, our proposed microfluidic biosensing platform in combination with the herein presented multi-parametric measurement scheme paves the way to explore the interdependent relationship between lipid bilayer properties and ion channel function.

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

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.

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

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.

Alkyl-substituted phenylamino derivatives of 7-nitrobenz-2-oxa-1,3-diazole as uncouplers of oxidative phosphorylation and antibacterial agents: involvement of membrane proteins in the uncoupling action.

In search for new effective uncouplers of oxidative phosphorylation, we studied 4-aryl amino derivatives of a fluorescent group 7-nitrobenz-2-oxa-1,3-diazol (NBD). In our recent work (Denisov et al., Bioelectrochemistry, 2014), NBD-conjugated alkyl amines (NBD-Cn) were shown to exhibit uncoupling activity. It was concluded that despite a pKa value being about 10, the expected hindering of the uncoupling activity could be overcome by insertion of an alkyl chain. There is evidence in the literature that the introduction of an aryl substituent in the 4-amino NBD group shifts the pKa to neutral values. Here we report the data on the properties of a number of 4-arylamino derivatives of NBD, namely, alkylphenyl-amino-NBD (Cn-phenyl-NBD) with varying alkyl chain Cn. By measuring the electrical current across planar bilayer lipid membrane, the protonophoric activity of Cn-phenyl-NBD at neutral pH grew monotonously from C1- to C6-phenyl-NBD. All of these compounds increased the respiration rate and reduced the membrane potential of isolated rat liver mitochondria. Importantly, the uncoupling action of C6- and C4-phenyl-NBD was partially reversed by glutamate, diethyl pyrocarbonate (DEPC), 6-ketocholestanol, and carboxyatractyloside, thus pointing to the involvement of membrane proteins in the uncoupling activity of Cn-phenyl-NBD in mitochondria. The pronounced recoupling effect of DEPC, an inhibitor of an aspartate-glutamate carrier (AGC), and that of its substrates for the first time highlighted AGC participation in the action of potent uncouplers on mitochondria. C6-phenyl-NBD produced strong antimicrobial effect on Bacillus subtilis, which manifested itself in cell membrane depolarization and suppression of bacterial growth at submicromolar concentrations.

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

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.

Expression and characterization of the Plasmodium translocon of the exported proteins component EXP2.

The malaria parasite Plasmodium falciparum requires the Plasmodium translocon of exported proteins (PTEX) to proliferate in human red blood cells. During the blood stages of malaria, several hundred parasite-encoded proteins are exported from the parasite into the cytosol of red blood cells. PTEX is the translocon for protein export and comprises 5 proteins: EXP2, PTEX150, PTEX88, Hsp101 and TRX2. Among them, EXP2 is thought to constitute the transmembrane pore, whereas the other components seem to play a role in unfolding the luggage proteins or providing a driving force. However, detailed functional and structural characterizations of PTEX proteins have not been performed. In this study, we expressed and characterized the membrane-associated component EXP2. Because expression of EXP2 is lethal to E. coli, EXP2 was expressed as a fusion protein with GST, and the recombinant EXP2 was obtained by protease digestion. The recombinant EXP2 formed pores in bilayer lipid membranes. The inner diameter of the pore was estimated to be approximately 3.5 nm based on electron microscopy images and channel currents. From this size and the molecular mass as determined by size exclusion chromatography and blue native polyacrylamide gel electrophoresis, we determined that the pore comprises approximately 10-12 EXP2 subunits. However, there is a possibility that the pore structure is different in the PTEX complex. These results provide important insights in the protein transport mechanism of PTEX, which will aid in developing new drugs targeting PTEX.

Biomimetic Membranes for Multi-Redox Center Proteins.

His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs). An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA) terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM). MRPs, such as the cytochrome c oxidase (CcO) from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs) from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced resonance Raman spectroscopy (SERRS), were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs). The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM) and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

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

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.

Dodecyl and octyl esters of fluorescein as protonophores and uncouplers of oxidative phosphorylation in mitochondria at submicromolar concentrations.

In our search for fluorescent uncouplers of oxidative phosphorylation, three esters of fluorescein, n-butyl-, n-octyl-, and n-dodecyl-oxycarbonyl-fluorescein (C4-FL, C8-FL, C12-FL) were synthesized and characterized. With increasing liposomal lipid content, the long-chain alkyl derivatives of fluorescein (C8-FL, C12-FL and commercially available C18-FL), but not C4-FL and unsubstituted fluorescein, exhibited an increase in fluorescence polarization reflecting the dye binding to liposomes. C12-FL induced proton permeability in lipid membranes, while C4-FL was inactive. In contrast to C4-FL and C18-FL, C12-FL and C8-FL increased the respiration rate and decreased the membrane potential of isolated rat liver mitochondria with half-maximal effective concentrations of 700nM and 300nM, respectively. The effect of Cn-FL on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by induction of mitochondria swelling in the potassium acetate medium. Binding of C8-FL to mitochondria depended on their energization, which was apparently associated with pH gradient generation across the inner mitochondrial membrane in the presence of a respiratory substrate. In wild-type yeast cells, C12-FL localized predominantly in plasma membrane, whereas in AD1-8 mutants lacking MDR pumps, it stained cytoplasmic organelles with some preference for mitochondria. Fluorescent uncouplers can be useful as a tool for determining their localization in a cell or distribution between different tissues in a living animal by fluorescent microscopy.