Membrane Ion Permeability Research Articles

Electrochemical characterization of mixed matrix heterogeneous cation exchange membrane modified by aluminum oxide nanoparticles: Mono/bivalent ionic transportation

Polyvinylchloride/aluminum oxide nanoparticles mixed matrix heterogeneous cation exchange membranes were prepared by solution casting technique. The effect of Al2O3 nanoparticles concentration on membrane electrochemical properties was studied. It was found that membrane water content was enhanced by increase of additive concentration in prepared membrane. Increase of aluminum oxide nanoparticles loading ratio in casting solution also caused to decrease in ion exchange capacity. Obtained results revealed that membrane permselectivity and transport number were improved initially in sodium chloride and barium chloride ionic solutions by increase of aluminum oxide nanoparticles concentration up to 2wt% in prepared membranes and then showed decreasing trend by more increase in additive content. Also membranes exhibited lower selectivity and transport number for bivalent ions in comparison with monovalent ones. Furthermore, membrane ionic permeability and flux were decreased initially by increase in aluminum oxide nanoparticles loading ratio up to 2wt% and then increased again by more additives concentration. Opposite trend was found for membrane areal electrical resistance. Results showed that modified membranes in this study are comparable with that of other commercial ones.

Ion permeability of artificial membranes evaluated by diffusion potential and electrical resistance measurements

In the present article, a novel model of artificial membranes that provides efficient assistance in teaching the origins of diffusion potentials is proposed. These membranes are made of polycarbonate filters fixed to 12-mm plastic rings and then saturated with a mixture of creosol and n-decane. The electrical resistance and potential difference across these membranes can be easily measured using a low-cost volt-ohm meter and home-made Ag/AgCl electrodes. The advantage of the model is the lack of ionic selectivity of the membrane, which can be modified by the introduction of different ionophores to the organic liquid mixture. A membrane treated with the mixture containing valinomycin generates voltages from -53 to -25 mV in the presence of a 10-fold KCl gradient (in to out) and from -79 to -53 mV in the presence of a bi-ionic KCl/NaCl gradient (in to out). This latter bi-ionic gradient potential reverses to a value from +9 to +20 mV when monensin is present in the organic liquid mixture. Thus, the model can be build stepwise, i.e., all factors leading to the development of diffusion potentials can be introduced sequentially, helping students to understand the quantitative relationships of ionic gradients and differential membrane permeability in the generation of cell electrical signals.

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.

Enhancing Effects on Vacuole-Targeting Fungicidal Activity of Amphotericin B

Invasive fungal infections are major threats for immunocompromised patients as well as for those undergoing cancer chemotherapy. Amphotericin B (AmB), a classical antifungal drug with a polyene macrolide structure, is widely used for the control of serious fungal infections. However, the clinical use of this antifungal drug is limited by its side effects and the emergence of drug-resistant strains. AmB lethality has been generally attributed to alterations in plasma membrane ion permeability due to its specific binding to plasma membrane ergosterol. Recent studies with Saccharomyces cerevisiae and Candida albicans reveal the vacuole disruptive action as another cause of AmB lethality on the basis of marked amplification of its activity in combination with allicin, an allyl-sulfur compound from garlic. The enhancing effect of allicin is dependent on the inhibition of ergosterol-trafficking from the plasma membrane to the vacuole membrane, which is considered to be a cellular response to protect against disintegration of the vacuole membrane. The polyol macrolide niphimycin (NM) also possesses vacuole-targeting fungicidal activity, which is greater than that of AmB and nystatin. The alkyl side chain attached to the macrolide ring of NM is considered to possess an allicin-like inhibitory effect on the intracellular trafficking of ergosterol. The vacuole-targeting fungicidal activity was additionally detected with a bactericidal cyclic peptide polymyxin B (PMB), and was markedly enhanced when administered together with allicin, monensin, or salinomycin. The synergistic fungicidal activities of AmB and allicin may have significant implications for the development of vacuole-targeting chemotherapy against fungal infections.

Preparation and characterization of poly (vinyl chloride)-blend-poly (carbonate) heterogeneous cation exchange membrane: Investigation of solvent type and ratio effects

In this research polyvinylchloride/polycarbonate blend heterogeneous cation exchange membranes were prepared by solution casting technique using cation exchange resin powder as functional groups agent. Tetrahydrofuran (THF) and dimethylformamide (DMF) were utilized as solvents. The effect of solvent type and ratio (THF/DMF mixture) on properties of prepared membranes was studied. SEM and SOM images showed relatively uniform particle distribution and also uniform surface for the membranes. Images showed that at high DMF ratio decrease of polymer conformation with particles surface reduces the compatibility of polymer-particle. The membrane ion exchange capacity and permeability were enhanced initially by increase of DMF ratio up to 5% (v/v) in casting solution and then they began to decrease with more DMF ratio. Results showed that membrane potential, transport number, selectivity and thermal stability all were decreased by DMF ratio increasing. Conversely, membrane water content, specific surface area and roughness showed opposite trends. Membrane electrical resistance initially declined by increase in DMF content up to 15% (v/v) and then it began to increase. The increase of electrolyte concentration also led to decrease in membrane transport number and selectivity. Membrane with (95:5) (v/v) solvent ratio (THF:DMF) exhibited more appropriate performance compared to others.

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

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

Direct Manipulation of a Single Potassium Channel Gate with an Atomic Force Microscope Probe

Ion channels are membrane proteins that regulate cell functions by controlling the ion permeability of cell membranes. An ion channel contains an ion-selective pore that permeates ions and a sensor that senses a specific stimulus such as ligand binding to regulate the permeability. The detailed molecular mechanisms of this regulation, or gating, are unknown. Gating is thought to occur from conformational changes in the sensor domain in response to the stimulus, which results in opening the gate to permit ion conduction. Using an atomic force microscope and artificial bilayer system, a mechanical stimulus is applied to a potassium channel, and its gating is monitored in real time. The channel-open probability increases greatly when pushing the cytoplasmic domain toward the membrane. This result shows that a mechanical stimulus at the cytoplasmic domain causes changes in the gating and is the first to show direct evidence of coupling between conformational changes in the cytoplasmic domain and channel gating. This novel technology has the potential to be a powerful tool for investigating the activation dynamics in channel proteins.

STEREOLOGY AND SOME STRUCTURAL CORRELATES OF RETINAL AND PHOTORECEPTOR CELL FUNCTION

The retina is the part of the eye which detects light, transduces it into nerve impulses and plays a significant role in visual perception. Sensitivity to light is multi-factorial and depends on the properties of photopigment molecules, their synthesis and incorporation into photoreceptor membranes and the neural circuitry between photoreceptor cells, bipolar neurons and ganglion neurons. In addition, it depends on structural factors such as the absolute and relative numbers of different types of photoreceptor neurons, their subcellular morphology, their distribution across the retina and the physical dimensions (especially surface areas) and spatial arrangements of their photoreceptor membranes. At the molecular level, these membranes harbour photosensitive pigment molecules comprising transmembrane glycoproteins (opsins, which vary between photoreceptor cells) and a non-protein chromophore. Phototransduction involves a conformational change in the chromophore and activation of an opsin. A transducer G protein, transducin, lowers levels of cGMP and triggers changes in membrane ion permeability including the closure of Na+ channels. This causes the plasmalemma to become less depolarized and the relative hyperpolarization stimulates ganglion cells whose axons form the optic nerve. Phosducin is a light-regulated phosphoprotein located in inner and outer segments of rod photoreceptor cells. It modulates phototransduction by binding to beta and gamma subunits of transducin. This review briefly illustrates ways in which stereology can contribute to our understanding of these processes by providing quantitative data on photoreceptor number, disk membrane surface area and the subcellular immunolocalisation of key molecules.

Structural, chemical surface and transport modifications of regenerated cellulose dense membranes due to low-dose γ-radiation

Modifications caused in commercial dense regenerated cellulose (RC) flat membranes by low-dose γ-irradiation (average photons energy of 1.23 MeV) are studied. Slight structural, chemical and morphological surface changes due to irradiation in three films with different RC content were determined by ATR-FTIR, XRD, XPS and AFM. Also, the alteration of their mechanical elasticity has been studied. Modification of membrane performance was determined from solute diffusion coefficient and effective membrane fixed charge concentration obtained from NaCl diffusion measurements. Induced structural changes defining new and effective fracture propagation directions are considered to be responsible for the increase of fragility of irradiated RC membranes. The same structural changes are proposed to explain the reduction of the membrane ion permeability through a mechanism involving either ion pathways elongation and/or blocking.

Preparation of chloromethylated/quaternized poly(phthalazinone ether ketone) anion exchange membrane materials for vanadium redox flow battery applications

Chloromethylated poly(phthalazinone ether ketone) (CMPPEK) was prepared from poly(phthalazinone ether ketone), with chloromethyl methyl ether (CME) as the chlromethylating reagent and concentrated sulfuric acid as the solvent. The effects of CME quantity, reaction temperature, and reaction time on degree of chloromethylation (DCM) were investigated. CMPPEK with DCM ranging from 0.73 to 2.32 mmol g −1 were obtained. CMPPEK were characterized with 1H NMR and TGA. CMPPEK membranes were prepared from CMPPEK/ N-methyl-2-pyrrolidinone casting solutions. Quaternized poly(phthalazinone ether ketone) (QAPPEK) anion exchange membranes were prepared from CMPPEK membranes with different DCM. The ion exchange capacity (IEC) and water content (Wc) of QAPPEK membranes were studied. QAPPEK membranes exhibited IEC ranging from 0.70 to 2.04 mmol g −1 and Wc ranging from 12.9 to 52.3%. IEC and Wc of QAPPEK increased with an increase in DCM of CMPPEK. The vanadium ion permeability of QAPPEK membranes was much lower than that of Nafion117 membrane. The performance of vanadium redox flow battery (VRB) single cell with QAPPEK membranes was investigated. Compare to VRB cell with Nafion117 membrane, the VRB single cell with QAPPEK membranes exhibited higher columbic efficiency. The results show that QAPPEK membranes could be promising anion exchange membranes for VRB applications.

Physical–chemical properties of plasma membrane and function of erythrocytes of cosmonauts after long-term space flight

We studied microfluidity and selective ion permeability of plasma membranes and O 2-binding properties of erythrocytes of cosmonauts during early rehabilitation after a long-term space flight (LTSF). Microfluidity of plasma membranes in surface regions was found to undergo a reversible decrease during 13–15 days following LTSF, which was accompanied by a reversible increase in relative cholesterol content. Cosmonauts’ erythrocytes revealed an increased activity of Na/H-exchanger and K Ca -channel as well as a decrease in number of discocytes and increase in number of echinocytes, stomatocytes and knizocytes. Total hemoglobin content as well as oxyhemoglobin content were lowered after the LTSF, while the affinity of hemoglobin to O 2 was advanced. It is suggested that the changes in Hb properties, microfluidity and selective permeability of plasma membranes following the elevated cholesterol content in the membranes can decrease tissue supply with O 2.

Understanding boron rejection by reverse osmosis membranes

Reverse osmosis (RO) membranes have high rejection for most of solutes in seawater except for boron. Therefore, boron rejection should be considered in the design and operation of the RO process for seawater desalination. In this paper, we investigated boron rejection and its relation to salt rejection using an irreversible thermodynamic model. Permeability constants for commercially-available RO membranes were obtained using theoretical model and the simulation data from membrane performance test program provided by membrane manufacturers. The effect of pH and concentration of the feed water on the boron rejection was also theoretically investigated under various operating conditions. The model calculations revealed that the rejection of boron follows a different mechanism from those of other ionic solutes and could not be readily correlated with ion rejections. To overcome the limit of mechanistic models, we explored an alternative approach for predicting boron permeability from membrane properties and ion permeability. It appears that this alternative approach can aid to achieve a better understanding of boron rejection by seawater RO membranes.

Preparation and characterization of ABS/HIPS heterogeneous cation exchange membranes with various blend ratios of polymer binder

In this research, acrylonitrile–butadiene–styrene (ABS)/high impact polystyrene (HIPS) blend heterogeneous cation exchange membranes were prepared by solution casting technique using tetrahydrofuran as solvent and cation exchange resin powder as functional group agent. The effect of blend ratio of polymers binder (ABS to HIPS) on properties of the prepared membranes was studied. Ultrasonic was used to appropriately disperse particles in the matrix of membrane. Scanning optical microscopy showed that sonication had a significant effect on the high distribution of resin particles in membranes. Moreover images showed a relatively uniform surface for membranes. The increment of HIPS ratio in casting solution led to a decline in water content of membranes. Ion exchange capacity, ion permeability, ionic flux, electrical conductivity and current efficiency of membranes were initially decreased by the increment of HIPS ratio to 10 wt% and then increased with increment from 10 to 80 wt%. However, they were decreased again in HIPS membrane. Opposite trend was found for energy consumption of membranes. Membrane potential, permselectivity, transport number and surface charge density of membranes were improved by improving the HIPS ratio to 80 wt%. In addition, the oxidative stability of membranes was increased and their mechanical strength was decreased by lowering the ABS ratio. Membrane with 20:80 (w/w) blend ratio of polymer binder (ABS to HIPS) exhibited appropriate efficiency and electrochemical properties in comparison with other prepared membranes in this research. However its mechanical strength was lower than the others. Moreover swelling of the prepared membranes was negligible.

Effect of entomocidal proteins from Bacillus thuringiensis on ion permeability of apical membranes of Tenebrio molitor larvae gut epithelium

Effects of entomocidal Cry-type proteins, delta-endotoxins Cry3A and Cry11A produced by Bacillus thuringiensis, on ion permeability of the apical membranes of intestinal epithelium from Tenebrio molitor larvae midgut were studied. Using potential-sensitive dyes safranine O and oxonol VI and DeltapH indicator acridine orange, it was shown that placing brush border membrane vesicles (BBMV) (loaded with Mg2+ during their preparation) into a salt-free buffer medium resulted in spontaneous generation of transmembrane electric potential on the vesicular membrane (negative inside the vesicles) accompanied by acidification of the aqueous phase inside the vesicles. The generation of transmembrane ion gradients on the vesicular membrane was a result of an electrogenic efflux of Mg2+ from the vesicles as shown by abolishing of the membrane potential by such agents as MgSO4 or CaCl2 in centimolar concentrations, a highly lipophilic cation tetraphenylphosphonium, and some blockers of cell membrane Ca2+-channels in submillimolar concentrations. A passive generation of membrane potential on the vesicular membrane (but positive inside the vesicles) was also observed upon addition of centimolar concentrations of K2SO4. Addition of delta-endotoxins Cry3A and Cry11A to the vesicle suspension in a salt-free buffer medium or in the same medium supplemented with centimolar concentrations of K2SO4 exerted a pronounced hyperpolarization of the vesicular membrane. This hyperpolarization was sensitive to the same agents, which abolished the membrane potential generation in the absence of delta-endotoxin. It is concluded that Cry proteins induced in BBMV from T. molitor opening pores or ion channels, which were considerably more permeable for alkaline- and alkaline-earth metal cations than for the accompanying anions.

Cisplatin impairs rat liver mitochondrial functions by inducing changes on membrane ion permeability: Prevention by thiol group protecting agents

Cisplatin (CisPt) is the most important platinum anticancer drug widely used in the treatment of head, neck, ovarian and testicular cancers. However, the mechanisms by which CisPt induces cytotoxicity, namely hepatotoxicity, are not completely understood. The goal of this study was to investigate the influence of CisPt on rat liver mitochondrial functions (Ca 2+-induced mitochondrial permeability transition (MPT), mitochondrial bioenergetics, and mitochondrial oxidative stress) to better understand the mechanism underlying its hepatotoxicity. The effect of thiol group protecting agents and some antioxidants against CisPt-induced mitochondrial damage was also investigated. Treatment of rat liver mitochondria with CisPt (20 nmol/mg protein) induced Ca 2+-dependent mitochondrial swelling, depolarization of membrane potential (Δ Ψ), Ca 2+ release, and NAD(P)H fluorescence intensity decay. These effects were prevented by cyclosporine A (CyA), a potent and specific inhibitor of the MPT. In the concentration range of up to 40 nmol/mg protein, CisPt slightly inhibited state 3 and stimulated state 2 and state 4 respiration rates using succinate as respiratory substrate. The respiratory indexes, respiratory control ratio (RCR) and ADP/O ratios, the Δ Ψ, and the ADP phosphorylation rate were also depressed. CisPt induced mitochondrial inner membrane permeabilization to protons (proton leak) but did not induce significant changes on mitochondrial H 2O 2 generation. All the effects induced by CisPt on rat liver mitochondria were prevented by thiol group protecting agents namely, glutathione (GSH), dithiothreitol (DTT), N-acetyl- l-cysteine (NAC) and cysteine (CYS), whereas superoxide-dismutase (SOD), catalase (CAT) and ascorbate (ASC) were without effect. In conclusion, the anticancer drug CisPt: (1) increases the sensitivity of mitochondria to Ca 2+-induced MPT; (2) interferes with mitochondrial bioenergetics by increasing mitochondrial inner membrane permeabilization to H +; (3) does not significantly affect H 2O 2 generation by mitochondria; (4) its mitochondrial damaging effects are protected by thiol group protecting agents. Based on these conclusions, it is possible to hypothesise that small changes on the redox-status of thiol groups, affecting membrane permeability to cations (Ca 2+ and H +) underlie CisPt-induced liver mitochondrial damage, putatively responsible for its hepatotoxicity. Therefore, we propose that CisPt-induced mitochondrial damage and consequent hepatotoxicity could be prevented by using thiol group protecting agents as therapeutic adjuvants.

Dielectrophoretic field-flow fractionation system for detection of aquatic toxicants.

Dielectrophoretic field-flow fractionation (dFFF) was applied as a contact-free way to sense changes in the plasma membrane capacitances and conductivities of cultured human HL-60 cells in response to toxicant exposure. A micropatterned electrode imposed electric forces on cells in suspension in a parabolic flow profile as they moved through a thin chamber. Relative changes in the dFFF peak elution time, reflecting changes in cell membrane area and ion permeability, were measured as indices of response during the first 150 min of exposure to eight toxicants having different single or mixed modes of action (acrylonitrile, actinomycin D, carbon tetrachloride, endosulfan, N-nitroso- N-methylurea (NMU), paraquat dichloride, puromycin, and styrene oxide). The dFFF method was compared with the cell viability assay for all toxicants and with the mitochondrial potentiometric dye assay or DNA alkaline comet assay according to the mode of action of the specific agents. Except for low doses of nucleic acid-targeting agents (actinomycin D and NMU), the dFFF method detected all toxicants more sensitively than other assays, in some cases up to 10 (5) times more sensitively than the viability approach. The results suggest the dFFF method merits additional study for possible applicability in toxicology.

Signal transduction mechanisms regulating ion fluxes in the sea urchin sperm

AbstractIon permeability changes involving ion channels and transporters are essential for fertilization, since they are key elements in sperm‐egg signaling and environmental sensing. Changes modulated by external factors and components from the outer layer of the homologous egg, like the sperm‐activating peptides and the fucose‐sulfate glycoconjugate, trigger complex signaling systems in the sperm that modulate how sperm swim, find the egg and fertilize it. These signaling systems depend on plasma membrane ion permeability and involve an alteration of second messenger levels, changes of membrane potential and intracellular Ca2+, Na+ and pH, along with changes in sperm morphology in the case of the acrosome reaction (AR). New procedures to elucidate the signaling pathways implicated in sperm ion transport have expanded the opportunities to dissect sperm‐egg signaling revealing how sperm ion channels participate in activation, motility, chemotaxis, and the AR. The sperm signaling pathways involve a large variety of ion channels and transporters, which are discretely localized and finely orchestrated to play specific roles that define the elaborate performance of sperm and allow a successful fertilization. In this review we will focus on the signaling molecules involved in the final aim of the sperm, to reach and fertilize the egg.

Involvement of Vesicular H+-ATPase in Insulin-Stimulated Glucose Transport in 3T3-F442A Adipocytes

In secretory cells, osmotic swelling of secretory granules is proposed to be an intermediate step in exocytic fusion of the granules with the plasma membrane. For osmotic swelling of the granule, a H (+) gradient generated by vacuolar-type H (+) -ATPase (V-ATPase) may be a driving force for accumulation of K (+) via its exchange with H (+) , concurrent with accumulation of Cl (-) and H(2)O. Here, we investigated whether a similar chemiosmotic mechanism is involved in the insulin-stimulated recruitment of GLUT4 to the plasma membrane in 3T3-F442A adipocytes. Incubating cells in a hypo-osmotic medium significantly increased 2-deoxy glucose (2-DG) uptake and the plasma membrane GLUT4 content (possibly via induction of osmotic swelling of GLUT4-containing vesicles (G4V)) and also potentiated the insulin-stimulated 2-DG uptake. Promotion of the G4V membrane ionic permeability using nigericin, an electroneutral K (+) /H (+) exchange ionophore, increased 2-DG uptake and the plasma membrane GLUT4 content. However, co-treatment with nigericin and insulin did not show an additive effect. Bafilomycin A(1), a diagnostically specific inhibitor of V-ATPase, inhibited insulin- and nigericin-stimulated 2-DG uptake. Immunoadsorption plus immunoblotting demonstrated that GLUT4 and V-ATPase co-localize in the same intracellular membranes. Together, these results indicate that V-ATPases in the G4V membrane may play an important role in the insulin-stimulated exocytic fusion of G4V with the plasma membrane via its participation in osmotic swelling of the vesicle.

Viroporin potential of the lentivirus lytic peptide (LLP) domains of the HIV-1 gp41 protein

BackgroundMechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development. Virally induced changes in membrane ionic permeability induced by lytic viruses of many families contribute to cytopathogenesis. HIV-1 induces disturbances in plasma membrane ion transport. The carboxyl terminus of TM (gp41) contains potential amphipathic α-helical motifs identified through their structural similarities to naturally occurring cytolytic peptides. These sequences have been dubbed lentiviral lytic peptides (LLP) -1, -2, and -3.ResultsPeptides corresponding to the LLP domains (from a clade B virus) partition into lipid membranes, fold into α-helices and disrupt model membrane permeability. A peptide corresponding to the LLP-1 domain of a clade D HIV-1 virus, LLP-1D displayed similar activity to the LLP-1 domain of the clade B virus in all assays, despite a lack of amino acid sequence identity.ConclusionThese results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin. Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs.

Simultaneous Monitoring of Ionophore- and Inhibitor-mediated Plasma and Mitochondrial Membrane Potential Changes in Cultured Neurons

Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV.