Alamethicin incorporation in lipid bilayers: a thermodynamic study

Interaction of the peptide antibiotic alamethicin with phospholipid vesicles has been monitored by changes in its circular dichroic and fluorescent properties. The data are consistent with an incorporation of the peptide in the lipid bilayer. Aggregation of alamethicin in the membrane phase is evident from a characteristic concentration dependence of the incorporation, reflecting the existence of a critical concentration. The data can be fully understood in terms of a theoretical approach that includes aggregation and thermodynamic nonideality. Thermodynamic parameters of the peptide-lipid interaction have been evaluated under a variety of conditions of temperature, ionic strength, and lipid type (saturated and unsaturated fatty acid chains). From the results obtained in this study, one can extrapolate to the incorporation behavior of alamethicin at low concentrations, as they are typical for measurements of conductance across planar lipid films. This leads to a simple explanation of the voltage-gating mechanism of alamethicin in a straightforward way.     

Fluorescent alamethicin fragments A study of membrane activity and aqueous phase aggregation

The linear polypeptide antibiotic alamethicin is known to form channels in artificial lipid membranes. Synthetic 13- and 17-residue alamethicin fragments, labelled with a fluorescent dansyl group at the N-terminus, have been shown to translocate divalent cations across phospholipid membranes and to uncouple oxidative phosphorylation in rat liver mitochondria, in a manner analogous to the parent peptides. From studies of the aqueous phase aggregation behavior of the peptides, as well as their interaction with rat liver mitochondria, it is concluded that the interaction of the peptides with membranes is a complex process, probably involving both aqueous and membrane phase aggregation.     

Voltage-dependent conductance for alamethicin in phospholipid vesicles. A test for the mechanism of gating.

The ion currents induced by alamethicin were investigated in unilamellar vesicles using electron paramagnetic resonance probe techniques. The peptide induced currents were examined as a function of the membrane bound peptide concentration, and as a function of the transmembrane electrical potential. Because of the favorable partitioning of alamethicin to membranes and the large membrane area to aqueous volume in vesicle suspensions, these measurements could be carried out under conditions where all the alamethicin was membrane bound. Over the concentration range examined, the peptide induced conductances increased approximately with the fourth power of the membrane bound peptide concentration, indicating a channel molecularity of four. When the alamethicin induced currents were examined as a function of voltage, they exhibited a superlinear behavior similar to that seen in planar bilayers. Evidence for the voltage-dependent conduction of alamethicin was also observed in the time dependence of vesicle depolarization. These observations indicate that the voltage-dependent behavior of alamethicin can occur in the absence of a voltage-dependent phase partitioning. That is, a voltage-dependent conformational rearrangement for membrane bound alamethicin leads to a voltage-dependent activity.     

Effect of the protein kinase C inhibitor on changes in Ca(2+) level in pig granulosa cells induced by prolactin

The effect of the PKC inhibitor on Ca2+ responses to prolactin in the pig granulosa cells was studied using fluorescent dye and chlortetracycline. The effect was shown to be connected with activation of the PKC. The Ro 31-8220 increased penetration of extracellular calcium and exit of calcium from intracellular stores. The data obtained suggest an involvement of the PKC in changes of calcium contents in the pig granulosa cells activated by prolactin.     

Role of membrane potential and calcium in chemotactic sensing by bacteria

Changes in Escherichia coli membrane potential in response to a wide variety of chemotactic stimuli were measured using a permeant, lipophilic cation (tetraphenylphosphonium chloride) and a fluorescent dye (3-propyl-2-(5-(3-propyl-2(3H)-benzothiazolylidene)-1,3-pentadienyl)iodide). Some attractants and repellants caused permanent, monotonic depolarizations or hyperpolarizations. Not all chemoeffectors, however, produced potential changes, and the direction of change did not correlate with physiological responses to these compounds. Moreover, changes were observed in a number of chemotactic mutants. From these results, we conclude that perturbations in membrane potential effected by chemical stimuli are not related to chemotactic sensing. These findings, and the close correlation between cytoplasmic ionic conditions and membrane potential, led us to examine the role of calcium in chemotaxis. By growing cells in the presence of a calcium chelator, we were able to lower cellular calcium levels over tenfold, with no change in behavior. These results indicate that sensory transduction in these cells is not mediated by this cation.     

Differential gel electrophoresis and transgenic mitochondrial calcium reporters demonstrate spatiotemporal filtering in calcium control of mitochondria

Mitochondria must adjust both their intracellular location and their metabolism in order to balance their output to the needs of the cell. Here we show by the proteomic technique of time series difference gel electrophoresis that a major result of neuroendocrine stimulation of the Drosophila renal tubule is an extensive remodeling of the mitochondrial matrix. By generating Drosophila that were transgenic for both luminescent and fluorescent mitochondrial calcium reporters, it was shown that mitochondrial calcium tracked the slow (minutes) but not the rapid (<1 s) changes in cytoplasmic calcium and that this resulted in both increased mitochondrial membrane polarization and elevated cellular ATP levels. The selective V-ATPase inhibitor, bafilomycin, further enhanced ATP levels, suggesting that the apical plasma membrane V-ATPase is a major consumer of ATP. Both the mitochondrial calcium signal and the increase in ATP were abolished by the mitochondrial calcium uniporter blocker Ru360. By using both mitochondrial calcium imaging and the potential sensing dye JC-1, the apical mitochondria of principal cells were found to be selectively responsive to neuropeptide signaling. As the ultimate target is the V-ATPase in the apical plasma membrane, this selective activation of mitochondria is clearly adaptive. The results highlight the dynamic nature and both spatial and temporal heterogeneity of calcium signaling possible in differentiated, organotypic cells and provide a new model for neuroendocrine control of V-ATPase.     

Excitation and detection of action potential-induced fluorescence changes through a single monomode optical fiber

An optical probe capable of detecting intracellular potential changes in individual cells, in vitro, which has the potential for in vivo applications, has been developed. A single-mode optical fiber directs laser light onto cells stained with the voltage-sensitive fluorescent dye, WW781 and also returns part of the resulting fluorescence to a detection system. Frog cardiac cells in vitro were used in these initial experiments. The fractional change in fluorescent intensity of 10(-3) for a 50 mV shift in transmembrane potential obtained from a heart immobilized in zero calcium Ringer's solution is comparable to that reported for other optical methods. For hearts in normal calcium Ringer's solutions, very large reproducible motion related artifacts were detected.     

Quantitation of lymphocyte intracellular free calcium signals using indo-1

The calcium-responsive fluorescent dye indo-1 has been used in lymphocyte suspensions to measure changes in internal free calcium concentration, [Ca2+]i, in response to crosslinking of cell surface immunoglobulin. The quantitation of [Ca2+]i requires that indo-am ester used to load the cells be completely hydrolyzed to the indo-1 form inside the cells. This is shown to be greatly facilitated in the lymphocyte by the detergent Pluronic F-127. The quantitation of [Ca2+]i transients also requires an estimate of the fraction of the cells which contribute to the observed changes. The use of excessive amounts of intracellular dye can buffer [Ca2+]i transients and this effect has been used to estimate the size of the pool of calcium which is available for release when the B cell is stimulated by anti-immunoglobulin.     

Calcium regulates the mode of exocytosis induced by hypotonic shock in isolated neuronal presynaptic endings

A decrease in the osmolarity of incubation medium is accompanied by calcium influx in neuronal presynaptic endings. We studied the influence of Ca2+ on exocytosis induced by hypotonic shock using the hydrophilic fluorescent dye acridine orange and the hydrophobic fluorescent dye FM2-10. It was shown using acridine orange that lowering of osmolarity to 230 mOsm/l induces exocytosis both in calcium-containing and calcium-free medium. By contrast, we were able to demonstrate calcium-dependence of exocytosis using styryl dye FM2-10. Lowering of osmolarity leads to increase of [3H]D-aspartate and [3H]GABA release in calcium-free medium. Addition of calcium inhibits hypotonic-induced neurotransmitter release. Decreasing of NaCl concentration to 92 mM in isotonic medium is able to induce d-aspartate and GABA release. Thus, our data suggest that hypotonic swelling induces calcium-independent exocytosis possibly by a "kiss and run" mechanism. Calcium influx mediated by stretch channels is able to provoke full fusion between plasma membrane and synaptic vesicles. [3H]D-aspartate and [3H]GABA released by hypotonic shock is determined by sodium lowering rather than by osmolarity decreasing itself.     

Real‐time calcium measurements of live optically trapped microorganisms

A system has been developed that allows for the real‐time measurement of calcium dynamics in swimming sperm. Specifically, the ratiometric dye Indo‐I is used as a fluorescent indicator of intracellular calcium dynamics. The dual emissions are collected by a high‐sensitivity back‐illuminated CCD camera coupled to a Dual‐View imaging system. From the CCD, the images are sent to a custom developed algorithm which processes the images and outputs the calcium measurements in real‐time. Additionally, sperm velocity and position data are processed and outputted in real‐time. The velocity and position data are obtained using a separate coupled red light (>670 nm) phase contrast imaging setup that does not optically interfere with the fluorescent imaging. Using this system the effects of optical trapping on calcium dynamics was determined. Optical trapping of sperm with a decaying focused laser power of 510 mW to 3 mW over 8 seconds causes a statistically insignificant change in calcium dynamics between in‐trap and out‐of‐trap conditions. Progesterone, a calcium activator, was added and sperm were trapped under the 8 second power decay conditions. Progesterone treated sperm has a statistically higher average calcium level than untreated sperm, but shows no statistical difference between progesterone treated in‐trap and out‐of‐trap conditions. Trapping at 16 seconds at 510 mW without decay, which have been shown to decrease sperm motility, shows a statistical difference between baseline pre‐trap and in‐trap intracellular calcium levels. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Relation between the passive transport of calcium into vesicles of the myocardial sarcolemma and membrane potential

The effect of membrane potential on the passive 45Ca2+ uptake by cardial sarcolemmal vesicles was investigated. Membrane potentials were generated by the K+ gradient in the presence of valinomycin and were measured using fluorescent dye diS-C3-(5). It was shown that the 45Ca2+ influx into vesicles increased twice after membrane depolarization. Evaluation of the 45Ca2+ influx over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. Passive 45Ca2+ transport was inhibited by 1 mM Cd2+ and Co2+. It is suggested that the voltage-dependent Ca2+ influx into vesicles occurs through Ca2+-channels.     

Release of calcium from intracellular stores in rat basophilic leukemia cells monitored with the fluorescent probe chlortetracycline.

Release of calcium from intracellular stores of rat basophilic leukemia cells was monitored using the fluorescent probe chlortetracycline. The ability of chlortetracycline to indicate release from intracellular calcium stores was initially validated. The decrease of chlortetracycline fluorescence upon antigen-stimulation was not the result of secretion of granule-associated dye or of changes in the properties of the membranes. The chlortetracycline fluorescence signal was not influenced by Ca2+ influx across the plasma membrane. Results obtained from these chlortetracycline fluorescence measurements corresponded well with 45Ca efflux data, an indirect measurement of release of calcium from stores. Chlortetracycline was used to examine the rate of antigen-induced release of calcium from stores, the depletion of intracellular calcium stores by EGTA, and the relationship between the antigen-stimulated release of stored calcium and exocytosis. Chlortetracycline was shown to be a useful qualitative indicator for the release of intracellular calcium with a relatively rapid response time.     

Calcium-induced inactivation of alamethicin in asymmetric lipid bilayers

This paper discusses a calcium-dependent inactivation of alamethicin- induced conductance in asymmetric lipid bilayers. The bilayers used were formed with one leaflet of phosphatidyl ethanolamine (PE) and one of phosphatidyl serine (PS). Calcium, initially confined to the neutral lipid (PE) side, can pass through the open alamethicin channel to the negative lipid (PS) side, where it can bind to the negative lipid and reduce the surface potential. Under appropriate circumstances, the voltage-dependent alamethicin conductance is thereby inactivated. We have formulated a model for this process based on the diffusion of calcium in the aqueous phases and we show that the model describes the kinetic properties of the alamethicin conductance under various circumstances. EGTA on the PS side of the membrane reduces the effects of calcium dramatically as predicted by the model.     

Membrane depolarization selectively inhibits receptor-operated calcium channels in human T (Jurkat) lymphoblasts

Jurkat lymphoblasts were stimulated by a monoclonal antibody against the CD3 membrane antigen and the evoked calcium signal was followed by the intracellular fluorescent calcium indicator indo-1. The technique applied allowed us to separately investigate the stimulus-induced intracellular calcium release and the calcium-influx pathways, respectively. In the same cells membrane potential was estimated by the fluorescent dye diS-C3-(5). The resting membrane potential of Jurkat lymphoblasts under normal conditions was between -55 and -60 mV. Membrane depolarization, obtained by increasing external K+ concentration, removing external Cl-, or by increasing the Na+/K+ leak permeability with gramicidin or PCMBS, did not induce calcium influx in the resting cells and did not influence the CD3 receptor-mediated internal calcium release, while strongly inhibited the receptor-mediated calcium influx pathway. Half-maximum inhibition of this calcium influx was observed at membrane potential values of about -35 to -40 mV and this inhibition did not depend on the external calcium concentration varied between 5 and 2500 microM. Membrane hyperpolarization by valinomycin did not affect either component of the calcium signal. The observed selective inhibition of the receptor-operated calcium influx pathway by membrane depolarization is probably an important modulator of calcium-dependent cell stimulation.     

Estimation of the mitochondrial calcium pool in rat brain synaptosomes using Rhod-2 AM fluorescent dye

The literature data on the role of synaptic mitochondria in the regulation of the cytosolic calcium level are contradictory. In the present paper calcium storage by mitochondria in rat brain synaptosomes using the fluorescent dye Rhod-2 has been investigated. The addition of 60 mM KCl increases Rhod-2 fluorescence. This effect is completely abolished by replacing K⁺ with Na⁺ or withdrawing Ca²⁺ from the incubation medium. A proton ionophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, and a mixture of rotenone/oligomycin mitochondrial toxins cause a two-fold decrease in Rhod-2 fluorescence. Thapsigargin, an inhibitor of endoplasmic reticulum ATPase (1 μM), but not bafilomycin, an inhibitor of ATPase in synaptic vesicles (500 nM) also leads to a mitochondrial calcium influx. The addition of calcium to synaptosomes with the retained plasma membrane potential increased Rhod-2 fluorescence; however, this effect is insensitive to carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. We have shown that mitochondria can serve as a calcium store in synaptosomes only in the case of a high cytosolic concentration of calcium.     

The Modulatory Effect of Calcium Ions Upon Alamethicin Monomers Uptake on Artificial Phospholipid Membranes

In this paper, we examined the influence exerted by calcium ions upon physical properties of lipids constituting an artificial membrane. Our strategy was to study changes on alamethicin oligomer kinetic features embedded into such an artificial membrane. At neutral pH and in the presence of calcium ions, we observed an increase in the number of alamethicin monomers that oligomerize within the membrane, forming a multi-substate nanopore. We make the argument that calcium ions binding within the interface between the hydrophobic and the hydrophilic regions of the biomembrane causes a sizeable alteration of the physical properties of neutral lipid membranes. This in turn is seen to influence the translocation rates of alamethicin monomers from the solution adjacent to the biomembrane and leads to an augmentation in the subunit composition of the alamethicin oligomers, leaving the electrical conductance of the substates and their kinetics mainly unchanged.     

Advantages of calcium green-1 over other fluorescent dyes in measuring cytosolic calcium in platelets.

Fluorescent indicators are widely used in the measurements of cytosolic calcium in many cell types for many purposes because they are relatively easy to use. Notwithstanding, they have some defects to prevent accurate measurements under certain conditions, such as significant dye leakage and UV-quenching effect. Menadione, a representative quinone derivative with antiaggregating effect, is also UV-absorbent. To investigate whether menadione can affect the change of cytosolic calcium in platelets by agonist, we measured the change of cytosolic calcium level using calcium green-1. Since this dye has not been used previously in platelets, we determined that the optimal loading of calcium green-1 to platelets was achieved using 3 microM dye incubated for 60 min at 37 degrees C. Our study compared the use of calcium green-1 with fura-2 and fluo-3 (two widely used dyes) in measurements of cytosolic calcium. Fura-2 is UV-excited, so when menadione was treated in fura-2-loaded cells, it had a quenching effect. Fluo-3, the other visible fluorescent indicator, leaked from platelets very rapidly and required the use of anion channel blockers which are known to affect physiological response of platelets. Our study demonstrated that changes in cytosolic calcium levels can be accurately measured without these problems by using calcium green-1. We therefore were able to demonstrate that menadione inhibited calcium increase by thrombin in a dose-dependent manner similar to menadione's antiaggregating effect in platelets.     

The measurement of the macrophage membrane potential by using an oxonol fluorescent probe]

Technical questions of macrophage (MP) membrane potential measuring with a probe bis(1,3-dibutyl barbiturate) trimethineoxonol (diBA-C4 (3)) have been elaborated. Measurements were made of single adherent cells. It was shown that at a high concentration of probe in the medium (900 nM) the fluorescent signal well traces the depolarization of membrane, whereas at a low concentration of probe (110 nM) the hyperpolarization is detected more effectively. To find out the reasons for this difference, measurements were made of dye distribution between the cell and the medium measured as well as of the kinetics of probe efflux from MP in the dye-free medium. The gradient of dye concentration on the cell-medium interface appeared to depend on the concentration of diBA-C4 (3) in the medium. Using gramicidin D and Na- and Cl-free solutions, the calibration of fluorescent signal was done; the value of K+ equilibrium potential of MP was -66 - -71 mV. The effect of quinidine and the binding of intracellular calcium result in a significant depolarization of MP membrane; a conclusion is made of the significant contribution of Ca(+)-dependent K(+)-channels to the maintenance of the MP resting potential.     

Primary neuronal cultures from the brains of late stage Drosophila pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.