Use of spectral characteristics of ryodipine in the study of dihydropyridine receptor complex of neuronal membranes]

A fluorescent dihydropyridine (DHP) derivative, ryodipine, was used to study structural characteristics of the DHP-sensitive Ca-channels in nerve terminals (synaptosomes) isolated from the rat cerebral cortex. It was found that an inductive resonance energy transfer from membrane proteins to ryodipine occurred in synaptosomal membranes. Two groups of membrane proteins differentially accessible to ryodipine were found by quenching of their own fluorescence. The percentage of group I proteins (20%) whose fluorescence was quenched by up to 1 microM ryodipine, was increased by 50% upon K(+)-depolarization and remained unchanged upon the addition of 100 microM Ni2+, whereas the addition of 100 microM Cd2+ prevented the increase induced by K(+)-depolarization. Nifedipine and nicardipine competed with ryodipine for the DHP receptor as evidenced by the change in percentage of group I proteins. The percentage of group II proteins (50% at 10 microM ryodipine) remained unchanged during various functional alterations of the synaptosomal membranes. Model experiments on proteoliposomes demonstrated that binding of ryodipine to synaptosomal membranes was due mainly to the hydrophobicity of DHP but not the ligand-receptor interaction. Nonetheless we that the membrane proteins-ryodipine system could be a qualitative test for the functional state of DHP-sensitive Ca-channels.     

Differentiation Between Alterations in Plasma and Mitochondrial Membrane Potentials in Synaptosomes Using a Carbocyanine Dye

The cationic potentiometric fluorescent probe 3,3'-diethylthiadicarbocyanine iodide [DiS-C2(5)] was used in synaptosomes to assess the relative contributions of plasma and mitochondrial membrane potentials (psi p and psi m, respectively) to overall fluorescence. Addition of synaptosomes to media containing 0.5 microM dye caused a decrease in fluorescence intensity due to dye accumulation, which equilibrated usually within 5 min. Depolarization of mitochondria by combined treatment with cyanide and oligomycin increased fluorescence by 42%, indicating significant prior accumulation of dye into intrasynaptosomal mitochondria. psi p was calculated to be -54 mV and was not altered significantly by prior depolarization of psi m with cyanide and oligomycin (hereafter referred to as "poisoned" synaptosomes). Similarly, the linear relationship between dye fluorescence and psi p was not altered by depolarization of psi m. Valinomycin, a K+ ionophore, caused a psi p-dependent increase in fluorescence in control (nonpoisoned) synaptosomes, but did not alter fluorescence of poisoned synaptosomes except when the extracellular concentration of K+ ([K+]e) was 2 mM, in which case valinomycin hyperpolarized psi p by about 5 mV. The pore-forming antibiotic gramicidin depolarized both psi p and psi m maximally. Under these conditions, Triton X-100 further increased fluorescence by 40%, indicating significant dye binding to synaptosomal components. In poisoned synaptosomes depolarized by 75 mM K+, gramicidin caused a decrease in fluorescence intensity (hyperpolarization of psi p). The organic solvent dimethyl sulfoxide, used as a vehicle for the hydrophobic ionophores, had voltage-dependent effects on psi p and psi m.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane effects of lysozyme amyloid fibrils

The influence of mature lysozyme fibrils on the structural and physical properties of model membranes composed of phosphatidylcholine (PC) and its mixtures with cardiolipin (CL) (10 mol%) and cholesterol (Chol) (30 mol%) was studied using fluorescent probes DPH, pyrene, Laurdan and MBA. Analysis of pyrene fluorescence spectra along with the measurements of DPH fluorescence anisotropy revealed that the structure of hydrocarbon chains region of lipid bilayer is not affected by the fibrillar aggregates of lysozyme. In contrast, probing the membrane effects by Laurdan and MBA showed the rise of both the generalized polarization of Laurdan and the MBA fluorescence anisotropy, suggesting that amyloid protein induces reduction of bilayer hydration and increase of lipid packing in the interfacial region of model membranes.     

Free calcium concentrations in the nerve endings in the brain of rats with spontaneous hypertension

Fluorescent indicator Quin-2 was used for the determination of free calcium (Ca2+in) in synaptosomes incubated in the normal medium and media where sodium is replaced by potassium or choline. At external calcium concentration of 1 mM, Ca2+in in all three media was 20-30% higher in synaptosomes of spontaneously hypertensive rats (SHR) than in control animals. At external calcium concentration of 5 mM, the increase in Ca2+in values induced by K+-depolarization in sodium- or choline-containing media was 50-80% higher in synaptosomes of SHR. These differences are suggested to be the basis for the mechanism of increased peripheral chain activity in the sympathetic nervous system in primary hypertension.     

Protein-dependent Reduction of the Pyrene Excimer Formation in Membranes

The presence of proteins in lipid bilayers always decreases the excimer formation rate of pyrene and pyrene lipid analogues in a way that is related to the protein-to-lipid ratio. Energy transfer measurements from intrinsic tryptophans to pyrene have shown (Engelke et al., 1994), that in microsomal membranes, the excimer formation rate of pyrene and pyrene fatty acids is heterogeneous within the membrane plane, because a lipid layer of reduced fluidity surrounds the microsomal proteins. This study investigates whether of not liposomes prepared from egg yolk phosphatidylcholine with incorporated gramicidin A give results comparable to those from microsomal membranes. The results indicate that the influence of proteins on the lipid bilayer cannot be described by one unique mechanism: Small proteins such as gramicidin A obviously reduce the excimer formation rate by occupying neighboring positions of the fluorescent probe and thus decrease the pyrene collision frequency homogeneously in the whole membrane plane, while larger proteins are surrounded by a lipid boundary layer of lower fluidity than the bulk lipid. The analysis of the time-resolved tryptophan fluorescence of gramicidin A incorporated liposomes reveals, that the tryptophan quenching by pyrene is stronger for tryptophans located closely below the phospholipid headgroup region because of the pyrene enrichment in this area of the lipid bilayer. Received: 29 December 1996/Revised: 15 May 1996     

Modulation of (Na+,K+)-ATPase activity by the lipid bilayer examined with dansylated phosphatidylserine

The fluorescent probe 8-(dimethylamino)naphthalene-1-sulfonylphosphatidylserine (Dns-PS) was incorporated into purified lamb kidney Na+- and K+-stimulated adenosinetriphosphatase (EC 3.6.1.3) [(Na+,K+)-ATPase] by using a purified phospholipid exchange protein. Phospholipase C was used to reduce phospholipid content. Up to 40% of the phospholipid could be hydrolyzed with only 10% inhibition of the (Na+,K+)-ATPase, but when 67% of the phospholipid was hydrolyzed, the enzyme was inhibited 53%. To examine the effect of protein on the phospholipid bilayer, the fluorescent parameters of the probe incorporated into the enzyme preparation were contrasted with the same parameters for the probe incorporated into the total lipid extract of the preparation. The polarization of fluorescence of the probe in the lipid extract was 0.118 while in the enzyme preparation it was 0.218. This reflected a decrease in fluidity of the glycerol region of the phospholipid bilayer which was mediated by the protein. This effect increased as the phospholipid content of the (Na+,K+)-ATPase preparation was reduced so that with maximal phospholipid reduction the polarization of fluorescence was 0.262. The protein caused a decrease in the transition temperature from gel to fluid states of the bilayer detected by polarization of the probe. The midpoint temperature transition of the enzyme preparation decreased from 33 degrees C when all phospholipids were present to 20 degrees C when 67% of the phospholipids were hydrolyzed. This decrease was not observed for the lipid extract of these samples. A direct correlation between the (Na+,K+)-ATPase specific activity and the polarization of fluorescence of Dns-PS was found. The reduction in phospholipid content did not affect the steady-state level of phosphorylation of the enzyme by ATP but did affect the rate of dephosphorylation which would require conformational changes of the enzymes. The data showed that the fluidity of the phospholipid bilayer can modulate the activity of the (Na+,K+)-ATPase.     

The structure and function of gramicidin A embedded in interdigitated bilayer

The effects of phase transition from normal to interdigitated lipid bilayer on the function and structure of membrane proteins were studied using linear gramicidin (gramicidin A) as a model. Interdigitated bilayer structure of dipalmitoylphosphatidylglycerol (DPPG) liposomes that was induced by atropine could not be changed notably by intercalating of gramicidin. The K+ transportation of gramicidin in both normal and interdigitated bilayer was assayed by measuring the membrane potential. Results showed that gramicidin in interdigitated bilayer exhibited lower transport capability. Intrinsic fluorescence spectrum of gramicidin in interdigitated bilayer blue-shifted 2.8 nm from the spectrum in normal bilayer, which means that interdigitation provides a more hydrophobic environment for gramicidin. Circular dichroism measurement results indicated that the conformation of gramicidin in interdigitated bilayer is not the typical beta6.3 helix as in the normal bilayer. The results suggested that the interdigitated lipid bilayer might largely affect the structure and function of membrane proteins.     

Effect of aminazine and triftazin on the synaptosome membranes of the rat cerebral cortex

Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene were employed in studying the effect of aminazine and triftazin versus that of imipramine on microviscosity of rat brain cortex synaptosomal membranes. Unlike imipramine, the neuroleptics decrease microviscosity of membrane's lipid bilayer. All drugs decrease fluorescence of endogenous tryptophan, but fail to change fluorescence of L-tryptophan in the solution. It is concluded that neuroleptics induce conformational perturbations in membrane-bound proteins modifying microviscosity of lipid bilayer whereas imipramine changes the surface electric charge of lipid bilayer of synaptosomal membranes.     

Effect of diamide on protein oxidation and physico-chemical properties of lipids in erythrocyte membranes

The effect of diamide on the physicochemical state of proteins and lipids of human erythrocyte membrane was studied. It was found that diamide at a concentration of 1 mM decreases the content of the SH-groups of membrane proteins by approximately 50%, resulting in enhanced vesiculation of erythrocytes upon metabolic exhaustion of cells. It was shown using fluorescein isothiocyanate-labeled concanavalin A and 4,4'-diisothiocyano-2,2'-stilbene disulfonate that diamide changes the structural state of the main integral protein of erythrocyte membranes, the band 3 protein. Changes in the microviscosity of the membrane lipid bilayer depending on diamide concentration were determined from the changes in the fluorescence parameters of the lipophilic probes (pyrene and 1,6-diphenyl-3,5-hexatriene). The level of lipid peroxidation products in membranes remained unchanged. It follows from these data that the SH-oxidizing agent diamide does not directly interact with the lipid bilayer of membrane and produces changes in the physicochemical state of lipids presumably by disrupting protein-lipid interactions that take place upon oxidation of the SH-groups and cross-linking of membrane proteins.     

Effects of oligomeric lysozyme on structural state of model membranes

The ability of oligomeric lysozyme to modify the molecular organization of the model bilayer membranes composed of phosphatidylcholine (PC) and its mixtures with phosphatidylglycerol (PG) or cholesterol (Chol) was assessed using fluorescent probes 6-propionyl-2-dimethylaminonaphthalene (Prodan), 4-dimethylaminochalcone (DMC), pyrene and 1,6-diphenyl-1,3,5-hexatriene (DPH). The observed changes in the fluorescence characteristics of polarity-sensitive probes Prodan and DMC, located in interfacial bilayer region, were interpreted due to the partial dehydration of the glycerol backbone, which was under the influence of aggregated protein. Cholesterol was found to prevent the perturbations of membrane polar part by lysozyme aggregates. Analysis of the pyrene excimerization data revealed an oligomer-induced reduction in bilayer free volume, presumably caused by an increased packing density of hydrocarbon chains. This effect proved to be virtually independent of membrane composition. It was demonstrated that membranotropic activity of oligomeric lysozyme markedly exceeds that of monomeric protein. The biological significance of the results obtained is twofold, implicating the general membrane-mediated mechanisms of oligomer toxicity and specific pathways of lysozyme fibrillogenesis in vivo associated with familial nonneuropathic systemic amyloidosis.     

Self-association of transmembrane alpha-helices in model membranes: importance of helix orientation and role of hydrophobic mismatch

Interactions between transmembrane helices play a key role in almost all cellular processes involving membrane proteins. We have investigated helix-helix interactions in lipid bilayers with synthetic tryptophan-flanked peptides that mimic the membrane spanning parts of membrane proteins. The peptides were functionalized with pyrene to allow the self-association of the helices to be monitored by pyrene fluorescence and Trp-pyrene fluorescence resonance energy transfer (FRET). Specific labeling of peptides at either their N or C terminus has shown that helix-helix association occurs almost exclusively between antiparallel helices. Furthermore, computer modeling suggested that antiparallel association arises primarily from the electrostatic interactions between alpha-helix backbone atoms. We propose that such interactions may provide a force for the preferentially antiparallel association of helices in polytopic membrane proteins. Helix-helix association was also found to depend on the lipid environment. In bilayers of dioleoylphosphatidylcholine, in which the hydrophobic length of the peptides approximately matched the bilayer thickness, association between the helices was found to require peptide/lipid ratios exceeding 1/25. Self-association of the helices was promoted by either increasing or decreasing the bilayer thickness, and by adding cholesterol. These results indicate that helix-helix association in membrane proteins can be promoted by unfavorable protein-lipid interactions.     

Action of neurotropic substances on gamma-aminobutyric acid release from brain synaptosomes during K+ depolarization

The effects of aminazine (0.25 mM), phthoracyzine (0.5 mM), trifluperidole (0.5 mM) and imipramine (0.5 mM) on GABA release from rat brain synaptosomes depolarized with K+ (50 mM) were investigated. Incubation of synaptosomes with aminazine led to a 2-fold and that with phthoracyzine, trifluperidole and imipramine to a 1.5-fold increase in GABA release from synaptosomes as compared with its basic level. The raising of K+ in the incubation medium to 50 mM brought about a 2-fold augmentation of GABA release. Exposure of synaptosomes to drugs and a higher K+ concentration at a time did not change GABA release as compared to its basic level. Introduction into the incubation medium of the Ga-ionophore A23187 together with 50 mM K+ and trifluperidole or with K+ and imipramine led to the same increase in GABA release from synaptosomes as that produced by the psychotropic drugs as regards native synaptosomes. It is assumed that the lack of the influence of the psychotropic drugs under study of GABA release from synaptosomes depolarized with K+ is caused by blockade of synaptic membrane conductibility for Ca2+.     

Effect of cholinergic ligands on the lipids of acetylcholine receptor-rich membrane preparations from torpedo californica

Ion permeation, triggered by ligand-receptor interaction, is associated with the primary events of membrane depolarization at the neuromuscular junction and synaptic connections. To explore the possible sites of ion permeation, the long-lived fluorescent probe pyrene (fluorescence lifetime ～400 nsec) has been inserted into the lipid phase of acetylcholine receptor-rich membrane (AcChR-M) preparations from Torpedo californica. The pyrene probe is susceptible to both fluidity and permeability changes in the lipid bilayer. These changes are detected by variations in the rate of decay of the excited singlet state of pyrene after pulsation with a 10-nsec ruby laser flash. Variations of these lifetimes in the membrane preparations alone or in the presence of quenchers show that binding of cholinergic agonists and antagonists, neurotoxins, and local anesthetics to AcChR-M produces varying effects on the properties of the pyrene probe in the lipid phase. It is concluded that binding of cholinergic ligands to the receptor does not significantly alter the fluidity or permeability of the lipids in the bilayer in contact with pyrene. On the other hand, local anesthetics do affect these properties.     

Use of the parallax-quench method to determine the position of the active-site loop of cholesterol oxidase in lipid bilayers

To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme. To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity. The loop-cysteine mutant was then labeled with acrylodan, an environment-sensitive fluorescence probe. The fluorescence increased and blue-shifted upon binding to lipid vesicles, consistent with a change into a more hydrophobic, i.e., lipid, environment. This acrylodan-labeled cholesterol oxidase was used to explore the pH, ionic strength, and headgroup dependence of binding. Between pH 6 and 10, there was no significant change in binding affinity. Incorporation of anionic lipids (phosphatidylserine) into the vesicles did not increase the binding affinity nor did altering the ionic strength. These experiments suggested that the interactions are primarily driven by hydrophobic effects not ionic effects. Using vesicles doped with either 5-doxyl phosphatidylcholine, 10-doxyl phosphatidylcholine, or phosphatidyl-tempocholine, quenching of acrylodan fluorescence was observed upon binding. Using the parallax method of London [Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45], the acrylodan ring is calculated to be 8.1 +/- 2.5 A from the center of the lipid bilayer. Modeling the acrylodan-cysteine residue as an extended chain suggests that the backbone of the loop does not penetrate into the lipid bilayer but interacts with the headgroups, i.e., the choline. These results demonstrate that cholesterol oxidase interacts directly with the lipid bilayer and sits on the surface of the membrane.     

The effects of ionic strength on the protein conformation and the fluidity of porcine intestinal brush border membranes. Fluorometric studies using N-[7-dimethylamino-4-methylcoumarinyl]maleimide and pyrene

The effects of ionic strength on the conformation around the SH groups of the proteins and the lipid fluidity of porcine intestinal brush border membranes were studied using two fluorescent dyes, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM) and pyrene. The extent of DACM labeling to the SH groups of the membrane proteins was accelerated depending on the KCl concentrations in medium. A quenching study of DACM-labeled membranes with acrylamide showed that the proximity of the quencher to the fluorescence-labeled SH groups in the membrane proteins is increased with increasing ionic strength of medium. An implication of the conformational changes around SH groups in the membrane proteins with increase of ionic strength was also obtained from the stimulation of guanidine effect on the fluorescence parameters of DACM-labeled membranes by addition of KCl. On the other hand, the results of the quenching study with KI, excimer fluorescence, and polarization measurements of pyrene-labeled membranes suggested an increase of membrane fluidity on addition of KCl to medium. The temperature dependence of polarization of the complex strongly suggested that the rotational freedom of pyrene molecules embedded into the lipid layers of the membranes is increased by addition of KCl. In fact, the harmonic means of the rotational relaxation times of pyrene molecules in the membranes with and without 100 mM KCl were estimated to be about 2900 and 9000 ns at 25 degrees C, respectively. Based on these results, the salt-induced alterations of the conformation in the vicinity of the bound dyes of the membrane proteins and of the membrane fluidity are discussed.     

Use of fluorescence probes 1-aniline-8-naphthalene sulfonate and pyrene for studying the localisation of proteins in inner membranes from wheat etioplasts

The fluorescence probes 1-aniline-8-naphthalene sulfonate (ANS) and pyrene were applied for characterisation of the light-induced changes in etioplast inner membranes (EPIMs) from 7 d-old dark-grown wheat seedlings (Triticum aestivum L. cv. Pobeda). The major aim was to obtain information about the localisation of membrane proteins in the EPIMs, using probes situated in different regions of the membranes. The quenching of tryptophan fluorescence showed tha the main parts of proteins were accessible to the pyrene buried in the lipid bilayer which suggests that most of the proteins also enter the lipid bilayer. The substantial quenching of the tryptophan fluorescence by the surface-situated ANS demonstrated that a part of the tryptophan residues was probably localised close to the membrane surface. The registered changes after irradiation could be explained by the presence of large aggregates of NADPH-protochlorophyllide oxidoreductase (POR), protochlorophyllide (PChlide) and NADPH in membranes that start to disconnect and redistribute along the prothylakoids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interaction of glucagon with dimyristoyl glycerophosphocholine

Glucagon can form amphipathic helices and can interact with dimyristoyl glycerophosphocholine at temperatures below the phase transition leading to a shift in the fluorescence emission maximum of tryptophan from 350 to 338 nm and a 3-fold enhancement of fluorescence intensity as well as a change in the polarization of fluorescence. The circular dichroism properties of the lipid-associated glucagon indicates that it has an increased content of alpha-helix. The phase transition temperature of the lipid as monitored by pyrene excimer fluorescence is not altered by interaction with glucagon although at higher glucagon/lipid ratios a decrease in excimer formation is noted at low temperature. Above the phase transition temperature, the addition of lipid has no effect on the fluorescence emission or circular dichroism of glucagon. Thus this hormone can interact with dimyristoyl glycerophosphocholine and this interaction is stronger below the phase transition temperature than above it.     

Glycoprotein-protein interaction examined by kinetic studies of pyrene transfer

The transfer of pyrene between ₁-acid glycoprotein, acethylcholinesterase and sonicated liposomes was used to monitor glycoprotein-protein interaction on the lipid bilayer. When a density solution of glycoprotein or protein labeled with pyrene was mixed with unlabeled suspension of free-phospholipid liposomes, or suspensions containing the complexes of glycoprotein-lipid, protein-lipid, or glycoprotein-protein-lipid, pyrene excimer fluorescence increased with a half-time of approximately 30–50 msec. Since the increase in excimer fluorescence indicates an increase in the microscope concentrations of pyrene, the observed fluorescence change reflects pyrene transfer. The half-times for the increase in excimer fluorescence were determined in the presence of glycoprotein and protein in the liposomes. On the basis of the determined half-times it was concluded that both, glycoprotein and protein are bound on the lipid bilayer. Our data also suggest that the thickness of the lipid bilayer is significantly changed in this case. The observation suggests strongly that the limiting step in the transfer of pyrene is not the dissociation of pyrene, but the uptake of the pyrene monomers by the lipid phase.     

Effects of in vitro hypoxia on depolarization-stimulated accumulation of inositol phosphates in synaptosomes

The effects of potassium and in vitro histotoxic hypoxia (i.e. KCN) on phosphatidylinositol turnover in rat cortical synaptosomes were determined. [2-3H] Inositol prelabelled rat synaptosomes were prepared from cerebral cortex slices that had been incubated with [2-3H] inositol. Depolarization with 60 mM KCl increased [2-3H] inositol phosphates in a time dependent manner. Depolarization with 60 mM KCl increased [2-3H] inositol trisphosphate transiently at 5 s. K+ induced rapid formation of [2-3H]-inositol bisphosphate and maintained an elevated level for at least 5 min. K+ stimulated gradual formation of [2-3H] inositol monophosphate with time. One minute of hypoxia enhanced potassium-stimulated [2-3H] inositol bisphosphate formation. However, 30 min of hypoxia impaired potassium-stimulated accumulation of [2-3H] inositol phosphates. The effects of histotoxic hypoxia were all dependent upon calcium in the medium and on K+-depolarization. Thus, hypoxia altered the K+-induced accumulation of inositol phosphates in prelabelled synaptosomes in a time dependent, biphasic manner that was calcium dependent.     

Interaction of S-100b protein with cardiolipin vesicles as monitored by electron spin resonance, pyrene fluorescence and circular dichroism

The interaction of S-100b protein with cardiolipin (CL) vesicles has been studied by electron spin resonance, pyrene fluorescence, and circular dichroism. Electron spin resonance and pyrene fluorescence data indicate that S-100b binds to the polar surface of vesicles Ca2+-independently. In the presence of Ca2+, S-100b potentiates the Ca2+-induced clustering of the polar headgroups of CL molecules and causes a further reduction in the Ca2+-dependent decrease in the lateral mobility of the pyrene inserted into the lipid bilayer, which points to an effect of the protein on the hydrophobic core of the lipid bilayer through a larger perturbation of its polar surface. Circular dichroism analyses indicate that CL vesicles cause a decrease in the alpha-helical content of S-100b, analogous to that produced by Ca2+ and that the effects of CL vesicles and of Ca2+ on the secondary structure of the protein are supra-additive. By this technique, we found that the affinity of Ca2+ for S-100b increases substantially in the presence of CL vesicles, even in the presence of physiologic concentrations of KCl, suggesting that once S-100b had interacted with CL vesicles it assumes a new conformation in which its Ca2+-binding properties are greatly enhanced. These results are discussed in relation to binding of S-100b proteins to natural membranes, and to a possible involvement of S-100b in the regulation of membrane structural organization.