Anion channels from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl₂ buffers (cis, 100mm/trans, 25mm), single channel conductances at –40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (P _Na:P _Ba:P _Cl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.230.041 and 0.050.031, respectively. Both channels exhibited characteristic voltage-dependent bursting activities. Open probability for type I channels had a maximum of 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from thecis side, while it blocked type II channels from either side.     

Time-dependent cadmium-neurotoxicity in rat brain synaptosomal plasma membranes

• 1.1. The modulation of lipid dynamics and lipid protein interactions were studied in rat brain synaptosomal plasma membranes (SPM) up to 24 hr after exposure to cadmium (Cd).
• 2.2. The activity of acetylcholinesterase and adenylate cyclase showed a considerable decrease after 6 hr of Cd exposure, followed by a progressive increase up to 24 hr.
• 3.3. SPM chemiluminescence showed a maximum decrease at 12 hr, demonstrating a considerable increase in lipid peroxidation.
• 4.4. SPM of Cd-exposed animals showed a statistical significant increase in fluorescence anisotropy parameter [(r₀/r) — 1]⁻¹ at 18 and 24 hr compared to SPM of the control, indicating a decrease of membrane fluidity.

     

Sensitivity of high-conductance potassium channels in synaptosomal membranes from the rat brain to intracellular pH

High-conductance potassium channels have been studied in inside-out patches excised from proteoliposomes reconstituted from giant liposomes and rat brain synaptosomes. Acid pH in the medium reduced single channel current amplitude and increased the mean open probability and the frequency of channel opening. This was accompanied by a shortening of the open time constant at positive potential and by shortening of the longer closed time constant. The decrease of channel amplitude, the increase of the open probability and the decrease in the longer closed time constant can be explained by neutralization of negative charges of the membrane and by a decrease in the surface membrane potential which mimics membrane depolarization. The shortening of the mean open time is apparently due to a channel blockade by protons. Correspondence to: H. Zemková     

A radiolabel and electron microscopy study of the interaction of liposomes with synaptosomal membranes

The quantitative and qualitative interaction of liposomes with synaptosomes isolated from rat brain was examined using radiolabeled phospholipids and electron microscopy. Liposomes were prepared by sonication and detergent dialysis. Binding (adsorption) of radiolabeled phospholipid to synaptosomes was saturable when liposomes were in the liquid-crystalline state, were electrically neutral (egg-phosphatidylcholine), or carried increasing fractions (10:2 and 10:4 molar ratio) of negatively charged phosphatidic acid. Analysis using the Langmuir isotherm equation indicated a biphasic adsorption behavior. Adsorption increased with increasing temperature (4 degrees C and 37 degrees C). Binding was nonsaturable when liposomes were positively charged with stearylamine or composed of dimyristoylphosphatidylcholine and phosphatidylinositol (10:2 molar ratio). Due to the latter composition's solid state at 4 degrees C, temperature dependency was inverse. Electron micrographs revealed disc-shaped areas of adsorption that were free of integral membrane particles which appeared to form a condensed layer surrounding the areas of liposome adsorption. Following interaction with stearylamine-containing liposomes the vesicular structure of synaptosomes appeared largely destroyed. It is concluded that both liposome surface charge and membrane fluidity determine the extent of interaction with biological membranes.     

Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes

Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.     

Influence of beta-adrenoceptor blocking drugs on lipid-protein interaction in synaptosomal membranes. An ESR study

The influence of the beta-adrenoceptor blocking drugs atenolol, doberol, propranolol and exaprolol on synaptosomal membranes was studied using ESR spectroscopy of stearic acid spin labeled at the 16th position. The drugs changed the ESR spectra of the label in the membranes, where in addition to changes of a fluid lipid component they increased the proportion of a motionally-restricted component. No motionally-restricted component was found in the samples prepared from brain total lipid liposomes treated with the drugs. The drug propensities at 20 mmol/l concentration to increase the proportion of the motionally-restricted component in the following order, control less than doberol approximately atenolol less than or equal to propranolol less than exaprolol did not correlate with their potency to influence the dynamics of the bulk lipid membrane phase. The motionally-restricted component induced by exaprolol increased with raising temperature and prolongation of time of the sample incubation. The results indicate that the beta-adrenoceptor blocking drugs influence lipid-protein interaction in the synaptosomal membranes, which could be important for elucidation of their mechanism of biological membrane activities.     

Spin label studies on synaptosomal membranes of rat brain cortex during aging

Membrane lipids of brain synaptosomes of 2, 12 and 24 months old rats were labeled by stearic acid spin probes with a doxyl group in the C-5 and C-16 position, respectively. We demonstrated that the hydrophobic region of synaptosomal membranes became more rigid during the life span studied, and the region located nearer the surface of membranes displays a significantly decreased fluidity only in the oldest group. Membrane proteins labeled with a nitroxide derivative of maleimide suggest that conformational changes take place during aging.     

Characterization of a pyruvate dehydrogenase modulator purified from insulin-treated rat brain plasma membranes

A factor able to stimulate pyruvate dehydrogenase when added to purified mitochondria was prepared from the supernatant of brain plasma membranes incubated with physiological concentrations of insulin (25 microU/ml). The factor completely reactivated pyruvate dehydrogenase previously inhibited with ATP and was active on pyruvate dehydrogenase from brain and liver mitochondria and from peripheral lymphocytes. The insulin-dependent stimulator of pyruvate dehydrogenase was heat and acid stable, was not absorbed on charcoal and displayed an isoelectric point of 5.5. The insulin mediator was purified by gel filtration, DEAE-cellulose and sulfonated polystyrene chromatography and, after dansylation, by high performance liquid chromatography. The purified mediator displayed a molecular weight of about 2800 and appeared as a peptide rich in glycine and serine and void of proline and sulfur containing aminoacids. It retained its stimulatory action on pyruvate dehydrogenase after dansylation and was completely inactivated by trypsin and chymotrypsin. Full reactivation of ATP-inhibited pyruvate dehydrogenase was attained when mitochondria were incubated with a mediator concentration of about 0.5 microM.     

Structural rearrangements in synaptosomal membranes of the brain]

Studies were carried out on structural state of synaptosomes and synaptic plasma membranes from brain of rats and guinea pigs at 5--50C. In membranes from guinea pig brain there were two breaks at 8 and at 18--20C in the curves of temperature dependence of light scattering. At the same temperatures adhesive properties of membranes had changed in jump-like manner when evaluating the degree of aggregation at pH4. The degree of aggregation in membranes from rat brain as well as rate of solubilization by 0.2% SDS (stoped-flow measurements) are characterized by single break point at 27C in Arrhenius curves. The breaks were interpreted as phase transitions with membrane surface involvement. Chlorpromazine and tetraciane (10(4)--10(3) M) abolished or modified these transitions. Because the number of negative charges and tryptophanyl and ANS fluorescence parameters were unchanged it was suggested that redistribution of ionogenic and hydrophobic goups took place at membrane surface during phase transitions.     

Preparation of chick brain synaptosomes and synaptosomal membranes

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome $\text{c}$: oxygen oxidoreductase (EC 1.9.3.1.), monoamine: oxygen oxidoreductase (deaminating) (EC 1.4.3.4), rotenoneinsensitive NADH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.3), NADPH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, $\text{(}\text{Na}{}^{\mathrm{+}}\text{?}\text{K}{}^{\mathrm{+}}\text{)-}\text{activated}$ ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.