Effects of local anesthetics on properties of tetrodotoxin-sensitive protein reconstituted into liposomes

The effects of some local anesthetics on properties of tetrodotoxin (TTX)-sensitive protein reconstituted into liposomes in such a manner that its TTX-sensitive center is located at the internal surface of the liposome membrane were studied. It was shown that tetracaine, lidocaine and its derivative QX-314 decreased the rate of efflux of radioactive sodium from the²²Na-preloaded proteoliposomes with the same efficiency as TTX acted from the inside of liposomes. The results confirm our earlier suggestion that TTX-sensitive protein is a soluble precursor of the protein forming voltage-dependent sodium channels.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 299–302, July–August, 1995.     

Effect of Lipids on the Activity of Calpain in Subcellular Fractions Obtained from the Rat Brain

We studied the effect of lipids on the activity of a neutral cysteine proteinase, calpain, in subcellular fractions obtained from the rat brain. Extraction of nearly 23% of membrane cholesterol from the coarse mitochondrial fraction did not result in modifications of specific activity of calpain in this fraction. Detergents (digitonin or Triton Х-100) used in 0.3% concentration enhanced the activity of calpain in the coarse mitochondrial fraction. Examination of the effects of preparations of different phospholipids on the activity of calpain in the cytoplasm demonstrated that only phosphatidylcholine, but not phosphatidylserine and/or cardiolipin, insignificantly increased the activity of calpain (independently of the size and structure of phospholipid vesicles). We hypothesize that the mechanisms underlying interaction between calpain and lipids are not universal; in native cells and model experiments, they can differ noticeably from each other and are modified depending on the corresponding conditions. Neirofiziologiya/Neurophysiology, Vol. 41, No. 1, pp. 3–9, January–February, 2009.     

Modification of the Membrane Components of Synaptosomes and the Fusion Process

A model system consisting of synaptic vesicles and synaptic membrane fragments isolated from brain synaptosomes was used for studying the role of the state of membrane components in membrane fusion. It is concluded that the state of proteins and lipids of biological membranes influences considerably the membrane's ability to fuse. This state can be changed by the regulation of cell enzyme systems (proteolytic enzymes, phospholipases), and regulation by nitric oxide is an important aspect of this control.     

Effect of potential-dependent potassium uptake on production of reactive oxygen species in rat brain mitochondria

The effect of potential-dependent potassium uptake on reactive oxygen species (ROS) generation in mitochondria of rat brain was studied. It was found that the effect of K⁺ uptake on ROS production in the brain mitochondria under steady-state conditions (state 4) was determined by potassium-dependent changes in the membrane potential of the mitochondria (ΔΨ_m). At K⁺ concentrations within the range of 0–120 mM, an increase in the initial rate of K⁺-uptake into the matrix resulted in a decrease in the steady-state rate of ROS generation due to the K⁺-induced depolarization of the mitochondrial membrane. The selective blockage of the ATP-dependent potassium channel (K _ATP ⁺ -channel) by glibenclamide and 5-hydroxydecanoate resulted in an increase in ROS production due to the membrane repolarization caused by partial inhibition of the potential-dependent K⁺ uptake. The ATP-dependent transport of K⁺ was shown to be ～40% of the potential-dependent K⁺ uptake in the brain mitochondria. Based on the findings of the experiments, the potential-dependent transport of K⁺ was concluded to be a physiologically important regulator of ROS generation in the brain mitochondria and that the functional activity of the native K _ATP ⁺ -channel in these organelles under physiological conditions can be an effective tool for preventing ROS overproduction in brain neurons.     

Fusion of synaptic vesicles and plasma membrane in the presence of synaptosomal soluble proteins

Fusion between synaptic vesicles and plasma membranes isolated from rat brain synaptosomes is regarded as a model of neurosecretion. The main aim of current study is to investigate whether the synaptosomal soluble proteins are essential members of Ca(2+)-triggered fusion examined in this system. Fusion experiments were performed using fluorescent dye octadecylrhodamine B, which was incorporated into synaptic vesicle membranes at self-quenching concentration. The fusion of synaptic vesicles, containing marker octadecylrhodamine B, with plasma membranes was detected by dequenching of the probe fluorescence. Membrane fusion was not found in Ca(2+)-supplemented buffer solution, but was initiated by the addition of the synaptosomal soluble proteins. When soluble proteins were treated with trypsin, they lost completely the fusion activity. These experiments confirmed that soluble proteins of synaptosomes are sensitive to Ca(2+) signal and essential for membrane fusion. The experiments, in which members of fusion process were treated with monoclonal antibodies raised against synaptotagmin and synaptobrevin, have shown that antibodies only partially inhibited fusion of synaptic vesicles and plasma membranes in vitro. These results indicate that other additional component(s), which may or may not be related to synaptobrevin or synaptotagmin, mediate this process. It can be assumed that fusion of synaptic vesicles with plasma membranes in vitro depends upon the complex interaction of a large number of protein factors.     

Effect of phosphorylation of latrotoxin-like brain protein on its fusogenic properties

A latrotoxin-like protein isolated from the bovine brain is shown to be a cAMP-dependent protein kinase substrate. The fusogenic activity of the protein was determined from the extent of mixing of content of the negatively charged liposomes in the presence of native or phosphorylated protein preparations. It was shown that phosphorylation of the protein considerably decreased its fusogenic activity at pH 7.5, but increased this activity at pH 6.0. The possible regulatory role of the phosphorylation in the functioning of latrotoxin-like protein is suggested.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 72–76, January–February, 1995.     

Some characteristics of fusogenic activity of cytoplasmic latrotoxin-like brain protein

We studied fusion of negatively charged artificial phospholipid vesicles (liposomes) in the presence of two electrophoretic fractions (molecular mass of about 90 and 50 kdalton) of latrotoxin-like (L) protein. It was shown that both fractions are capable of causing liposome fusion in acidic media. Treatment of native preparations of L protein with NEM depressed their fusogenic activity. Some common characteristics of L protein and well-known fusogenic proteins allow us to account for the possibility of participation of L protein in fusion of the membranes in the cell.