ABSTRACT
A panel of monoclonal antibodies (mAb) against alpha-latrotoxin (LT) has been produced and their main characteristics have been determined. The influence of mAb on the functional effects of LT in synaptosomes from rat brain and on the channel formation in bilayer lipid membrane has been investigated. These mAbs do not inhibit binding of LT to rat synaptosomes but modify LT-receptor interaction in terms of LT's channel-forming and secretogenic effects. Antibodies A6 and A24 block these effects, whereas A4 partially preserves the secretogenic action of LT and completely blocks its channel-forming action. Only antibodies A15 affect the LT ability to form cationic channels in BLM, inducing considerable decrease in the frequency of the channel formation. These data and their analysis allow to identify several functional (and, probably, structural) domains of LT responsible for: 1) toxin-receptor interaction; 2) channel-forming and related calcium-dependent secretogenic effects; 3) calcium-independent secretogenic effects; 4) formation of cationic channels in the artificial lipid bilayer.
Subject(s)
Spider Venoms/metabolism , Animals , Antibodies, Monoclonal , Brain/metabolism , Calcium/metabolism , Cations, Divalent , Immunochemistry , Ion Channels/metabolism , Lipid Bilayers , Rats , Synaptosomes/metabolism , gamma-Aminobutyric Acid/metabolismABSTRACT
The accessibility to trypsin of 125I-labeled latrotoxin bound to rat brain synaptosomes was investigated. It was shown that latrotoxin bound to synaptosomes in the cold can be practically completely removed by trypsin treatment. The resistance of latrotoxin to proteolysis increases during its incubation with synaptosomes (37 degrees C). Concanavalin A (10(-6) M) decreases toxin binding by 30%, but fully prevents internalization (incorporation). Moreover, latrotoxin is not incorporated into synaptosomal membrane fragments irrespective of duration and temperature of incubation. Latrotoxin incorporated into synaptosomal membranes undergoes degradation by endogenous proteases resulting in the formation of TCA-soluble products.
Subject(s)
Brain/metabolism , Spider Venoms/metabolism , Synaptosomes/metabolism , Animals , Binding Sites , Calcium/metabolism , Iodine Radioisotopes , Ion Channels/metabolism , Kinetics , Rats , TemperatureABSTRACT
The binding of [125I] alpha-latrotoxin to synaptosomes from the rat brain is studied. It is shown that the constant rate of toxin association with the synaptosome receptor at 37 degrees C is equal to 8.2 +/- 1.3 x 10(7) M-1.s-1, while that of synaptosomal membrane -7.6 +/- 2.7 x 10(6) M-1 s-1. Depolarization of the synaptosome membrane induced by 55 mM KCl decreases the binding rate of toxin to the receptor, the rate constant being equal to 3.9 +/- 1.5 x 10(7) m-1 s-1. The pattern of the dissociation process of the toxin-receptor complex of synaptosomes and of synaptosomal membrane is different. In the first case dissociation follows two stages with the rate constants 3.6 x 10(-3) s-1 and 1.2/10(-4) s-1, in the second case it follows one stage with the constant equalled 2.0 x 10(-5) s-1. The quantity of the toxin binding sites on synaptosomes may vary under the action of agents modifying the activity of calcium fluxes which are induced by alpha-latrotoxin. It is supposed that a decrease in the ATP level in synaptosomes as well as deenergy of the surface membrane leads to a change in the state of the alpha-latrotoxin receptor.
Subject(s)
Arthropod Venoms/metabolism , Brain/metabolism , Spider Venoms/metabolism , Synaptic Membranes/metabolism , Synaptosomes/metabolism , Animals , Binding, Competitive , Kinetics , Rats , Receptors, Neurotransmitter/metabolismABSTRACT
The paper deals with characteristics of relationship between synaptosomal calcium permeability induced by alpha-latrotoxin and cytosolic concentration of ATP. It is shown that reagents decreasing the ATP level in the synaptosomes (monoiodoacetate, papaverine) inactivate the toxin-induced ionic fluxes and, on the contrary, reagents increasing the ATP level in synaptosomes enhance the toxin-induced calcium influx. The treatment of synaptosomes with inhibitors of phosphodiesterase of cAMP and cAMP-dependent protein kinase has no effect on the alpha-latrotoxin-induced calcium influx.
Subject(s)
Adenosine Triphosphate/metabolism , Arthropod Venoms/pharmacology , Ion Channels/metabolism , Spider Venoms/pharmacology , Synaptosomes/metabolism , Animals , In Vitro Techniques , Ion Channels/drug effects , Papaverine/pharmacology , Rats , Succinates/pharmacologyABSTRACT
The aim of the present study is to elucidate the possible significance of metabolic control of the calcium permeability of synaptosomes induced by alpha-latrotoxin. The alpha-latrotoxin influence of processes of GABA release and reuptake is shown to depend on synaptosomal metabolism. It has been found that incubation by the synaptosomes during one hour at 37 degrees C or their treatment by 10(-3) M monoiodacetate during 10 min at 10 degrees C or at room temperature completely abolished or at least strongly decreased the ability of alpha-latrotoxin to induce the calcium influx. Under the same conditions the ability of alpha-latrotoxin to activate the GABA release and inhibit the GABA reuptake do not change. It is concluded that only formation of calcium channels by alpha-latrotoxin is strictly controlled by the level of synaptosomal metabolism.
