alpha-Latrotoxin affects mitochondrial potential and synaptic vesicle proton gradient of nerve terminals.

Ca(2+)-independent [(3)H]GABA release induced by alpha-latrotoxin was found to consist of two sequential processes: a fast initial release realized via exocytosis and more delayed outflow through the plasma membrane GABA transporters [Linetska, M.V., Storchak, L.G., Tarasenko, A.S., Himmelreich, N.H., 2004. Involvement of membrane GABA transporters in alpha-latrotoxin-stimulated [(3)H]GABA release. Neurochem. Int. 44, 303-312]. To characterize the toxin-stimulated events attributable to the transporter-mediated [(3)H]GABA release from rat brain synaptosomes we studied the effect of alpha-latrotoxin on membrane potentials and generation of the synaptic vesicles proton gradient, using fluorescent dyes: potential-sensitive rhodamine 6G and pH-sensitive acridine orange. We revealed that alpha-latrotoxin induced a progressive dose-dependent depolarization of mitochondrial membrane potential and an irreversible run-down of the synaptic vesicle proton gradient. Both processes were insensitive to the presence of cadmium, a potent blocker of toxin-formed transmembrane pores, indicating that alpha-latrotoxin-induced disturbance of the plasma membrane permeability was not responsible to these effects. A gradual dissipation of the synaptic vesicle proton gradient closely coupled with lowering the vesicular GABA transporter activity results in a leakage of the neurotransmitter from synaptic vesicles to cytoplasm. As a consequence, there is an essential increase in GABA concentration in a soluble cytosolic pool that appears to be critical parameter for altering the mode of the plasma membrane GABA transporter operation from inward to outward. Thus, our data allow clarifying what cell processes underlain a recruitment of the plasma membrane transporter-mediated pathway in alpha-LTX-stimulated secretion.

Effectiveness of extracellular lactate/pyruvate for sustaining synaptic vesicle proton gradient generation and vesicular accumulation of GABA.

The effects of extracellular monocarboxylates pyruvate and lactate on membrane potentials, acidification and neurotransmitter filling of synaptic vesicles were investigated in experiments with rat brain synaptosomes using [(3)H]GABA and fluorescent dyes, potential-sensitive rhodamine 6G and pH-sensitive acridine orange. In experiments investigating accumulation of acridine orange in synaptic vesicles within the synaptosomes, monocarboxylates, similarly to glucose, ensured generation of the vesicle proton gradient by available and recycled vesicles, and pyruvate demonstrated the highest efficacy. An increase in the level of proton gradient correlated with enhanced accumulation of [(3)H]GABA in synaptic vesicles and resulted in enlarged exocytosis and attenuated the transporter-mediated [(3)H]GABA release. Pyruvate added to glucose-contained medium caused more active binding of rhodamine 6G by synaptosomes that reflected mitochondrial membrane hyperpolarization, and this intensification of nerve terminal energy metabolism resulted in an increase in total ATP content by approximately 25%. Pyruvate also prolonged the state of metabolic competence of nerve terminal preparations, keeping the mitochondrial potential and synaptic vesicle proton gradient at steady levels over a long period of time. Thus, besides glucose, the extracellular monocarboxylates pyruvate and lactate can provide sufficient support of energy-dependent processes in isolated nerve terminals, allowing effective functioning of neurotransmitter release and reuptake systems.

Improvement of metabolic competence of isolated nerve terminals by extracellular pyruvate.

Neuronal activity is tightly coupled with brain energy metabolism. Numerous studies have proved that glucose is not a sole energy substrate for neurons; metabolic monocarboxylate intermediates derived from glucose (pyruvate and lactate) released by astrocytes are shown to be taken up and oxidized by neurons, and, moreover, could serve as neuroprotective agents. Herein, we presented the data that extracellular pyruvate (4 mM) in the presence of glucose caused the increase in synaptosomal ATP content from 3.48+/-0.30 to 4.38+/-0.23 nmol/mg of protein. This correlates with the enhanced accumulation of fluorescent dye acridine orange in the available and the recycling synaptic vesicles within the synaptosomes reflecting the improved generation of proton gradient through the synaptic vesicle membrane. We have also demonstrated the effect of extracellular pyruvate on distribution of [3H]GABA between synaptic vesicles and cytoplasm in loaded synaptosomes. To estimate [3H]GABA accumulation into the synaptic vesicles, Ca 2+-dependent 4-aminopyridine-triggered exocytotic neurotransmitter release was studied. Evaluation of cytosolic 1H]GABA pool was performed by measuring the Ca2+-independent transporter-mediated neurotransmitter release evoked by nipecotic acid or high K+. The presence of pyruvate resulted in doubled exocytotic release of [3H]GABA, and significantly attenuated Ca2+-independent release of cytosolic [3H]GABA. Together, these observations provide insight into the important role of glucose metabolic intermediate, pyruvate, in sustaining activity of vesicular inhibitory amino acid transporter and so normal inhibitory transmission. We propose to use pyruvate for keeping up synaptosomal preparations in state of metabolic stability.

Phenylarsine oxide is able to dissipate synaptic vesicle acidic pool.

Phenylarsine oxide (PAO) has a number of targets in the neurons, one of them is exocytotic process. In this study, we have focused on the mechanisms of phenylarsine oxide action on Ca(2+)-dependent and Ca(2+)-independent neurotransmitter release from rat brain synaptosomes. We investigated the influence of phenylarsine oxide on: (i) l-[(14)C]glutamate and [(3)H]GABA release and uptake; (ii) plasma membrane potential using a potential-sensitive fluorescent probe rhodamine 6G; (iii) exo/endocytotic process using a pH-sensitive fluorescent probe acridine orange (AO). It has been found that phenylarsine oxide induced deacidification of synaptic vesicles. This effect was completely abolished by preliminary treatment of synaptosomes with a protonophore FCCP indicating that both reagents injured a proton electrochemical gradient. Dissipation of the proton gradient by low concentrations of phenylarsine oxide (not exceed 1 microM) did not prevent KCl-triggered exocytotic response, but essentially modified endocytotic one. At higher concentrations of phenylarsine oxide (up to 10 microM), the proton gradient dissipation was intensified and the exocytotic response was fully abolished. The reagent did not change plasma membrane potential, but depolarized mitochondria. It also caused potent inhibition of the Ca(2+)-stimulated l-[(14)C]glutamate and [(3)H]GABA release and increase the Ca(2+)-independent release of l-[(14)C]glutamate, but not of [(3)H]GABA. Disulfide-reducing reagents (dithiothreitol and beta-mercaptoethanol) completely prevented phenylarsine oxide-evoked injuries. They could also restore the initial levels of the mitochondrial potential, the exocytotic response to KCl and the release and uptake of neurotransmitters. Our data provide the evidence that phenylarsine oxide causes dissipation of synaptic vesicle acidic pool resulting in the reduction of vesicle filling and as consequence in attenuation of Ca(2+)-stimulated neurotransmitter release.

Involvement of membrane GABA transporter in alpha-latrotoxin-stimulated [3H]GABA release.

Alpha-latrotoxin evokes massive [3H]GABA release from rat brain synaptosomes by stimulating exocytosis and outflow from non-vesicular pool. In the present study, GABA transporter-mediated [3H]GABA release was shown to be involved in alpha-latrotoxin-triggered release of [3H]GABA from non-vesicular pool. The following agents have been exploited as tools: (1) a protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and bafilomycin A1 for evoking depletion of synaptic vesicle [3H]GABA and enlargement of non-vesicular pool; (2) a non-substrate high-affinity GABA transport blocker NO-711 for determining participation of GABA carrier in the toxin-stimulated GABA release; (3) a competitive inhibitor of GABA reuptake nipecotic acid for heteroexchange [3H]GABA release. As shown by the experiments with nipecotic acid, FCCP and bafilomycin A1 considerably increase the content of non-vesicular [3H]GABA. The treatment of the synaptosomes with these agents modified the response to alpha-latrotoxin, particularly to its subnanomolar concentrations: the lack or substantial lowering of the toxin-evoked release during the first 2 min after the toxin addition and substantial enhancement of release up to the 5th minute were observed. Only the step of enhanced release was sensitive to GABA transporter blocker NO-711. Distinct sensitivity to NO-711 was shown to be characteristic for different steps of alpha-latrotoxin-stimulated [3H]GABA release from the control, untreated synaptosomes: lack of any effect of NO-711 during the first 2 min and powerful inhibition in 10 min after the toxin application. Taken together these data appear to indicate that the toxin non-simultaneously from vesicular and non-vesicular origins releases the neurotransmitter, the first rapid step reflects exocytosis stimulation, and the second tardy step is at least in part due to the release mediated by GABA transporters. The incomplete inhibition with NO-711 of the tardy step of the release evoked by nanomolar toxin concentrations suggests the participation not only of the GABA transporters.

Phenylarsine oxide inhibits alpha-latrotoxin-stimulated [3H]GABA release from rat brain synaptosomes.

Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of membrane-trafficking processes, including exocytosis of neurotransmitters. However, there are contradictory findings concerned ability of phenylarsine oxide (PAO), an inhibitor of phosphatidylinositol 4-kinase, to affect exocytotic release of different types of neurotransmitters. We bent our efforts to a detailed analysis of action of PAO on Ca(2+)-dependent and Ca(2+)-independent [3H]GABA release produced by exposure of rat brain synaptosomes to different concentrations of alpha-latrotoxin. We also compared PAO action on alpha-latrotoxin- and 4-aminopyridine (4-AP)-evoked [3H]GABA release. The experiments have shown that release of [3H]GABA evoked by the depolarization with 4-AP was decreased by 80% as a result of action of 3 microM PAO and the complete inhibition of release was observed with 10 microM PAO. When alpha-latrotoxin as a stimulant was applied, release of [3H]GABA was increased as toxin concentration used was elevated from 0.5 to 3.0 nM, however, concomitantly, the response of the toxin-induced [3H]GABA release to PAO became attenuated: 10 microM PAO led to almost complete inhibition of the effect of 0.5 nM alpha-latrotoxin and only partly decreased (by 40%) the response to 3.0 nM alpha-latrotoxin. To test whether the efficacy of PAO depended on the toxin-induced outflow of cytosolic [3H]GABA, synaptosomes with depleted cytosolic [3H]GABA pool were also exploited. Depletion was performed by means of heteroexchange of cytosolic [3H]GABA with nipecotic acid. The experiments have shown that treatment of loaded synaptosomes with nipecotic acid resulted in some increase of [3H]GABA release evoked by 0.5 nM alpha-latrotoxin, but in the two-fold decrease of the response to 3.0 nM alpha-latrotoxin. PAO essentially inhibited [3H]GABA release from depleted synaptosomes irrespective of alpha-latrotoxin concentration used. Therefore, the amount of [3H]GABA released from cytosolic pool determined, in considerable degree, the insensitivity of alpha-latrotoxin action to PAO. Thus, our data show that subnanomolar concentrations of alpha-latrotoxin may be used for stimulation of exocytotic release of [3H]GABA. Exposure of synaptosomes with nanomolar toxin concentrations leads not only to stimulation of exocytosis, but also to leakage of [3H]GABA from cytosolic pool. PAO potently inhibits exocytotic release of [3H]GABA and its inhibitory effectiveness is diminished as far as the outflow of [3H]GABA is elevated.

Does extracellular calcium determine what pool of GABA is the target for alpha-latrotoxin?

Presynaptic neurotoxin alpha-latrotoxin, from the venom of Latrodectus mactans tredecimguttatus, causes massive [(3)H]GABA release from rat brain synaptosomes, irrespective of calcium presence in the extracellular medium. Whether the binding of alpha-latrotoxin to Ca(2+)-dependent (neurexin 1 alpha) or to Ca(2+)-independent (latrophilin) receptor triggers [(3)H]GABA release by the same mechanisms or different ones, inducing either exocytotic process or outflow by mobile membrane GABA transporter, is unknown. We examined alpha-latrotoxin-evoked [(3)H]GABA release from synaptosomes which cytosolic [(3)H]GABA pool was depleted either by applying competitive inhibitors of the GABA transporter, nipecotic acid and 2,4-diaminobutyric acid, or by permeation with digitonin. We also compared the effect of the GABA transporter inhibitors on depolarisation-evoked and alpha-latrotoxin-evoked [(3)H]GABA release using as depolarising agents 4-aminopyridine and high KCl in the Ca(2+)-containing and in Ca(2+)-free medium, respectively. Incubation of synaptosomes with nipecotic acid induced the essential acceleration of unstimulated [(3)H]GABA release and deep inhibition of high KCl-evoked Ca(2+)-independent [(3)H]GABA release. In contrast, at the similar conditions the effect of alpha-latrotoxin was greatly augmented with respect to the control response. Another way to assay what GABA pool was involved in alpha-latrotoxin-induced release lays in an analysis of the effects of depolarisation and alpha-latrotoxin in consecutive order. The preliminary 4-aminopyridine-stimulated [(3)H]GABA release attenuated the toxin effect. But when depolarisation occurred in Ca(2+)-free medium, no influence on alpha-latrotoxin effect was revealed. Employing digitonin-permeated synaptosomes, we have shown that alpha-latrotoxin could stimulate [3H]GABA release in the medium with 1mM EGTA, this effect of the toxin was blocked by concanavalin A and was ATP-dependent. The latter suggests that alpha-latrotoxin-released neurotransmitter has the vesicular nature. We assume that the type of the toxin membrane receptor does not determine the mechanisms of [(3)H]GABA release evoked by alpha-latrotoxin.

Okadaic acid and cyclosporin A modulate [(3)H]GABA release from rat brain synaptosomes.

Rat brain synaptosomes were used to investigate the effect of okadaic acid, an inhibitor of protein phosphatase 1 and 2A, and cyclosporin A, an inhibitor of protein phosphatase 2B (calcineurin), on [(3)H]GABA release. Release of [(3)H]GABA was evoked by 4-aminopyridine in the presence of calcium and by alpha-latrotoxin in the presence and absence of calcium. Pretreatment of synaptosomes with 1 microM okadaic acid reduced [(3)H]GABA release evoked by 4-aminopyridine by about 40%. The effect of alpha-latrotoxin on [(3)H]GABA release was stimulated by okadaic acid. This stimulation was equal in both media. The stimulating effect of 4-aminopyridine and alpha-latrotoxin on [(3)H]GABA release was activated when synaptosomes were pretreated with cyclosporin A. Activation of 4-aminopyridine-evoked [(3)H]GABA release was observed at 1 microM cyclosporin A, but the toxin effect was enhanced only when concentration of cyclosporin A was increased to 10 microM. The level of cyclosporin A activation depended on alpha-latrotoxin concentrations used - a higher stimulating effect of cyclosporin A was observed with lower toxin concentration. These results suggest that in calcium medium 4-aminopyridine- and alpha-latrotoxin-evoked [(3)H]GABA release was realized by different mechanisms.

Differential effect of protein kinase inhibitors on calcium-dependent and calcium-independent [14C]GABA release from rat brain synaptosomes.

Rat brain synaptosomes were isolated to study the effects of protein kinase inhibitors (sphingosine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide, staurosporine) on Ca2+-dependent and Ca2+-independent [14C]GABA release. The Ca2+-dependent [14C]GABA release was stimulated by depolarization with a K+-channel blocker, 4-aminopyridine, or high K+ concentration. It has been shown that 4-aminopyridine-evoked [14C]GABA release strongly depends on extracellular Ca2+ while K+-evoked [14C]GABA release only partly decreases in the absence of calcium. The substitution of sodium by choline in Ca2+-free medium completely abolished Ca2+-independent part of K+-evoked [14C]GABA release. So the main effect of 4-aminopyridine is the Ca2+-dependent one while high K+ is able to evoke [14C]GABA release in both a Ca2+-dependent and Na+-dependent manner. In experiments with protein kinase inhibitors, 4-aminopyridine and high K+ concentration were used to study the Ca2+-dependent and the Ca2+-independent [14C]GABA release, respectively. In addition, the Ca2+-independent [14C]GABA release was studied using alpha-latrotoxin as a tool. Pretreatment of synaptosomes with protein kinase inhibitors tested, except of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, resulted in a marked inhibition of 4-aminopyridine-stimulated Ca2+-dependent [14C]GABA release. The inhibitory effects of N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide and staurosporine on [14C]GABA release were not due to their effects on 4-aminopyridine-promoted 45Ca2+ influx into synaptosomes. Only sphingosine (100 microM) reduced the 45Ca2+ influx. All the inhibitors investigated were absolutely ineffective in blocking the Ca2+-independent [14C]GABA release stimulated by alpha-latrotoxin. Three of them, except for sphingosine, did not affect the Ca2+-independent [14C]GABA release stimulated by high potassium. The inhibitory effect of sphingosine was equal to 30%. Thus, if [14C]GABA release occurred in a Ca2+-independent manner irrespective of whether alpha-latrotoxin or high K+ stimulated this process, it was not inhibited by the drugs decreased the Ca2+-dependent [14C] GABA release. Given the above points it is therefore not unreasonable to assume that the absence of Ca2+ in the extracellular medium created the conditions in which the activation of neurotransmitter release was not accompanied by Ca2+-dependent dephosphorylation of neuronal phosphoproteins, and as a consequence the regulation of exocytotic process was modulated so that the inhibition of protein kinases did not disturb the exocytosis.

[Phosphorylation of rat brain synaptosomal proteins stimulated by alpha-latrotroxin]. / Fosforilirovanie belkov sinaptosom golovnogo mozga krys, stimuliruemoe alph-latrotoksinom.

alpha-Latrotoxin (LTX) is a presynaptic neurotoxin from black widow spider (Latrodectus mactans tredecimguttatus) venom; it causes massive Ca(2+)-independent neurotransmitter release. The effect of LTX on phosphorylation of synaptosomal proteins was studied including synapsin I, synaptotagmin, and dynamin. Experiments were performed in Ca(2+)-supplemented medium containing 1 mM CaCl2 or nominally Ca(2+)-free medium (without added Ca2+ and EGTA) using intact synaptosomes isolated from rat brain and prelabeled with 32P(i). The data indicate the exposure of synaptosomes to 10 nM LTX for 10 sec stimulates 32P(i) incorporation into synapsin I up to 143% versus control in Ca(2+)-supplemented medium and up to 130% in Ca(2+)-free medium. LTX (20 nM) significantly stimulated synapsin I phosphorylation (up to 173% versus control) only in Ca(2+)-free medium. Under these conditions, dephosphorylation of dynamin was not observed. Exposure of synaptosomes to LTX in Ca(2+)-supplemented medium for 10 sec did not affect 32P(i) incorporation into synaptotagmin but increase in incubation time up to 30 sec results in dephosphorylation of synaptotagmin down to 70% versus control level. In Ca(2+)-free medium, LTX stimulates the 32Pi incorporation into synaptotagmin (up to 140% versus control) and the effect was not time-dependent. Thus, LTX-stimulated neurosecretion in Ca(2+)-supplemented and Ca(2+)-free medium requires phosphorylation of synapsin I. Phosphorylation-dephosphorylation cycle of synaptotagmin can be important for regulation of recyclization of synaptic vesicles.

alpha-Latrotoxin-stimulated GABA release can occur in Ca(2+)-free, Na(+)-free medium.

We have studied [14C]GABA release from synaptosomes induced by native and monoclonal antibodies-modified alpha-latrotoxin (LTX). Modification of LTX eliminates the toxin's ability to increase [Ca2+]i influx into synaptosomes. It has been shown that native LTX does not change 22Na influx into rat brain synaptosomes. Both toxin forms studied, native and modified by monoclonal antibodies, stimulate [14C]GABA release from synaptosomes in divalent-free medium where sodium was substituted by equimolar concentrations of choline chloride. Native toxin induces a more rapid stimulation of [14C]GABA release than the modified one. It was suggested that the difference in the mediator release rates is not accounted for by the inability of modified toxin to form active ion channels in synaptosomal plasmalemma, but most probably by the state of toxin-receptor complexes.

Modulation of functional activities of the neurotoxin from black widow spider venom.

We have studied the action of an alpha-latrotoxin (alpha-LTX) complex of two polypeptides (LTX 130 kDa and low molecular weight protein (LMWP) 8 kDa) and the action of a venom fraction containing LTX with excess LMWP on calcium influx into synaptosomes and PC12 cells as well as on [14C]GABA release from synaptosomes. Both preparations considerably activate calcium influx and stimulate [14C]GABA release from synaptosomes. Preincubation of both preparations with antibodies against a 14 amino acid residue C-terminal peptide of LMWP differentially modulates these effects. Antibodies inhibit induced calcium influx and enhance induced GABA release.

[Functioning of latrotoxin channels during pH changes]. / Funktsionirovanie latrotoksinovykh kanalov pri izmenenii pH.

Using the fluorescent probe BCECP, the pH dependence of Ca2+ transport in synaptosomes along alpha-latrotoxin-formed channels, was studied. It was found that the pH value in synaptosomes is equivalent to 7.16 +/- 0.09. Acidification or alkalinization of the intracellular medium by 0.1-0.3 pH units had no appreciable influence on the Ca2+ influx along latrotoxin-formed channels. Alteration of external pH caused a parallel shift in the cytoplasmic pH in the synaptosomes. The pH decrease in the external medium down to 6.0 caused the inhibition of Ca2+ fluxes along latrotoxin-formed channels. Dissipation of the proton gradient by high concentrations of KCl in the presence of nigericin decreased the latrotoxin ability to form ionic channels without any loss in the activity of the preformed channels. The influx of bivalent cations along latrotoxin-formed channels led to alkalinization of the synaptosomal cytoplasm to pH of the external medium. This pH change did not depend on the presence of Na+ in the external medium and was blocked by cadmium.

[Immunochemical and functional study of the latrotoxin molecule]. / Immunokhimicheskoe i funktsional'noe issledovanie molekuly latrotoksina.

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.

[A high level of cytoplasmic calcium inactivates ion channels, formed by alpha-latrotoxin in the rat brain synaptosomes]. / Vysokii uroven' tsitoplazmaticheskogo kal'tsiia inaktiviruet ionnye kanaly, formiruemye alpha-latrotoksinom v sinaptosomakh mozga krys.

Effect of alpha-latrotoxin on the concentration level of free calcium [( Ca2+]in) in the rat brain synaptosomes and dependence of the activity of "latrotoxin" channels on [Ca2+]in were studied using fluorescent calcium probe quin-2. It is shown that alpha-latrotoxin exerts effect on calcium permeability of plasmalemma and does not induce calcium ejection from the intracellular compartments. A lag-period is characteristic of alpha-latrotoxin action. A degree of the [Ca2+]in increase in synaptosomes depends on the toxin concentration. When [Ca2+]in increases as a result of preliminary potassium depolarization of plasmalemma of synaptosomes, the amount of incoming calcium ions followed by the toxin effect as well as the calcium input rate considerably decrease. Inactivation of calcium-transferring channels induced by alpha-latrotoxin is not a result of a change in the potential on the membrane, as during the blockage of potential-depending calcium channels by D-600, an increase of KCl in the incubation medium does not influence the alpha-latrotoxin action. Differences in the properties of alpha-latrotoxin channels are discussed in synaptosomes and BLM.

[Latrotoxin channels. Permeability for divalent cations]. / Latrotoksinovye kanaly. Pronitsaemost' dlia dvukhvalentnykh kationov.

The dependence of Ca2+ transport in synaptosomes along the channels formed by alpha-latrotoxin on [Ca2+]in and the feasibility of transport along these channels of other bivalent cations were studied. It was found that the concentration dependence of Ca2+ influx is nonlinear and is described by the Michaelis-Menten kinetics (Km = 1.07 +/- 0.19 mM). Mg2+, Ba2+, Sr2+, Mn2+ and Co2+ competitively inhibited the Ca2+ influx via latrotoxin channels. Studies with the use of the fluorescent Ca2+ probes, Quin-2 and Fura-2, revealed that these cations can also penetrate inside synaptosomes via latrotoxin channels. The bivalent cation influx via latrotoxin channels caused a decrease of the membrane potential of synaptosomes. The similarity of properties of latrotoxin and endogenous Ca2+ channels is discussed.

[Interaction of alpha-latrotoxin with synaptosomes; metabolic control of the induction of calcium channels]. / Vzaimodeistvie alpha-latrotoksina s sinaptosomami; metabolicheskii kontrol' induktsii kal'tsievykh potokov.

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.

[Transmembrane localization of cytosol proteins from the heart muscle in proteoliposomes]. / O transmembrannoi lokalizatsii belkov tsitozolia serdechnoi myshtsy v proteoliposomakh.

It was shown that soluble cytoplasmic proteins of rabbit cardiac muscle were able to penetrate into phospholipid bilayer during preincubation with liposomes. The incorporation of cytoplasmic proteins into the liposomic membrane induced the sodium permeability that was sensitive to neurotoxins (veratridine and tetrodotoxin). This effect was partly eliminated by addition of tetrodotoxin to the external medium and completely eliminated in the presence of tetrodotoxin on the both sides of the liposomic membrane. It was found also that some of the proteins incorporated into the liposomes become resistant to pronase digestion when the enzyme is added to external medium. At the same time pronase included in the internal volume of the proteoliposomes was able to digest partly proteoliposomic proteins. All these data support the ideas of the integral incorporation of soluble cytoplasmic proteins into the liposomic membrane and to the existence of cytoplasmic precursor of voltage-dependent sodium channel.

[Interaction of tetrodotoxin-sensitive structures of soluble cardiac muscle proteins with liposomes]. / O vzaimodeistvii tetrodotoksin-chuvstvitel'nykh struktur rastvorimykh belkov serdechnoi myshtsy s liposomami.

It is studied how conditions of soluble myocardium proteins interaction with liposomes provide formation of the channel-like structures with tetrodotoxin- and veratrine-dependent sodium permeability. The temperature is shown to influence the formation rate of neurotoxin-sensitive proteoliposomes. The veratrine effect on sodium permeability of proteoliposomes intensifies with the rise of temperature. Pretreatment of liposomes by veratrine prevents formation of tetrodotoxin sensitivity in proteoliposomes.