Brigitte Stecher

The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from permeabilized adrenal chromaffin cells

The heavy and light chains of botulinum A toxin were separated by anion exchange chromatography. Their intracellular actions were studied using bovine adrenal chromaffin cells permeabilized with streptolysin O. Purified light chain inhibited the Ca2+‐stimulated [3H]noradrenaline release with a half‐maximal effect at about 1.8 nM. The inhibition was incomplete. Heavy chain up to 28 nM was neither effective by itself nor did it enhance the inhibitory effect of light chain. It is concluded that the light chain of botulinum A toxin contains the functional domain responsible for the inhibition of exocytosis.

Reductive chain separation of botulinum A toxin — a prerequisite to its inhibitory action on exocytosis in chromaffin cells

Cleavage of the disulfide bond linking the heavy and the light chains of tetanus toxin is necessary for its inhibitory action on exocytotic release of catecholamines from permeabilized chromaffin cells [(1989) FEBS Lett. 242, 245–248; (1989) J. Neurochem., in press]. The related botulinum A toxin also consists of a heavy and a light chain linked by a disulfide bond. The actions of both neurotoxins on exocytosis were presently compared using streptolysin O‐permeabilized bovine adrenal chromaffin cells. Botulinum A toxin inhibited Ca2+‐stimulated catecholamine release from these cells. Addition of dithiothreitol lowered the effective doses to values below 5 nM. Under the same conditions, the effective doses of tetanus toxin were decreased by a factor of five. This indicates that the interchain SS bond of botulinum A toxin must also be split before the neurotoxin can exert its effect on exocytosis.

Exploring the functional domain and the target of the tetanus toxin light chain in neurohypophysial terminals

The tetanus toxin light chain blocks calcium induced vasopressin release from neurohypophysial nerve terminals. Here we show that histidine residue 233 within the putative zinc binding motif of the tetanus toxin light chain is essential for the inhibition of exocytosis, in the rat. The zinc chelating agent dipicolinic acid as well as captopril, an inhibitor of zinc-dependent peptidases, counteract the effect of the neurotoxin. Synthetic peptides, the sequences of which correspond to motifs present in the cytoplasmic domain of the synaptic vesicle membrane protein synaptobrevin 1 and 2, prevent the effect of the tetanus toxin light chain. Our results indicate that zinc bound to the zinc binding motif constitutes the active site of the tetanus toxin light chain. Moreover they suggest that cleavage of synaptobrevin by the neurotoxin causes the inhibition of exocytotic release of vasopressin from secretory granules.

Exocytotic membrane fusion as studied in toxin-permeabilized cells

Publisher Summary This chapter discusses the exocytotic membrane fusion as studied in toxin- permeabilized cells. Permeabilized cells have been widely used in the analysis of exocytotic membrane fusion or intracellular Ca 2+ regulation. They allow the study of the function of intracellular organelles in situ under conditions that are close to the physiological situation in intact cells. Toxin-permeabilized preparations have also been instrumental in analyzing the intracellular glucose metabolism in hepatocytes, the chain of events leading to smooth muscle contraction, and the regulation of intracellular Ca 2+ sequestration. In most of the studies dealing with exocytosis from permeabilized cells, an “intracellular medium” containing potassium as a main cation and glutamate as an anion I was used. Because the free Ca 2+ concentration within the cells under resting conditions, as well as during stimulation, is in the micromolar range, this ion must be carefully controlled in the buffers used.

Functional characterization of the catalytic site of the tetanus toxin light chain using permeabilized adrenal chromaffin cells

The molecular events underlying the inhibition of exocytosis by tetanus toxin were investigated in permeabilized adrenal chromaffin cells. We found that replacement of amino acid residues within the putative zinc binding domain of the tetanus toxin light chain such as of histidine (position 233) by cysteine or valine, or of glutamate (position 234) by glutamine completely abolished the effect of the light chains on Ca2+ induced catecholamine release. Dipicolinic acid, a strong chelating agent for zinc, also prevented the effect of the tetanus toxin light chain. Zn2+ and, less potently Cu2+ and Ni2+, but not Cd2+ and Co2+, restored the activity of the neurotoxin. These data show that zinc and the putative zinc binding domain constitute the active site of the tetanus toxin light chain. Neither captopril, an inhibitor of synaptobrevin cleavage nor peptides spanning the site of synaptobrevins cleaved by the tetanus toxin in neurons, prevented the inhibition of Ca2+ induced catecholamine release by the tetanus toxin light chain. This suggests that synaptobrevins are not a major target of tetanus toxin in adrenal chromaffin cells.

Noradrenaline release from permeabilized synaptosomes is inhibited by the light chain of tetanus toxin

Noradrenaline release from rat brain cortical synaptosomes permeabilized with streptolysin O can be triggered by μM concentrations of free Ca2+. This process was inhibited within minutes by tetanus toxin and its isolated light chain, but not by its heavy chain. The data demonstrate that the effect of tetanus toxin on NA release from purified synaptosomes is caused by the intraterminal action of its light chain.

Involvement of a 65 kDa phosphoprotein in the regulation of membrane fusion during exocytosis in Paramecium cells

Antisera were raised against a phosphoprotein of 65 kDa (PP65) from Paramecium cells (shown before to be selectively dephosphorylated during synchronous exocytosis) and specified by immunoblotting. By immunofluorescence PP65 has been localized within the cortex, beneath the cell membrane. This corresponds to data obtained by cell fractionation, applying SDS‐PAGE autoradiography to cortices prepared from 32P‐prelabeled cells. Antisera against PP65 inhibit exocytosis in vivo (microinjection). Applying anti‐PP65 antisera in vitro to cortices we could demonstrate inhibition not only of exocytosis, but also of PP65 dephosphorylation. We conclude that PP65 is involved in the regulation of membrane fusion during exocytosis.

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

Abstract: The inositol 1,4,5‐trisphosphate (IP3)‐induced Ca2+ release was studied using streptolysin O‐permeabilized bovine adrenal chromaffin cells. The IP3‐induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3‐induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 μM GTP fully restored the sensitivity to IP3. Guanosine 5′‐O‐(3‐thio)triphosphate (GTPγS) could not replace GTP but prevented the action of GTP. The effects of GTP and GTPγS were reversible. Neither GTP nor GTPγS induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 μM free Ca2+, a half‐maximal Ca2+ release was elicited with ∼0.1 μM IP3. At 1 μM free Ca2+, no Ca2+ release was observed with 0.1 μM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 μM) were required to evoke Ca2+ release. At 8 μM free Ca2+, even 0.25 μM IP3 failed to induce release of Ca2+ from the store. The IP3‐induced Ca2+ release at constant low (0.2 μM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. Depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.

Enzymatic Regulation of Membrane Fusion During Synchronous Exocytosis in Paramecium Cells

We have used Paramecium tetraurelia cells to analyze parameters relevant for membrane fusion regulation during exocytosis for different reasons: This cell type secretes trichocysts synchronously (1 sec); there exist secretory mutants; finally, the cell cortex can be isolated and used to analyze exocytosis in vitro. We have found with SDS-FAGE-ARG from 32P-prelabeled cells that the selective dephosphorylation of a 65 kD PP always parallels exocytosis. Its extent depends on the amount of trichocysts actually released (dose-dependence in “normal” cells; none in non-discharge mutations; variable in cells during replenishment of the trichocyst pool after a preceding trigger). SDS-PAGE-ARG or immuno labeling reveal 65 kD PP in the cell cortex, which also contains CaM (localized by affinity- or immuno-labeling specifically on the preformed exocytosis sites) and a CaN-like protein (shown on nitrocellulose blots). Ca+CaM+CaN stimulates exocytosis in vitro, as it does, after microinjection, in vivo. (Alkaline phosphatase can mimic this effect). Concomitantly, αCaM- or αCaN-AB suppress exocytosis. We could also show that Ca+CaM+CaN causes 65 kD PP dephosphorylation during exocytosis. The Ca2+-CaM triggered dephosphorylation of the 65 kD PP, mediated by a CaN-type phosphatase, is therefore considered as the/a primary step in fusion regulation during exocytosis. ATP possibly reduces the fusion capacity, at least in part, via protein rephosphorylation. In our system we could also ascertain that ATP consumption as well as protein phosphorylation occur only ≳5 seconds after synchronous exocytosis and protein dephosphorylation. The synchronicity of our system allows to avoid any possible overlaps of these phenomena.