Clostridial neurotoxins, including tetanus toxin and the seven serotypes of botulinum toxin (A-G), are produced as single chains and cleaved to generate toxins with two chains joined by a single disulphide bond (Fig. 1). The heavy chain (M(r) 100,000 (100K)) is responsible for specific binding to neuronal cells and cell penetration of the light chain (50K), which blocks neurotransmitter release. Several lines of evidence have recently suggested that clostridial neurotoxins could be zinc endopeptidases. Here we show that tetanus and botulinum toxins serotype B are zinc endopeptidases, the activation of which requires reduction of the interchain disulphide bond. The protease activity is localized on the light chain and is specific for synaptobrevin, an integral membrane protein of small synaptic vesicles. The rat synaptobrevin-2 isoform is cleaved by both neurotoxins at the same single site, the peptide bond Gln 76-Phe 77, but the isoform synaptobrevin-1, which has a valine at the corresponding position, is not cleaved. The blocking of neurotransmitter release of Aplysia neurons injected with tetanus toxin or botulinum toxins serotype B is substantially delayed by peptides containing the synaptobrevin-2 cleavage site. These results indicate that tetanus and botulinum B neurotoxins block neurotransmitter release by cleaving synaptobrevin-2, a protein that, on the basis of our results, seems to play a key part in neurotransmitter release.

Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin

The protein tyrosine kinase PYK2, which is highly expressed in the central nervous system, is rapidly phosphorylated on tyrosine residues in response to various stimuli that elevate the intracellular calcium concentration, as well as by protein kinase C activation. Activation of PYK2 leads to modulation of ion channel function and activation of the MAP kinase signalling pathway. PYK2 activation may provide a mechanism for a variety of short- and long-term calcium-dependent signalling events in the nervous system.

Protein tyrosine kinase PYK2 involved in Ca 2+ -induced regulation of ion channel and MAP kinase functions

▪ Abstract Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intr...

Membrane Fusion and Exocytosis

Nerve terminals are specific sites of action of a very large number of toxins produced by many different organisms. The mechanism of action of three groups of presynaptic neurotoxins that interfere directly with the process of neurotransmitter release is reviewed, whereas presynaptic neurotoxins acting on ion channels are not dealt with here. These neurotoxins can be grouped in three large families: 1) the clostridial neurotoxins that act inside nerves and block neurotransmitter release via their metalloproteolytic activity directed specifically on SNARE proteins; 2) the snake presynaptic neurotoxins with phospholipase A(2) activity, whose site of action is still undefined and which induce the release of acethylcholine followed by impairment of synaptic functions; and 3) the excitatory latrotoxin-like neurotoxins that induce a massive release of neurotransmitter at peripheral and central synapses. Their modes of binding, sites of action, and biochemical activities are discussed in relation to the symptoms of the diseases they cause. The use of these toxins in cell biology and neuroscience is considered as well as the therapeutic utilization of the botulinum neurotoxins in human diseases characterized by hyperfunction of cholinergic terminals.

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.2.717

Neurotoxins Affecting Neuroexocytosis

Neurotransmitter release is potently blocked by a group of structurally related toxin proteins produced by Clostridium botulinum. Botulinum neurotoxin type B (BoNT/B) and tetanus toxin (TeTx) are zinc-dependent proteases that specifically cleave synaptobrevin (VAMP), a membrane protein of synaptic vesicles. Here we report that inhibition of transmitter release from synaptosomes caused by botulinum neurotoxin A (BoNT/A) is associated with the selective proteolysis of the synaptic protein SNAP-25. Furthermore, isolated or recombinant L chain of BoNT/A cleaves SNAP-25 in vitro. Cleavage occurred near the carboxyterminus and was sensitive to divalent cation chelators. In addition, a glutamate residue in the BoNT/A L chain, presumably required to stabilize a water molecule in the zinc-containing catalytic centre, was required for proteolytic activity. These findings demonstrate that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25. Thus, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.

Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25

The clostridial neurotoxins tetanus and botulinum toxin type A are known to block transmitter release from nerve terminals1–3, probably by interfering with some essential process controlling exocytosis3,4 after the entry of Ca2+ ions Although exocytosis occurs in many secretory cells, these toxins show a high specificity for neurones and the secretory response of cultured bovine adrenal medullary cells is not inhibited by exposure to medium containing tetanus or botulinum toxin type A (although it is by botulinum toxin type D)4 Here we report that when tetanus toxin and botulinum neurotoxin type A are injected intracellularly into chromaffin cells they strongly inhibit secretion, as revealed by the measurement of cell capacitance5 These results indicate that these toxins are normally ineffective in chromaffin cells because they are not bound and internalized, so do not reach their site of action Furthermore, we have localized the secretion-blocking effects of the toxin to a fragment comprising the light chain covalently linked to part of the heavy chain, suggesting that this part of the molecule contains the active site

Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells

1.

Extracellular recordings of potential changes under the perineural sheath of nerve bundles close to some of the nerve terminals were performed using the M. triangularis sterni of the mouse.




2.

The nerve signals consisted of a predominant doublepeaked negativity which was often preceded by a small positive deflection. While the first negative peak is related to the propagating nerve action potential, the second negative deflection can be attributed to a potassium conductance since it was selectively blocked by tetraethylammonium (TEA) or 3,4-diaminopyridine (3,4-DAP).




3.

Combined application of TEA and 3,4-DAP gave rise to a prolonged positive-going wave which was blocked by Cd2+, thus, indicating its underlying cause to be a Ca current.




4.

Ionophoretic application of TEA and Cd2+ to the endplates affected potassium and calcium components of the subendothelial signals, respectively, thus indicating their presynaptic origin. This finding is supported by the decrease of the amplitude of these components with increasing distance from the endplate region.




5.

Maximal effects on K conductance attainable with 3,4-DAP could still be potentiated by TEA, indicating the presence of at least two distinct sets of K channels.




6.

The prolonged positive potential induced by TEA and 3,4-DAP consisted of a fast and slow component, both of which can be attributed to Ca conductances with different characteristics.




7.

The fast positive signal component is attributed to the voltage-dependent Ca channel, responsible for the initiation of transmitter release. Its amplitude and duration depend on extracellular Ca2+-concentration. The fast component is still present when Ca2+ is substituted by Sr2+ or Ba2+. It is blocked by Cd2+ and Mn2+ in the millimolar range, but remains unaffected by organic Ca-antagonists.




8.

The slow positive signal component, whose physiological role remains to be elucidated, also depends on extracellular Ca2+-concentration. It is reduced by frequent nerve stimulation. The slow component can be carried by Sr2+ or Ba2+ and it is blocked by Cd2+ and Mn2+ in the micromolar range. In contrast to the fast ‘Ca potential’ the slow one is reduced by verapamil and diltiazem but not by the 1,4-dihydropyridines nitrendipine and nisoldipine. Concentration-response curves can best be described assuming two dissociation constants.

Two different presynaptic calcium currents in mouse motor nerve terminals

Botulinum a neurotoxin inhibits non-cholinergic synaptic transmission in mouse spinal cord neurons in culture

1.

The blocking effect of tetanus toxin on the neuromuscular junction of the mouse phrenic nervehemidiaphragm preparation exposed to the toxin (0.05–20 μg/ml) in the organ bath was studied and compared with the action of botulinum A toxin.




2.

The time course of the paralysis of the diaphragm could be divided into a latent and a manifest period. Still during the latent period the effect of the toxin became progressively resistant to washing and, with some delay, to antitoxin.




3.

Between 25 and 41°C the time until paralysis strongly depended on temperature with Q10 of about 2.7.




4.

Procedures increasing the transmitter release shortened, and procedures depressing it prolonged the time until paralysis.




5.

4-Aminopyridine and guanidine temporarily restored the contraction of the partially paralyzed diaphragm, indicating the persistence of activatable calcium and acetylcholine pools. Raising the external Ca2+-concentration and application of the Ca-Ionophore A 23187 were ineffective in the doses applied.




6.

About 80 min after exposure to the toxin (10 μg/ml), the m.e.p.p. activity decreased by a factor of 30. Parallel to this, paralysis of nerve evoked muscle contraction developed.




7.

Neuraminidase treatment did not prevent tetanus toxin poisoning.




8.

The paralysis is produced by tetanus toxin itself and not by contaminants as shown by the parallel decrease of toxicity and paralysis following treatment with either antitoxin or brain homogenate, or by the use of spontaneously inactivated toxin.




9.

Tetanus toxin was compared with botulinum A toxin as to the shape of its dose-response curve, time course of paralysis, temporary reversal by 4-aminopyridine and behaviour against Ca-ionophore. In any case, both toxins were indistinguishable, albeit botulinum A neurotoxin was calculated to be about 2000 times more potent than tetanus toxin.

Tetanus toxin blocks the neuromuscular transmission in vitro like botulinum A toxin.

After the first detailed description of the delayed outward potassium current in squid axon by Hodgkin and Huxley (1952) it took electrophysiologists more than 20 years to realize that in addition to it several types of K+ currents can exist in the same cell and that they have a number of functions including modulation of cell excitability and modulation of integrating neuronal function. That as late as 1985 an international symposium on membrane control of cellular activity completely excluded K+ currents highlights the comparatively late interest of electrophysiologists in these ion currents. Moreover, our knowledge of the structure and function of K+ channels was very limited compared with what we know about Na2+ and Ca2+ channels and the acetylcholine receptor. One major reason has been the lack of suitable ligands that act on K+ channels with high affinity and selectivity such as tetrodotoxin on Na+ channels, dihydropyridines on one type of Ca2+ channels and α-bungarotoxin on nicotinic acetylcholine receptors. This has now profoundly changed mainly due to the development of the patch-clamp recording technique by Neher and Sakmann (Hamill et al. 1981), giving the tool to differentiate between an increasing number of receptor- and/or voltage-operated ion channels. Particularly the K+ channels have now mushroomed into a large, branching family of at least 11 members (Cook 1988). The dilemma is now that cell membranes, not only excitable ones, are equipped with a variety of K+ channels differing in their gating properties, which often complicates the interpretation of whole-cell K+ currents and their modulation by drugs.

Florian Dreyer

Papers

Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells

Two different presynaptic calcium currents in mouse motor nerve terminals

Botulinum a neurotoxin inhibits non-cholinergic synaptic transmission in mouse spinal cord neurons in culture

Tetanus toxin blocks the neuromuscular transmission in vitro like botulinum A toxin.

Peptide Toxins and Potassium Channels