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The seven serotypes (A-G) of botulinum neurotoxin (BoNT) are proteins produced by Clostridium botulinum and have multifunctional abilities: (i) they target cholinergic nerve endings via binding to ecto-acceptors (ii) they undergo endocytosis/translocation and (iii) their light chains act intraneuronally to block acetylcholine release. The fundamental process of quantal transmitter release occurs by Ca2+-regulated exocytosis involving sensitive factor attachment protein-25 (SNAP-25), syntaxin and synaptobrevin. Proteolytic cleavage by BoNT-A of nine amino acids from the C-terminal of SNAP-25 disables its function, causing prolonged muscle weakness. This unique combination of activities underlies the effectiveness of BoNT-A haemagglutinin complex in treating human conditions resulting from hyperactivity at peripheral cholinergic nerve endings. In vivo imaging and immunomicroscopy of murine muscles injected with type A toxin revealed that the extended duration of action results from the longevity of its protease, persistence of the cleaved SNAP-25 and a protracted time course for the remodelling of treated nerve-muscle synapses. In addition, an application in pain management has been indicated by the ability of BoNT to inhibit neuropeptide release from nociceptors, thereby blocking central and peripheral pain sensitization processes. The widespread cellular distribution of SNAP-25 and the diversity of the toxin's neuronal acceptors are being exploited for other therapeutic applications.

Voltage-gated K+ currents expressed in two rat prostate cancer ("Dunning") cell lines of markedly different metastatic ability were characterised using electrophysiological, pharmacological and molecular approaches. Whole-cell patch-clamp recordings showed that both strongly metastatic MAT-LyLu and weakly metastatic AT-2 cell lines possessed outward (delayed-rectifier type) K+ currents, which activated at around -40 mV. From the parameters measured, several characteristics of the two cell lines were similar. However, a number of statistically significant differences were noted for MAT-LyLu versus the AT-2 cells as follows: (1) current densities were smaller; (2) the slope factor for channel activation was smaller; (3) the voltage at which current was half-inactivated, and the slope factor for channel inactivation were greater; (4) the time constants for current decay at -20 and 0 mV were smaller; and (5) the residual peak current was larger following 60 s of repetitive voltage pulses for stimulation frequencies in the range 0.05-0.2 Hz. On the other hand, the K+ currents in both cell lines showed similar pharmacological profiles. Thus, the currents were blocked by 4-aminopyridine, tetraethylammonium, verapamil, margatoxin, and charybdotoxin, with highly similar IC(50)s for given blockers. The electrophysiological and pharmacological data taken together suggested expression of voltage-gated K+ channels of the Kv1 family, expression of the Kv1.3 subunit being predominant. Western blot and RT-PCR tests both confirmed that the cells indeed expressed Kv1.3 and to a lesser extent Kv1.4 and Kv1.6 channel alpha-subunits. In view of the similarity of channel expression in the two cell lines, voltage-gated K+ channel activity may not be a primary determinant of metastatic potential in the rat model of prostate cancer, but the possible contribution of K+ channel activity to the metastatic process is discussed.

Pure alpha-latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the alpha-latrotoxin pores are permeable to Ca(2+) and small molecules including fluorescein isothiocyanate and norepinephrine. Bound alpha-latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin (in the presence of Ca(2+)). Interestingly, on subsequent removal of Ca(2+), alpha-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-alpha-latrotoxin antibodies. Our results indicate that (i) alpha-latrotoxin is a pore-forming toxin, (ii) receptors that bind alpha-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) alpha-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require alpha-latrotoxin interaction with other neuronal proteins.

The multiple roles of voltage-sensitive K(+) channels (Kv1 subfamily) in brain are served by subtypes containing pore-forming alpha (1.1-1.6) and auxiliary beta subunits, usually in an (alpha)(4)(beta)(4) stoichiometry. To facilitate structure/activity analysis, combinations that are prevalent in neurones and susceptible to alpha-dendrotoxin (alphaDTX) were reproduced in mammalian cells, using Semliki Forest virus. Infected Chinese hamster ovary cells expressed N-glycosylated Kv1.1 and 1.2 alpha subunits (M(r) approximately 60 and 62 K) that assembled and bound [(125)I]-alphaDTX with high affinity; an appreciable proportion appeared on the cell surface, with Kv1.2 showing a 5-fold enrichment in a plasma membrane fraction. To obtain 'native-like' alpha/beta complexes, beta1.1 or 2.1 (M(r) approximately 42 and 39 K, respectively) was co-expressed with Kv1.1 or 1.2. This slightly enhanced N-glycosylation and toxin binding, most notable with beta2. 1 and Kv1.2. Solubilization of membranes from cells infected with Kv. 1.2 and beta2.1, followed by Ni(2+) chromatography, gave a purified alpha1.2/beta2.1 complex with a size of approximately 405 K and S(20, W) = 15.8 S. Importantly, these values indicate that four alpha and beta subunits co-assembled as in neurones, a conclusion supported by the size ( approximately 260 K) of the homo-tetramer formed by Kv1.2 alone. Thus, an authentic K(+) channel octomer has been reconstructed; oligomeric species were also found in plasma membranes. To create 'authentic-like' hetero-oligomeric channels, Kv1.1 and 1.2 were co-expressed and shown to have assembled by the precipitation of both with IgGs specific for either. Consistently, confocal microscopy of cells labeled with these antibodies showed that the relatively low surface content of Kv1.1 was increased by Kv1.2. [(125)I]-alphaDTX binding to these complexes was antagonized by DTX(k), a probe selective for Kv1.1, in a manner that mimicks the pattern observed for the Kv1.1/1.2-containing channels in neuronal membranes.

We have examined the effects of co-expression of Kvbeta1.1 and Kvbeta2.1 subunits on the gating of rat brain Kv1.4 channels, expressed in Xenopus oocytes. Expression of Kv1.4 subunits alone produced a rapidly inactivating "A" type current, which activated at potentials beyond -60 mV in a solution containing high levels of rubidium. Current activation curves obtained from tail current measurements were fitted with a Boltzmann function, with V1/2 = -47 mV and k = 10 mV. Neither the Kvbeta1.1 nor Kvbeta2.1 subunits altered the voltage dependence of activation. Both subunits accelerated the activation time constant of Kv1.4, without affecting its voltage dependence. Surprisingly, the Kvbeta2.1 subunit, which lacks an N-terminal inactivation domain, was almost as effective as the Kvbeta1.1 subunit in speeding up Kv1.4. Steady-state inactivation of Kv1.4 was unchanged upon co-expression with either Kvbeta1.1 or Kvbeta2.1 subunits. Kv1.4 recovered from inactivation with two time constants; apart from an approximately 50% lengthening of the slow time constant with a high Kvbeta2.1 injection ratio, neither time constant was altered by either the Kvbeta1.1 or Kvbeta2.1 subunits, suggesting little interaction with recovery from C-type inactivation. Clearly, beta subunits have the potential to modify the gating of Kv1.4 channels in the brain more subtly than has been suggested previously.

Structural and functional diversity of voltage-gated Kv1-type potassium channels in rat brain is enhanced by the association of two different types of subunits, the membrane-bound, poreforming alpha-subunits and a peripheral beta-subunit. We have cloned a beta-subunit (Kv beta 1) that is specifically expressed in the rat nervous system. Association of Kv beta 1 with alpha-subunits confers rapid A-type inactivation on non-inactivating Kv1 channels (delayed rectifiers) in expression systems in vitro. This effect is mediated by an inactivating ball domain in the Kv beta 1 amino terminus.

Voltage-dependent cation channels are large heterooligomeric proteins. Heterologous expression of cDNAs encoding the alpha subunits alone of K+, Na+, or Ca2+ channels produces functional multimeric proteins; however, coexpression of those for the latter two with their auxiliary proteins causes dramatic changes in the resultant membrane currents. Fast-activating, voltage-sensitive K+ channels from brain contain four alpha and beta subunits, tightly associated in a 400-kDa complex; although molecular details of the alpha-subunit proteins have been determined, little is known about the beta-subunit constituent. Proteolytic fragments of a beta subunit from bovine alpha-dendrotoxin-sensitive neuronal K+ channels yielded nine different sequences. In the polymerase chain reaction, primers corresponding to two of these peptides amplified a 329-base-pair fragment in a lambda gt10 cDNA library from bovine brain; a full-length clone subsequently isolated encodes a protein of 367 amino acids (M(r) approximately 40,983). It shows no significant homology with any known protein. Unlike the channels' alpha subunits, the hydropathy profile of this sequence failed to reveal transmembrane domains. Several consensus phosphorylation motifs are apparent and, accordingly, the beta subunit could be phosphorylated in the intact K+ channels. These results, including the absence of a leader sequence and N-glycosylation, are consistent with the beta subunit being firmly associated on the inside of the membrane with alpha subunits, as speculated in a simplified model of these authentic K+ channels. Importantly, this first primary structure of a K(+)-channel beta subunit indicates that none of the cloned auxiliary proteins of voltage-dependent cation channels, unlike their alpha subunits, belong to a super-family of genes.

Mamba dendrotoxins, 7K M(r) polypeptides with three disulfide bonds, selectively inhibit certain fast-activating, voltage-sensitive neuronal K+ channels and have been instrumental in their identification, localization, and purification. However, derivatives with more refined specificity are essential to define the structural and functional properties of the multiple subtypes known to reside in the nervous system. Hence, utilizing a constructed cDNA library from the venom glands of the black mamba (Dendroaspis polylepis), the gene encoding dendrotoxin K was isolated, amplified, and expressed as a maltose-binding fusion protein in the periplasmic space of Escherichia coli. After cleavage of the chaperone from the affinity-purified product, a recombinant protein was isolated and shown to be identical to native dendrotoxin K in its N-terminal sequence, chromatographic behavior, convulsive-inducing activity, and binding to voltage-activated K+ channels in bovine synaptic membranes. This successful expression of refolded active toxin, in adequate yield, makes possible for the first time the preparation of mutants with specificity tailored for each K+ channel subtype, based both on the recently derived three-dimensional structure of alpha-dendrotoxin and the identified binding site on cloned K+ channels.

1. In the rat olfactory cortex, unmyelinated axons give rise to synapses en passant. This tissue was used to study the pharmacology of axonal K(+)-currents. Responses were measured from a group of these axons as unclamped field currents, with a polarizable suction electrode. 2. A single stimulus to the axons elicited a tetrodotoxin-sensitive Na(+)-dependent transient K(+)-currents were revealed by positive polarization of the suction electrode and were manifest as a negative current following the Na(+)-component. 3. In the presence of tetraethylammonium (TEA, 5 mM) and Cd2+ (100 microM), the K(+)-component was depressed by 3,4-diaminopyridine (3,4-DAP; 1 to 20 microM; IC50 2.0 +/- 0.4 microM). alpha-Dendrotoxin (DTX; 15-1500 nM) also attenuated the aminopyridine-sensitive component (IC50 93 +/- 4 nM). At the highest DTX concentration, depression of the K(+)-current was incomplete, the residual K+ current being reduced by 3,4-DAP (0.1 to 5 microM). 4. These results indicate the presence of two aminopyridine-sensitive K+ currents in this preparation distinguished by their susceptibility to DTX.

Neuronal acceptors for alpha-dendrotoxin (alpha-DTX) have recently been purified from mammalian brain and shown to consist of two classes of subunit, a larger (approximately 78,000 M(r)) protein (alpha) whose N-terminal sequence is identical to that of a cloned, alpha-DTX-sensitive K+ channel, and a novel M(r) 39,000 (beta) polypeptide of unknown function. However, little information is available regarding the oligomeric composition of these native molecules. By sedimentation analysis of alpha-DTX acceptors isolated from bovine cortex, two species have been identified. A minority of these oligomers contain only the larger protein, while the vast majority possess both subunits. Based on accurate determination of the molecular weights of these two forms it is proposed that alpha-DTX-sensitive K+ channels exist as alpha 4 beta 4 complexes because this combination gives the best fit to the experimental data.

High affinity acceptors for alpha-dendrotoxin, a selective probe for certain fast activating voltage-dependent K+ channels, were purified approximately 4,000-fold from synaptic plasma membranes of bovine cerebral cortex. Although the preparation possessed a low content of high affinity sites for beta-bungarotoxin, antagonism of alpha-dendrotoxin binding by the latter required high concentrations; this indicates that more than one acceptor subtype has been purified. After deglycosylation of the acceptor, the sizes of its subunits were determined electrophoretically to be Mr 65,000 and Mr 39,000. Solid phase microsequencing of these isolated subunits showed that the smaller one had a blocked N terminus, but the Mr 65,000 protein gave a sequence of 27 residues. This is virtually identical to the N-terminal sequence deduced from cDNA of RCK 5, a K+ channel protein from rat brain known to be susceptible to alpha-dendrotoxin. This first report on the partial sequence of any K+ channel protein confirms that the extensive information acquired to date on the alpha-dendrotoxin acceptors is pertinent to functional neuronal K+ channels.

With use of biologically active 125I-labelled delta-dendrotoxin, a putative K+-channel ligand, homogeneous, noninteracting, high-affinity acceptors (KD = 0.32 +/- 0.07 nM; Bmax = 0.33 +/- 0.04 pmol/mg) were observed in synaptosomes from guinea-pig cortex. This binding was antagonised noncompetitively by alpha-dendrotoxin, an inhibitor of certain fast-activating, voltage-gated K+ channels. Chemical cross-linking of the delta-dendrotoxin-acceptor complex in synaptosomes yielded two specifically labeled polypeptides with molecular masses of 69 and 82 kilodaltons. Although alpha-dendrotoxin prevents the labelling of both these bands, it cross-linked only a single protein with a molecular mass of 69 kilodaltons. It is concluded that delta-dendrotoxin interacts with a distinct site on the oligomeric acceptors for alpha-dendrotoxin.

Treatment of rat cerebrocortical synaptosomes with botulinum toxin types E and C1 or tetanus toxin removed the majority of intact SNAP-25, syntaxin 1A/1B, and synaptobrevin and diminished Ca(2+)-dependent K+ depolarization-induced noradrenaline secretion. With botulinum toxin type E,10% of intact SNAP-25 remained and K(+)-evoked release of glutamate and GABA was inhibited. The large component of noradrenaline release evoked within 120 s by inclusion of the Ca2+ ionophore A23187 with the K+ stimulus was also attenuated by these toxins; additionally, botulinium neurotoxin type E blocked the first 60 s of ionophore-induced GABA and glutamate exocytosis. However, exposure to A23187 for longer periods induced a phase of secretion nonsusceptible to any of these toxins (120 s for noradrenaline;60 s for glutamate or GABA). Most of this late phase of release represented exocytosis because of its Ca2+ dependency, ATP requirement, and sensitivity to a phosphatidylinositol 4-kinase inhibitor. Based on these collective findings, we suggest that the ionophore-induced elevation of [Ca2+]i culminates in the disassembly of complexes containing nonproteolyzed SNAP receptors protected from the toxins that can then contribute to neuroexocytosis.

Previous electrophysiological evidence has indicated that astrocytes and oligodendrocytes express inwardly rectifying K(+) channels both in vitro and in vivo. Here, for the first time, we have undertaken light microscopic immunohistochemical studies demonstrating the location of one such channel, Kir4.1, in both cell types in regions of the rat CNS. Some astrocytes such as those in the deep cerebellar nuclei, Bergmann glia, retinal Müller cells, and a subset in hippocampus express Kir4.1 immunoreactivity, but not others including those in white matter. Oligodendrocytes also express this protein, strongly in perikarya and to a lesser extent in their processes. Expression of Kir4.1 in astrocytes and oligodendrocytes would enable these cells to clear extracellular K(+) through this channel, whereas nonexpressors might use other mechanisms.

α–latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G–protein–coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin–G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with Gα q/11 and Gα o but not with Gα s , Gα i or Gα z , indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX–evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca 2+ , LTX triggers vesicular exocytosis because botulinum neurotoxins E, C1 or tetanus toxin inhibit the Ca 2+ –dependent component of the toxin–evoked release. Based on (i) the known involvement of Gα q in the regulation of inositol–1,4,5–triphosphate generation and (ii) the requirement of Ca 2+ in LTX action, we tested the effect of inhibitors of Ca 2+ mobilization on the toxin–evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca 2+ –dependent toxin's action. Thapsigargin, which depletes intracellular Ca 2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca 2+ . On the other hand, clostridial neurotoxins or drugs interfering with Ca 2+ metabolism do not inhibit the Ca 2+ –independent component of LTX–stimulated release. In the absence of Ca 2+ , the toxin induces in the presynaptic membrane non–selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LTX stimulates norepinephrine exocytosis only in the presence of external Ca 2+ provided intracellular Ca 2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca 2+ , which then triggers secretion.

α‐Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca 2+ ‐dependent and ‐independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca 2+ ‐dependent LTX‐evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10‐fold more sensitive to external Ca 2+ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca 2+ and is blocked by drugs depleting Ca 2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca 2+ ‐independent LTX‐stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca 2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin9s effects and suggest that LTX‐stimulated exocytosis depends upon the co‐operative action of external and intracellular Ca 2+ involving G proteins and PLC, whereas the Ca 2+ ‐independent release is largely non‐vesicular.

Dendrotoxin K (DTXK) is a 57-residue protein from mamba venom that blocks certain non-inactivating, voltage-activated K+ currents in neurones. In order to pinpoint the residues responsible for its specificity, structure-activity relations of DTX(K) were investigated by mutagenesis. A previously cloned gene encoding this toxin [Smith et al. (1993) Biochemistry 32, 5692-5697] was used to make single mutations; after expression in Escherichia coli as fusion proteins and enzymatic cleavage of the conjugates isolated from the periplasmic space, nine toxins were purified. Structural analysis of the native DTXK and representative mutants by circular dichroism showed that no significant differences were detectable in their folded structures. The biological activity of the mutants, which contained alterations of positively charged and other amino acids, was determined from their abilities to compete for the binding of 125I-labeled DTX(K) to K+ channels in synaptic plasma membranes from rat cerebral cortex. Mutants with residues substituted in the alpha-helix near the C-terminus (R52A or R53A) yielded binding parameters similar to those of wild-type and native DTX(K). In the case of the beta-turn (residues 24-28), however, altering single amino acids reduced binding to the high-affinity site of K+ channels, with the rank order of decreases being K26A >> W25A > K24A = K28A. Also, substitutions made in the 3(10)-helix (residues 3-7), a region located close to the beta-turn, produced equivalent effects (K3A > K6A). Thus, it is deduced that residues in the distorted beta-turn and neighboring 3(10)-helix of DTX(K) are critical for its interaction with neuronal K+ channels.

Tetanus toxin (TeTX) has been demonstrated to inhibit transmitter release by two mechanisms: Zn(2+)-dependent proteolytic cleavage of synaptobrevin and activation of a neuronal transglutaminase. Herein, attenuation of TeTX-induced blockade of noradrenaline release from synaptosomes was achieved by prior disassembly of microfilaments with cytochalasin D or breakdown of microtubules by colchicine or nocodazole. These drugs and monodansylcadaverine, a transglutaminase inhibitor, displayed some additivity in antagonizing the inhibitory effect of the toxin on synaptosomal transmitter release; as none of them reduced synaptobrevin cleavage, all appear to work independently of the toxin's proteolytic action. Prior stabilization of microtubules with taxol prevented the antagonism seen with colchicine, highlighting that this cytoskeletal component is the locus of the effect of colchicine. Replacement of Ca2+ with Ba2+ caused disappearance of the fraction of evoked secretion whose inhibition by TeTX is reliant on polymerized actin but did not alter the blockade by toxin that is dependent on microtubules. Two temporally distinguished phases of release were reduced by TeTX, and colchicine lessened its effects on both. Blockade of the fast phase (or = 10 s) of secretion by TeTX was unaffected by cytochalasin D, but it clearly antagonized the toxin-induced inhibition of the slow (10-s toor = 5-min) component; it is notable that such antagonism was accentuated during a second bout of evoked release. These findings are consistent with sustained release requiring dissociation of synaptic vesicles from the microfilaments, a step that seems to be perturbed by TeTX.

Botulinum neurotoxin type A, a di-chain protein produced by Clostridium botulinum and responsible for botulism, blocks acetylcholine release from peripheral nerves by binding to the terminals, undergoing internalization and proteolyzing a protein essential for exocytosis. As butolinum neurotoxin is being used clinically for the treatment of dystonias and certain spasticities, deciphering the details of its specific targeting to cholinergic nerve endings has assumed great importance. Thus, interaction of butolinum neurotoxin type A with murine motor nerve terminals-a prime target in vivo-was investigated. Autoradiographic analysis revealed saturable, high-affinity interaction of radioiodinated toxin (0.4 nM) with two ecto-acceptor types, distinguished by an excess of the toxin's heavy chain which prevented only a fraction of this binding. Botulinum neurotoxin was also biotinylated through its free sulfhydryl groups, known not to be essential for neurotoxicity. Similar binding of this active derivative was, likewise, partially blocked by heavy chain, confirming the above results. This binding that is resistant to heavy chain equates to botulinum neurotoxin interacting with productive ecto-acceptors, leading to delivery to its cytosolic site of action, because heavy chain proved unable to antagonize toxin-induced neuromuscular paralysis. In contrast, it is deduced that botulinum neurotoxin bound to heavy chain-susceptible sites has a different fate, presumably due to trafficking via another route, and thus would be inefficient in causing neuroparalysis.

Specific proteolysis by the tetanus toxin light chain of a vesicle-associated membrane protein (VAMP) involved in exocytosis is thought to underlie its intracellular blockade of neurotransmitter release. To substantiate this mechanism, recombinant light chain was expressed as a maltose binding protein-light chain fusion product in Escherichia coli. After purification of affinity chromatography and cleavage with factor Xa, the resultant light chain was isolated and its identity confirmed by Western blotting and N-terminal sequencing. It exhibited activity similar to that of the native light chain in proteolyzing its target in isolated bovine small synaptic vesicles and in hydrolyzing a 62-residue synthetic polypeptide spanning the cleavage site of the substrate. The importance of Glu234 in the catalytic activity of the light chain, possibly analogous to Glu143 of thermolysin, was examined using site-directed mutagenesis. Changing Glu234 to Ala abolished the protease activity of the light chain, but its ability to bind the polypeptide substrate was retained. Each recombinant light chain could be reconstituted with the heavy chain of tetanus toxin, yielding the same level of disulfide-linked species as the two native chains. Whereas the toxin formed with wild-type light chain exhibited appreciable neuromuscular paralysis activity and mouse lethality, the equivalent dichain material containing the Ala234 mutant lacked neurotoxicity in both the in vitro and in vivo assays. Thus, these results demonstrate directly, for the first time, that the lethality of tetanus toxin and its inhibition of exocytosis in intact neurons are attributable largely, if not exclusively, to endoprotease activity.

The epileptogenic and neurodegenerative effects induced by intra-hippocampal injection of a selective K + channel inhibitor, α-dendrotoxin (DTx), were investigated in normal rats and those bearing a monolateral surgical lesion of the Schaffer collaterals that causes degeneration of their nerve terminals and also, isolates the CA3 area. In addition, these effects have also been studied in rats pretreated with NBQX, an AMPA receptor antagonist. Injection of DTx (35 pmol) into one dorsal hippocampus induced motor and electrocortical (ECoG) seizures in all the treated animals that were rapid in onset (within 2–3 min). The seizures were accompanied at 24 h by significant neuronal cell loss which occurred in the CA1, CA3 and CA4 pyramidal cell layers of the hippocampus, ipsilateral to the side of injection. This neuronal loss was paralleled by a significant decrease in the density of radioiodinated DTx labelled acceptors. Lesioning of the excitatory afferents to the CA1 pyramidal cells, gave a substantial reduction in the density of radioiodinated DTx labelled acceptors in the strata oriens and radiatum, revealing that a proportion of these K + channels are present on the Schaffer collateral terminals. Under these conditions, motor and ECoG seizures persisted. As expected, the lesion prevented loss of the isolated CA3 pyramids, normally produced by the administration of DTx, leaving unaffected CA1 and CA4 pyramidal cell damage, consistent with an observed diminuition of DTx binding sites in the latter areas. In unlesioned rats pre-treated with NBQX (30 mg/kg i.p.), subsequent injection of DTx evoked epileptogenic effects after a latency of 15 min and caused significant cell loss in the CA1 but not in the CA3 and CA4 pyramidal cell layers, ipsilateral to the side of toxin injection. A lower dose of NBQX (15 mg/kg i.p.) proved ineffective. In conclusion, these data together with our published results on NMDA antagonists indicate that motor and ECoG seizures and CA1 pyramidal cell loss elicited by intra-hippocampal injection of the K + channel blocker, DTx, are independent from mechanisms involving glutamate-mediated excitotoxicity whereas CA3 and CA4 pyramidal cell loss may be the consequence of excessive activation of AMPA receptors.

Despite the fundamental importance of quantal transmitter release in neural communication, there is only limited information available on the detailed mechanism(s) involved (Valtorta et al. 1990; Tauc and Poulain 1991). Clearly, progress with this challenging problem ought to be achieved by identifying molecular components in nerve terminals concerned with the process or its regulation. As several neurotoxins have been characterized with abilities to perturb transmitter release in very selective ways (Table 1), using them as probes to discover such functional entities is proving to be a successful experimental strategy (reviewed by Dolly 1990, 1991). Of course, antibodies to particular proteins involved in synaptic vesicle trafficking (e.g synaptophysin; Valtorta et al. 1988) or thought to be concerned with acetylcholine (ACh) release (e.g. mediatophore; Israel et al. 1990) can also provide useful tools, but few such macromolecules have been identified. Moreover, when antibodies are raised, they often bind to non-essential epitopes (or their activity cannot be detected in screening due to the lack of suitable assays). In contrast, neurotoxins are by definition designed to cause malfunction of their targets and, fortunately for research purposes, these can represent a series of proteins (e.g. Fig. 1) that function in the multi-step process of synaptic transmission. In other cases, a family of homologous toxins (e.g. dendrotoxins) exhibit subtle differences in specificities for variants of voltage-dependent, fast-activating K+ channels (cf. Awan and Dolly 1991).

Voltage-gated K-channels of the Shaker related subfamily have two subunits, membrane integrated alpha- and peripheral beta-subunits. alpha-Subunits may assemble as tetramers and form in in vitro expression systems functional K-channels. beta-Subunits cannot from channels by themselves. Like for alpha-subunits, the rat nervous system apparently expresses a family of beta-subunit proteins. We have demonstrated that one rat K-channel beta-subunit, Kv beta 1, contains an inactivating domain. Upon association of alpha- and Kv beta 1-subunits, delayed-rectifier type K-channels are converted to rapidly inactivating A-type K-channels. The beta-subunit inactivation domain acts via a ball and chain type mechanism previously proposed for N-type inactivation of alpha-subunits. The association of alpha- and beta-subunits endows the nervous system with an unprecedented flexibility and diversity of K-channels which may play an important role in the regulation of nervous excitability.

Botulinum neurotoxin type A consists of a disulfide-linked light and heavy chain, with an intradisulfide present within the C-terminal half of the latter. The functional consequences of reducing these bonds and alkylating the thiols were investigated. Modification of free cysteine residues had no effect on the toxicity in mouse bioassays or on acetylcholine release in the mouse nerve-diaphragm and the buccal ganglion of Aplysia californica. However, reduction of the toxin prior to alkylation drastically decreased neuroparalytic potency; yet, this derivative inhibited transmitter release if injected directly into a presynaptic neuron in the Aplysia ganglion or added to bovine permeabilized adrenal chromaffin cells. Its antagonism of the action of botulinum neurotoxin A at mammalian motor nerve endings and Aplysia neurons indicates retention of the ability to bind to the toxin's productive ecto-acceptors. Thus, the abolition of the toxicity of extracellularly applied botulinum neurotoxin A by the cleavage of both disulfides, and the alkylation of the half-cystines involved, results from ineffective uptake. Modified forms of the isolated chains of botulinum neurotoxin A were utilized to determine which of the disulfides were necessary for internalization. Alkylation of the cysteines in the light and heavy chains, including those involved in the interchain bond but excluding those of the intact disulfide in the heavy chain, revealed that the intermolecular bond must be present, or the thiols concerned unmodified, for botulinum neurotoxin A to undergo membrane translocation into Aplysia neurons.

The neuroparalytic activities of botulinum neurotoxin type A (BoNT A), tetanus toxin (TeTx), or homologous and heterologous combinations of their constituent polypeptides were examined at cholinergic and non-cholinergic synapses of Aplysia californica. When applied extracellularly, BoNT A or a mixture of its heavy (HC) and light (LC) chains were far more potent in blocking transmitter release at cholinergic than non-cholinergic synapses. The reverse was true for TeTx or a mixture its constituent chains. Such selectivity was assigned to differences in neuronal targetting and uptake of the neurotoxins since both exhibited similar potencies when injected directly into the cell body of either cell type. When bath-applied, heterologous combinations of the toxins' HC and LC appeared as effective as the parent neurotoxins from whence each HC was derived. Moreover, targetting/internalization was attributable to the analogous N-terminal moieties, H2 and beta 2, of the HC from BoNT A and TeTx. Thus, it may be postulated that the latter regions possess two functional domains, one being distinct and responsible for the divergent neuronal specificity, whereas the other serves a common role in translocating the LC of either toxin. Also, it was shown that the C-terminal portion of the HC of TeTx is unable to play the intracellular role of its counterpart in BoNT A.

The inhibitory effects of botulinum neurotoxins types A and B on Ca2(+)-dependent evoked release of [3H]noradrenaline from rat cerebrocortical synaptosomes were compared and their molecular basis investigated. A23187, a Ca2+ ionophore, proved more efficacious in reversing the blockade produced by type A than that by B, whereas the actions of neither were changed by increasing intraterminal cyclic GMP levels using 8-bromo-cyclic GMP of nitroprusside. Disruption of the actin-based cytoskeleton with cytochalasin D did not alter the inhibition seen subsequently with either toxin. However, prior disassembly of microtubules with colchicine, nocodazole, or griseofulvin reduced the potency of type B toxin, but not that of type A toxin; stabilization of the microtubules with taxol counteracted this effect of colchicine. Because colchicine treatment of synaptosomes did not interfere with the measurable binding of type B toxin or its apparent uptake, it appears to act intracellularly. Collectively, these data suggest that botulinum neurotoxins types A and B inactivate transmitter release by interaction at different sites in the process. Based on the consistent results observed with four different drugs known to affect selectively microtubules, their involvement in the action of the type B neurotoxin is proposed.

High affinity acceptors for alpha-dendrotoxin, a selective probe for certain fast activating voltage-dependent K+ channels, were purified approximately 4,000-fold from synaptic plasma membranes of bovine cerebral cortex. Although the preparation possessed a low content of high affinity sites for beta-bungarotoxin, antagonism of alpha-dendrotoxin binding by the latter required high concentrations; this indicates that more than one acceptor subtype has been purified. After deglycosylation of the acceptor, the sizes of its subunits were determined electrophoretically to be Mr 65,000 and Mr 39,000. Solid phase microsequencing of these isolated subunits showed that the smaller one had a blocked N terminus, but the Mr 65,000 protein gave a sequence of 27 residues. This is virtually identical to the N-terminal sequence deduced from cDNA of RCK 5, a K+ channel protein from rat brain known to be susceptible to alpha-dendrotoxin. This first report on the partial sequence of any K+ channel protein confirms that the extensive information acquired to date on the alpha-dendrotoxin acceptors is pertinent to functional neuronal K+ channels.

1. A large-scale purification procedure has been developed for Clostridium botulinum type F neurotoxin. Commencing with 160 litres of bacterial culture, 101 mg of purified type F neurotoxin with a specific toxicity of 2 x 10(7) mouse LD50 (median lethal dose).mg-1 were obtained. 2. Purified type F neurotoxin was labelled to high specific radioactivity (900-1360 Ci/mmol) without loss of biological activity using a chloramine-T procedure. Of the two neurotoxin subunits, the heavy chain was preferentially radiolabelled. 3. Radiolabelled type F neurotoxin displayed specific saturable binding to rat synaptosomes. At least two pools of acceptors were evident: a low content of high-affinity acceptors sites [KD approximately 0.15 nM; Bmax (maximal binding) 20 fmol/mg] and a larger pool of lower-affinity sites (KD greater than 20 nM; Bmax greater than 700 fmol/mg). Both pools of acceptors were sensitive to trypsin and neuraminidase treatment, which suggests that protein and sialic acid residues are components of the synaptosomal acceptors. 4. Experiments investigating competition among botulinum neurotoxin types A, B, E and F for acceptors on rat brain synaptosomes showed that type F neurotoxin binds to acceptor molecules which are completely distinct from those of the other three neurotoxins.

Using pharmacological (Simpson, L.L., 1980, J. Pharmacol. Exp. Ther. 212:16-21) and autoradiographic techniques (Black, J.D., and J.O. Dolly, 1986, J. Cell Biol., 103:521-534), it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target. In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography. It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin. In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis. These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake. This proposal is supported further by the fact that lysosomotropic agents, which are known to interfere with the endocytic pathway, retard the onset of BoNT-induced neuroparalysis and also affect the distribution of silver grains at nerve terminals treated with 125I-BoNT. Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors. On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target.

Dendrotoxin is a snake polypeptide that blocks selectively and potently certain voltage-sensitive, fast-activating K+ channels in the nervous system, where it binds with high affinity to membranous acceptors. Herein, the acceptor protein for dendrotoxin in bovine synaptic membranes is solubilized in active form and its complete purification achieved by affinity chromatography, involving a novel elution procedure. This putative K+-channel constituent is shown to be a large oligomeric glycoprotein containing two major subunits, with Mr values of 75,000 and 37,000.

1. The acute effects of antibodies (both polyclonal and monoclonal) raised against the acetylcholine receptor were studied at mouse and rat end-plates. Isolated muscles were incubated in solutions containing antibody for 2 1/4 to 3 1/2 h. Intracellular microelectrode techniques were then used to record miniature end-plate potentials (MEPPs) and voltage noise. 2. Most antibody preparations investigated did not reduce MEPP amplitudes as compared with controls. One monoclonal (C7) and one polyclonal (J) preparation irreversibly reduced MEPP amplitudes. Both preparations caused reductions in acetylcholine-induced depolarization and associated channel opening frequency (from voltage noise analysis). Single-channel depolarization was not altered by these antibodies. 3. On the basis of these and previous results, four antibody binding regions on the receptor surface were distinguished according to whether channel function and/or alpha-bungarotoxin binding were affected. Although most antibody preparations did not affect channel function, monoclonal antibody C7 appeared to alter function by acting on the channel itself so as to prevent channel opening.

The labeling patterns produced by radioiodinated botulinum neurotoxin (125I-BoNT) types A and B at the vertebrate neuromuscular junction were investigated using electron microscopic autoradiography. The data obtained allow the following conclusions to be made. 125I-BoNT type A, applied in vivo or in vitro to mouse diaphragm or frog cutaneous pectoris muscle, interacts saturably with the motor nerve terminal only; silver grains occur on the plasma membrane, within the synaptic bouton, and in the axoplasm of the nerve trunk, suggesting internalization and retrograde intra-axonal transport of toxin or fragments thereof. 125I-BoNT type B, applied in vitro to the murine neuromuscular junction, interacts likewise with the motor nerve terminal except that a lower proportion of internalized radioactivity is seen. This result is reconcilable with the similar, but not identical, pharmacological action of these toxin types. The saturability of labeling in each case suggested the involvement of acceptors; on preventing the internalization step with metabolic inhibitors, their precise location became apparent. They were found on all unmyelinated areas of the nerve terminal membrane, including the preterminal axon and the synaptic bouton. Although 125I-BoNT type A interacts specifically with developing terminals of newborn rats, the unmyelinated plasma membrane of the nerve trunk is not labeled, indicating that the acceptors are unique components restricted to the nerve terminal area. BoNT types A and B have distinct acceptors on the terminal membrane. Having optimized the conditions for saturation of these binding sites and calibrated the autoradiographic procedure, we found the densities of the acceptors for types A and B to be approximately 150 and 630/micron 2 of membrane, respectively. It is proposed that these membrane acceptors target BoNT to the nerve terminal and mediate its delivery to an intracellular site, thus contributing to the toxin's selective inhibitory action on neurotransmitter release.

Three stable hybrid cell lines have been established that secrete monoclonal antibodies of G(1) sub-class to dendrotoxin, a convulsant polypeptide (M(r) = 7000). Using [(125)I]labelled dendrotoxin the resultant ascitic fluids were found to show no cross-reactivity with homologous toxins (toxins 1, B and E from Dendroaspis polylepis, toxin Dv-14 from Dendroaspis viridis and ?-bungarotoxin from Bungarus multicinctus). In contrast, polyclonal antibodies raised against dendrotoxin cross-reacted to varying degrees with its congeners; most importantly, the rank order of cross-reactivities was in accordance with their potencies in eliciting convulsions when injected intracerebroventricularly into rat brain. All the antibodies prevented significantly the binding of dendrotoxin to its protein acceptor in brain synaptic membranes. Moreover, when they were injected into rat brain together with lethal doses of dendrotoxin they delayed, or in some cases prevented, the onset of convulsive symptoms. Ultracentrifugation of the complexes formed by [(125)I]labelled dendrotoxin and one or more of the monoclonal antibodies showed only a single peak of radioactivity with an S(20.w) of 7S, indicating that all these mono-specific antibodies are directed to the same or overlapping epitope(s). Conversely, polyclonal antisera produced larger complexes with the antigen, revealing the presence of at least two determinants on this molecule. Such antibodies are proving helpful in identifying regions of the toxin responsible for the neurotoxicity and associated interaction with its acceptor, a putative constituent of A-current K(+)-channels.

An alpha-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest of the chick brain which can also bind alpha-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the alpha-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors.

A neurotoxic, [(3)H]labelled derivative of ?-bungarotoxin, known to inhibit neurotransmitter release and to be free of phospholipase activity, was used to demonstrate autoradiographically the distribution and ultrastructural location of its saturable binding component in brain. Light-microscope autoradiography of rat cerebellum and hippocampus showed that it resides primarily in synaptic-rich areas, with much lower densities of sites in other regions containing cell bodies; also, little binding was associated with myelinated tracts. Ultrastructural localisation and sub-fractionation studies on purified cerebrocortical synaptosomes showed that [(3)H]toxin binding sites are located predominantly on brain synaptosomal membranes, consistent with their possible association with transmitter release.

The interaction between two presynaptically acting agents, Lambert-Eaton myasthenic syndrome (LEMS) immunoglobulin G (IgG) and purified botulinum neurotoxin (BoNT) type A, was studied. Intracellular microelectrode recordings were carried out on mouse muscles after injection with LEMS IgG. BoNT was either injected before recordings were made or applied in vitro. The time course of the in vitro actions of BoNT on miniature end-plate potential and end-plate potential parameters were not affected by pretreatment with LEMS IgG. After in vivo injection of BoNT, end-plate potential quantal content was reduced to less than 2% of control values, whether or not LEMS IgG had also been previously given. Quantitative electron-microscope autoradiographical analysis showed that neither the binding of 125I-BoNT to acceptors on the nerve terminal membrane nor the pattern of its internalisation were affected by pretreatment with LEMS IgG. We conclude that the effects of BoNT are not affected by LEMS IgG, suggesting different presynaptic binding sites for the two agents.

Immunization of rabbits with homogeneous preparations of acetylcholine receptor from denervated muscle of cat and chicken, which contained single or multiple sizes of polypeptides respectively, induced myasthenic-like symptoms. One of the resultant antisera, and the IgG fraction thereof, reduced significantly and irreversibly the amplitude of miniature endplate potentials in murine muscle; the effect was not abolished by heat inactivation of complement. This antiserum also retarded the binding of α-bungarotoxin to a solubilised extract of denervated muscle containing homologous receptor. The other five antibody preparations were unable to affect these miniature potentials but many of them did reduce the binding of α-bungarotoxin to denervated muscle receptor in solution and, in some cases, decreased the effectiveness of the latter in blocking neuromuscular transmission. Although inoculation with each of the four individual subunits from the receptor of Torpedo marmorata electroplax did not produce muscle weakness in rabbits, antibodies to α- or β-polypeptides lowered, to a significant extent, the amplitude of spontaneous synaptic potentials in mouse diaphragm muscle. It is concluded that antibodies with direct blocking actions on the receptor-ion channel complex are not common in such immunized animals and their presence cannot be correlated readily with the induction of physical disability. The majority of the antibody species bind to loci distant from the acetylcholine recognition site. Antiserum from one of the immunized rabbits reacted preferentially with receptor from denervated rather than innervated cat and rat muscle, indicating some dissimilarity.

Botulinum neurotoxin, purified to homogeneity from Clostridium botulinum (Type A), was found to be highly neurotoxic (>8 x 10 7 mouse ld 50 /mg protein). Labelling of this pure neurotoxin with 125 I-iodine to high specific radioactivity was achieved without appreciable loss of biological activity. This was used to demonstrate saturable binding sites for this toxin at the neuromuscular junction, following in vivo administration into mice. A demonstrable inhibitory effect of the neurotoxin on release of acetylcholine from rat cerebrocortical synaptosomes indicates that it affects synapses in the central nervous system. Kinetic studies on the binding of 125 I-labelled neurotoxin to brain synaptosomes yielded an association rate constant of 2.3 x 10 5 M −1 s −1 ; dissociation plots were biphasic and the predominant species showed a rate constant of 1.2 x 10 −4 s −1 . The saturable binding component is heatsensitive and inactivated by trypsin. Preliminary studies showed that botulinum neurotoxin associates with plasma membrane fractions of synaptosomes and that binding does not result in any gross structural changes, at least in the majority of the toxin molecules.

The beta-bungarotoxin-induced depolarization of the synaptosomal plasma membrane monitored by the efflux of 86Rb+ is potentiated by raising the albumin in the incubation, is Ca2+-dependent and is due neither to inhibition of the (Na+ + K+)-dependent ATPase nor to activation of the voltage-dependent Na+ channel. Occupancy of the beta-bungarotoxin-binding site by dendrotoxin inhibits partially the action of beta-bungarotoxin. The efflux of 86Rb+ is parallelled by a release of lactate dehydrogenase from the synaptosome, and the two processes are maximal with 2 nM-toxin. Digitonin induces a release of 86Rb+ and lactate dehydrogenase closely similar to that seen with beta-bungarotoxin. It is concluded that the toxicity of beta-bungarotoxin for mammalian nerve terminals can be largely accounted for by specific site-directed phospholipase A2-induced permeabilization of the plasma membrane.

The radiation inactivation method was used to study the size of acetylcholine receptors in the intact membrane-bound state. This technique was reinvestigated, and modifications were made which remove substantial difficulties affecting previous applications of it to such proteins. The molecular size was deduced here by reference to a set of protein standards: an inactivation ratio was defined relative to a given internal enzyme molecular weight standard, and a linear calibration plot for the inactivation ratios of the protein standards was constructed and applied. The acetylcholine receptor in Torpedo electric organ, cat denervated muscle, and chick embryonic muscle was found by this method to exist in the membrane as a homogeneous population of the same size in each case. This receptor, when identified thus by the alpha-neurotoxin-binding target structure, has an apparent molecular weight of 300000 or a molecular volume of about 350 nm3. In comparison, the molecular weight of the cat muscle receptor when solubilized, as analyzed by gel electrophoresis after extensive cross-linking, was found to be 270000 +/- 20000. These two values are thought to be equivalent by virtue of the situation and structure of the receptor protein in the cell membrane. If a disulfide-bridge dimeric receptor exists in the membrane (as other evidence has indicated for Torpedo), each monomer acts independently there in binding alpha-neurotoxin, since the monomers can be inactivated independently by irradiation in the Torpedo membrane. In the muscle membrane no evidence for the existence of receptor dimers, of any kind, has been found.

Botulinum neurotoxin (BoNY) type A, a causative agent of botulism, is a di-chain protein (molecular weight 140,000) from Clostridium botulinum, and the most neurotoxic substance known. Some cases of sudden infant cot deaths have been attributed to such a neuroparalytic condition. BoNT inhibits irreversibly the release of acetylcholine from peripheral nerves in a highly selective manner. Hence, it is potentially an invaluable probe for studying the mechanism of transmitter release. Here we demonstrate specific labelling of murine motor nerve terminals with neurotoxic, 125I-labelled BoNT (type A) by autoradiography. We observed saturable, temperature-sensitive binding of BoNT to sites which reside solely on the nerve terminal membrane; these were distributed on all unmyelinated areas, at an average density of 150-500 per micron2 of membrane. The binding was mediated by the larger subunit of the toxin and was inhibited partially by tetanus toxin, another microbial protein. No specific binding was detectable on any other cell types examined, including noradrenergic terminals. Following binding, internalization of radioactivity was observed; this process was energy-dependent as it could be prevented totally by azide or dinitrophenol (DNP). This direct demonstration of separable steps, including highly selective binding and acceptor-mediated internalization, is reconcilable with the unique potency and the multiphasic inhibitory action of BoNT on transmitter release, as shown electrophysiologically.

1. Large-scale production and purification of complexes between Clostridium botulinum neurotoxin and haemagglutinin have been achieved. 2. Haemagglutinin-free neurotoxic protein of the complexes was purified to high specific neurotoxicity by affinity chromatography, on p-aminophenyl beta-D-thiogalactopyranoside coupled to Sepharose 4B, followed by chromatography on DEAE-Sephacel. 3. The resultant neurotoxin was homogeneous on isoelectric focussing (pI = 6.3) and on dodecylsulphate/polyacrylamide gel electrophoresis under non-reducing conditions when its Mr was 1.4 X 10(5); after reduction two polypeptides (Mr = 9.9 and 5.5 X 10(4) were present. 4. On double-immunodiffusion gels, using antiserum against neurotoxin-haemagglutinin complex, the neurotoxin showed a single, sharp precipitin line that was immunologically distinct from a relatively non-toxic protein (Mr = 1.3 X 10(5), which co-purifies with the neurotoxin but is removed by the ion-exchange chromatography step. 5. Application of the neurotoxin to animals in vitro or in vivo produced near complete and irreversible blockade of neurotransmission. Botulinisation of rat leg muscles reduced spontaneous transmitter release; the amplitude of miniature end-plate potentials was altered from the normal 'bell-shaped" to a skewed distribution. 6. In normal muscle, a large transient increase in frequency of the miniatures was produced by beta-bungarotoxin. In contrast, with botulinised muscle the latter induced a much smaller increase in the absolute frequency; in addition, the mean amplitude was increased somewhat but the distribution remained skewed. The results show botulinisation of muscle modifies the action of beta-bungarotoxin.

Three polypeptides (β, γ and δ) homologous to α-dendrotoxin, an inhibitor of certain voltage-activated K + channels, were found to elevate the cytosolic free concentration of calcium ([Ca 2+ ] c ) in isolated central nerve terminals. Relative to α-dendrotoxin (EC 50 − 2.1 nM), the β-, γ- and δ-toxins were 790-, 214- and 5.7-fold less effective; no additivity was apparent in the toxins' effects on [Ca 2+ ] c . Each toxin antagonized the high affinity binding of 125 I-labelled α- and δ-dendrotoxin to synaptosomes but with different potencies. The mutual interaction of α- and δ-dendrotoxin with the acceptor appeared complex, the inhibition curves being noticeably extended. For the inhibition of binding of α- and δ-dendrotoxin, the respective K i 's (nM) observed for α, β, γ and δ toxins were 0.78, 50, 99, 8, and 2.3, 95, 61, 0.53. Apparently, interactions of α- and δ-, but not β- or γ-dendrotoxin with the acceptor are closely coupled to an inhibition of K + channel(s) as observed indirectly by elevation of [Ca 2+ ] c .

Under optimised conditions for intoxication, botulinum neurotoxin type A was shown to inhibit approximately 90% of Ca2+-dependent K+-evoked release of [3H]acetylcholine, [3H]noradrenaline, and [3H]dopamine from rat cerebrocortical synaptosomes; cholinergic terminals were most susceptible. In each case, the dose-response curve for the neurotoxin was extended, with about 50% of evoked release being inhibited at approximately 10 nM whereas 200 nM was required for the maximal blockade. This may suggest some heterogeneity in the release process. The action of the toxin was time and temperature dependent and appeared to involve binding and sequestration steps prior to blockade of release. The neurotoxin failed to exert any effect on synaptosomal integrity or on Ca2+-independent release of the transmitters tested; it produced only minimal changes in neurotransmitter uptake although small secondary effects were detected with cholinergic terminals. Blockade by the neurotoxin of Ca2+-dependent resting release of transmitter was apparent; Sr2+, Ba2+, or high concentrations of Ca2+ restored the resting release of 3H-catecholamine but not [3H]acetylcholine. Interestingly, none of the latter conditions or 4-aminopyridine could reverse the toxin-induced blockade of evoked release. This lack of specificity in its action on synaptosomes, and other published findings, lead to the conclusion that toxin-sensitive component(s) exist in all nerve terminals that are concerned with transmitter release.

Dendrotoxin, a small single-chain protein from the venom of Dendroaspis angusticeps, is highly toxic following intracerebroventricular injection into rats. Voltage-clamp analysis of CA1 neurons in hippocampal slices, treated with tetrodotoxin, revealed that nanomolar concentrations of dendrotoxin reduce selectively a transient, voltage-dependent K conductance. Epileptiform activity known to be induced by dendrotoxin can be attributed to such an action. Membrane currents not affected directly by the toxin include (i) Ca-activated K conductance; (ii) noninactivating voltage-dependent K conductance; (iii) inactivating and noninactivating Ca conductances; (iv) persistent inward (anomalous) rectifier current. Persistence of the effects of the toxin when Cd was included to suppress spontaneous transmitter release indicates a direct action on the neuronal membrane. Using biologically active, 125I-labeled dendrotoxin, protein acceptor sites of high affinity were detected on cerebrocortical synaptosomal membranes and sections of rat brain. In hippocampus, toxin binding was shown autoradiographically to reside in synapse-rich and white matter regions, with lower levels in cell body layers. This acceptor is implicated in the action of toxin because its affinities for dendrotoxin congeners are proportional to their central neurotoxicities and potencies in reducing the transient, voltage-dependent K conductance.

The action of type A and type B botulinum neurotoxin on neurotransmitter release was studied on identified ganglionic synapses of Aplysia. Using this model, we have shown that botulinum neurotoxins at concentrations used in vertebrate preparations had the same specificity of action and that both heavy and light chains of these toxins are intracellularly required to inhibit neurotransmitter release.