Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors

At present the cryo-electron microscopy structure at 4 Å resolution is known for the Torpedo marmorata nicotinic acetylcholine receptor (nAChR), and high-resolution X-ray structures have been recently determined for bacterial ligand-gated ion channels which have the same type of spatial organization. Together all these structures provide the basis for better understanding functioning of muscle-type and neuronal nAChRs, as well as of other Cys-loop receptors: 5HT3-, glycine-, GABA-A and some other. Detailed information about the ligand-binding sites in nAChRs, necessary both for understanding the receptor functioning and for rational drug design, became available when the X-ray structures were solved for the acetylcholine-binding proteins (AChBP), excellent models for the ligand-binding domains of all Cys-loop receptors. Of special value in this respect are the X-ray structures of AChBP complexes with agonists and antagonists. Among the latter are the complexes with polypeptide and peptide antagonists, that is with protein neurotoxins from snake venoms and peptide neurotoxins (α-conotoxins) from poisonous marine snails of Conus genus. The role of a bridge between the AChBP and nAChRs is played by the X-ray structure of the ligand-binding domain of α1 subunit of nAChR in the complex with α-bungarotoxin.The purpose of this review is to show the role of well-known and new polypeptide and peptide neurotoxins, from the earlier days of nAChRs research until present time, in identification of different nAChR subtypes and mapping their binding sites.     

Characterization of nicotinic receptors inducing noradrenaline release and absence of nicotinic autoreceptors in human neocortex

Presynaptic facilitatory nicotinic receptors (nAChRs) on noradrenergic axon terminals were studied in slices of human or rat neocortex and of rat hippocampus preincubated with [3H]noradrenaline ([3H]NA). During superfusion of the slices, stimulation by nicotinic agonists for 2 min only slightly increased [3H]NA outflow in the rat neocortex, but caused a tetrodotoxin-sensitive. Ca(2+)-dependent release of [3H]NA in rat hippocampus and human neocortex. In both tissues a similar rank order of potency of nicotinic agonists was found: epibatidine > DMPP > nicotine approximately cytisine > or = acetylcholine; choline was ineffective. In human neocortex, the effects of nicotine (100 microM) were reduced by mecamylamine, methyllycaconitine, di-hydro-beta-erythroidine (10 microM, each) and the alpha3beta2/alpha6betax-selective alpha-conotoxin MII (100/200 nM). The alpha3beta4 selective alpha-conotoxin AuIB (1 microM), and the alpha7 selective alpha-conotoxin ImI (200 nM) as well as alpha-bungarotoxin (125 nM) were ineffective. Glutamate receptor antagonists (300 microM AP-5, 100 microM DNQX) acted inhibitory, suggesting the participation of nAChRs on glutamatergic neurons. On the other hand, nAChR agonists were unable to evoke exocytotic release of [3H]acetylcholine from human and rat neocortical slices preincubated with [3H]choline. In conclusion: (1) alpha3beta2 and/or alpha6 containing nAChRs are at least partially responsible for presynaptic cholinergic facilitation of noradrenergic transmission in human neocortex; (2) nicotinic autoreceptors were not detectable in rat and human neocortex.     

Behavioural and anticonvulsant effects of Ca2+ channel toxins in DBA/2 mice

This study investigated the behavioural and anticonvulsant effects of voltage-sensitive calcium channel blockers in DBA/2 mice.-Conotoxin MVIIC (0.1, 0.3 g ICV/mouse) and-agatoxin IVA (0.1, 0.3, 1 g ICV), which act predominantly at P- and/or Q-type calcium channels, prevented clonic and tonic sound-induced seizures in this animal model of reflex epilepsy (ED₅₀ values with 95% confidence limits for protection against clonic sound-induced seizures were 0.09 (0.04–0.36) g ICV and 0.09 (0.05–0.15) g ICV, respectively and against tonic seizures 0.07 (0.03–0.16) g ICV and 0.08 (0.04–0.13) g ICV, respectively). The N-type calcium channel antagonists-conotoxin GVIA and-conotoxin MVIIA were also tested in this model.-Conotoxin GVIA was anticonvulsant in DBA/2 mice, but only at high doses (3 g ICV prevented tonic seizures in 60% of the animals; 10 g ICV prevented clonic seizures in 60% and tonic seizures in 90% of the animals), whereas-conotoxin MVIIA did not inhibit sound-induced seizures in doses up to 10 g ICV. Both-conotoxin GVIA and-conotoxin MVIIA induced an intense shaking syndrome in doses as low as 0.1 g ICV, whereas-conotoxin MVIIC and-agatoxin IVA did not produce shaking at any of the doses examined. Finally,-conotoxin GI (0.01–1 g ICV) and-conotoxin SI (0.3–30 g ICV), which both act at acetylcholine nicotinic receptors, were not anticonvulsant and did not induce shaking in DBA/2 mice. These results confirm that blockers of N- and P-/Q-type calcium channels produce different behavioural responses in animals. The anticonvulsant effects of-conotoxin MVIIC and-agatoxin IVA in DBA/2 mice are consistent with reports that P- and/or Q-type calcium channel blockers inhibit the release of excitatory amino acids and are worthy of further exploration.     

Presence of multiple binding sites on α9α10 nAChR receptors alludes to stoichiometric-dependent action of the α-conotoxin,Vc1.1

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in fast synaptic transmission. nAChRs are pentameric receptors formed from a combination of different or similar subunits to produce heteromeric or homomeric channels. The heteromeric, α9α10 nAChR subtype is well-known for its role in the auditory system, being expressed in cochlear hair cells. These nAChRs have also been shown to be involved in immune-modulation. Antagonists of α9α10 nAChRs, like the α-conotoxin Vc1.1, have analgesic effects in neuropathic pain. Unlike other nAChR subtypes there is no evidence that functional receptor stoichiometries of α9α10 exist. By using 2-electrode voltage clamp methods and maintaining a constant intracellular Ca²⁺ concentration, we observed a biphasic activation curve for ACh that is dependent on receptor stoichiometry. Vc1.1, but not the α9α10 antagonists RgIA or atropine, inhibits ACh-evoked currents in a biphasic manner. Characteristics of the ACh and Vc1.1 activation and inhibition curves can be altered by varying the ratio of α9 and α10 mRNA injected into oocytes, changing the curves from biphasic to monophasic when an excess of α10 mRNA is used. These results highlight the difference in the pharmacological profiles of at least two different α9α10 nAChR stoichiometries, possibly (α9)₃(α10)₂ and (α9)₂(α10)₃. As a result, we infer that there is an additional binding site for ACh and Vc1.1 at the α9–α9 interface on the hypothesized (α9)₃(α10)₂ nAChR, in addition to the α10–α9 and or α9–α10 interfaces that are common to both stoichiometries. This study provides further evidence that receptor stoichiometry contributes another layer of complexity in understanding Cys-loop receptors.     

Characterization of a novel psi-conotoxin from Conus parius Reeve.

The M-superfamily of conotoxins currently comprises three major groups of peptides (the mu-, kappaMu-, and psi-families) that share a key structural characteristic, the six-cysteine motif CC-C-C-CC, but differ with respect to their molecular targets. The psi-family consists of M-superfamily conotoxins that are nicotinic acetylcholine receptor (nAChR) antagonists. To date, only two psi-conotoxins, PIIIE and PIIIF, are known, both of which were isolated from a single Conus species, Conus purpurascens. In this paper, we report the discovery and initial characterization of a psi-conotoxin from another Conus species, Conus parius, which we designated as PrIIIE. Its amino acid sequence, inferred from a cloned cDNA, differed significantly from those of PIIIE and PIIIF. Its bioactivity was investigated by using the synthetic form of the peptide in mice and fish bioassays. At 2.5 nmol, the synthetic peptide induced flaccid paralysis in goldfish in ca. 4 min but did not induce any remarkable behavior in mice (after i.c. and i.p. injection of up to 10 nmol of peptide) and did not block action potential in directly stimulated frog muscle preparations. Electrophysiological experiments carried out to measure inhibition of ion currents through mouse nAChR receptors expressed in oocytes revealed that PrIIIE (IC(50) approximately 250 nM) was significantly more potent than PIIIE (IC(50) approximately 7000 nM) and that PrIIIE showed higher inhibition potency against the adult-type than the fetal-type nAChR. In similar electrophysiological assays, PrIIIE showed no inhibitory effects against the mouse muscle subtype Na(+) channel isoform Na(v) 1.4. The discovery of this psi-conotoxin from a Conus species that belongs to the subgenus Phasmoconus, which is distinct from and larger than the clade in which C. purpurascens belongs, suggests that greater structural and functional diversity of psi-conotoxins remains to be discovered from the members of this subgenus.     

Cholinergic agonists decrease quantal output at the frog neuromuscular junction by targeting a calcium channel blocked by ω-conotoxin

 Nicotinic cholinergic agonists are known to decrease synchronous evoked quantal output at the frog neuromuscular junction [Van der Kloot 1993, J Physiol (Lond) 468:567–589]. Here we also show that carbachol decreases the frequency of miniature endplate potentials (F _MEPP) in solutions containing elevated levels of K⁺ and Ca²⁺. Carbachol did not decrease F _MEPP in hypertonic solutions or in solutions containing the Ca²⁺ ionophore ionomycin and Ca²⁺. We conclude that the nicotinic agonists decrease Ca²⁺ influx through voltage-gated Ca²⁺ channels. Carbachol did not alter two-pulse facilitation. A blocker of N-type Ca²⁺ channels, ω-conotoxin GVIA, antagonized the nicotinic agonist-induced decrease in evoked quantal output. The effect of carbachol was not altered by ω-conotoxin MVIIC, a blocker of P-type and certain other Ca²⁺channels. The Ca²⁺ channel targeted by the nicotinic agonists appears to be of the N-type. Received: 6 March 1997 / Received after revision: 6 May 1997 / Accepted: 4 June 1997     

Involvement of different calcium channels in K+- and veratridine-induced increases of cytosolic calcium concentration in rat cerebral cortical synaptosomes

Intracellular calcium ion concentrations ([Ca²⁺]_i) in rat cerebral cortical synaptosomes were measured, using the calcium chelating fluorescence dye fura-2. The synaptosomes were depolarized by elevation of the extracellular K⁺ concentration or by addition of veratridine, which opens voltage-dependent Na⁺-channels and prevents their inactivation. Both enhancement of the concentration of extracellular K⁺ (up to 60 mM) and veratridine (1–100 μM) increased the [Ca²⁺]_i in a concentration-dependent manner. In the absence of extracellular Ca²⁺, the K⁺- and veratridine-induced increases in [Ca²⁺]_i were abolished, indicating that the increase in [Ca²⁺]_i was due to an influx of extracellular Ca²⁺. Tetrodotoxin (TTX), a blocker of the voltage-dependent Na⁺ channel, inhibited the veratridine-induced (10 μM) Ca²⁺ influx by more than 80%, while the K⁺-evoked (30 mM) increase of [Ca²⁺]_i was TTX-resistant. Both the K⁺- and the veratridine-induced Ca²⁺ influx were not reduced by nifedipine (1 μM), a blocker of L-type Ca²⁺ channels. Blockade of the voltage dependent N-type Ca²⁺ channels with ω-conotoxin GVIA (ω-CTx GVIA; 0.1 μM) and of the voltage-dependent P/Q-type channels with ω-agatoxin IVA (ω-AgaTx IVA; 0.2 μM) inhibited the K⁺-induced increase in [Ca²⁺]_i by about 30 and 55%, respectively; these effects were additive. ω-Conotoxin MVIIC (ω-CTx MVIIC) at a concentration of 0.2 μM, which may be assumed to block predominantly the Q-type Ca²⁺ channel, inhibited the K⁺-induced increase in [Ca²⁺]_i by 50%. The veratridine-induced increase in [Ca²⁺]_i was reduced by about 25% by ω-CTx GVIA (0.1 μM), but was resistant to ω-AgaTx IVA (0.2 μM) and ω-CTx MVIIC (0.2 μM). Mibefradil (former designation Ro 40-5967), a Ca²⁺ antagonist which blocks all types of voltage-dependent Ca²⁺ channels including the T and R channels, led to a concentration-dependent inhibition of the K⁺- and veratridine-induced increase in [Ca²⁺]_i (abolition at 10 μM mibefradil). Ifenprodil, another non-specific blocker of voltage-dependent Ca²⁺ channels, also inhibited the K⁺- and veratridine-induced increase in [Ca²⁺]_i in concentration-dependent manner and abolished it at 320 μM ifenprodil. In contrast, KB-R 7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 μM), a highly potent and selective inhibitor of the Na⁺/Ca²⁺ exchanger (NCX1), failed to inhibit the K⁺- and veratridine-induced increase in [Ca²⁺]_i. It is concluded that the K⁺-induced increase in free cytosolic Ca²⁺ results from Ca²⁺ influx through voltage-dependent N- and, above all, Q-type Ca²⁺ channels. N-type Ca²⁺ channels also play a minor role in the veratridine-induced increase in [Ca²⁺]_i, but P/Q-type channels do not appear to be involved at all. The inhibition of the veratridine-induced, ω-CTx GVIA- and ω-AgaTx IVA-resistant increase in [Ca²⁺]_i by mibefradil and the failure of KB-R 7943 to inhibit this response are compatible with the suggestion that in rat cerebral cortical synaptosomes, Ca²⁺ influx via the R-type Ca²⁺ channel and/or another so far uncharacterized Ca²⁺ channel may substantially contribute to the veratridine-induced increase in [Ca²⁺]_i. Received: 7 March 1997 / Accepted: 9 September 1997     

ω-Conotoxin MVIIA inhibits amygdaloid kindled seizures in Sprague–Dawley rats

ω-Conotoxin MVIIA (ω-CTX MVIIA) is a reversible and potent antagonist of N-type voltage-dependent calcium channels (VDCCs) in neurons. In this study, we evaluated the effect of a fusion form of ω-CTX MVIIA with glutathione S-transferase (GST) on amygdaloid kindled seizures. Intracerebraventricular (i.c.v.) injection of the fusion protein of GST–ω-CTX MVIIA significantly decreased seizure stage and shortened afterdischarge duration and generalized seizure duration in a dose-dependent and time-related manner. In addition, GST–ω-CTX MVIIA significantly increased the GABA levels in the cortex and glycine levels in the brainstem. In contrast, GST alone did not have any effect on seizure behavior or neurochemical levels. These findings firstly demonstrate that the N-type VDCC is a potential therapeutic target for temporal lobe epilepsy. The mechanism of the anticonvulsant function of ω-CTX MVIIA is related to the blockade of N-type VDCC-mediated neurotransmitter release in the brain.     

A comparison of the mu-conotoxins by [3H]saxitoxin binding assays in neuronal and skeletal muscle sodium channel

Sodium channels from rat brain, rat skeletal muscle, chick brain, and eel electroplax were compared by using the mu-conotoxins GIIIA, PIIIA, and StIII and [3H]saxitoxin. Rat skeletal muscle and eel electroplax sodium channels are equally sensitive to GIIIA, PIIIA, and StIII, displacing >90% of the [3H]saxitoxin binding sites in rat skeletal muscle and eel electroplax membranes and exhibiting inhibitory concentrations at half-maximal percentage specific binding (IC50) of 0.97 nM for GIIIA, 1.3 nM for PIIIA in rat skeletal muscle, and concentrations of 3.5 nM for GIIIA and 2.8 nM for PIIIA in eel electroplax. PIIIA and GIIIA at all concentrations inhibit only up to 10% of [3H]saxitoxin binding sites in chick brain membranes. Mu-conotoxin StIII at all concentrations inhibits only up to 10% of [3H]saxitoxin binding sites in rat brain membranes and displays two-site binding inhibition of the [3H]saxitoxin binding sites in rat skeletal muscle. PIIIA displaces >60% of the [3H]saxitoxin binding sites (IC50 of 44 nM) while GIIIA blocks out only 30% of the binding sites in rat brain sodium channels (IC50 of 69 nM). Thus, sodium channel subtypes can be classified into two categories: mu-conotoxin sensitive (i.e., subtypes predominantly found in rat skeletal muscle and eel electroplax) and insensitive (i.e., subtypes predominantly found in rat and chick brain).