ABSTRACT
Weak neurotoxins belong to the superfamily of three-finger toxins from snake venoms. In general, weak toxins have a low toxicity and, contrary to other three-finger toxins, their molecular targets are not well characterized: in vitro tests indicate that these may be nicotinic acetylcholine receptors. Here, we report the influence of intraperitoneal and intravenous injections of weak neurotoxin from Naja kaouthia venom on mouse behaviour. Dose-dependent suppression of orientation-exploration and locomotion activities as well as relatively weak neurotropic effects of weak neurotoxin were observed. The myorelaxation effect suggests a weak antagonistic activity against muscle-type nicotinic acetylcholine receptors. Neurotoxic effects of weak neurotoxin were related to its influence on peripheral nervous system. The symptomatology of the intoxication was shown to resemble that of muscarinic agonists. Our data suggest that, in addition to interaction with nicotinic acetylcholine receptors observed earlier in vitro, weak neurotoxin interacts in vivo with some other molecular targets. The results of behavioural experiments are in accord with the pharmacological profile of weak neurotoxin effects on haemodynamics in mice and rat indicating the involvement of both nicotinic and muscarinic acetylcholine receptors.
Subject(s)
Behavior, Animal/drug effects , Cobra Neurotoxin Proteins/toxicity , Neurotoxicity Syndromes/physiopathology , Neurotoxins/toxicity , Acoustic Stimulation , Animals , Chromatography, High Pressure Liquid , Cobra Neurotoxin Proteins/administration & dosage , Cobra Neurotoxin Proteins/isolation & purification , Defecation/drug effects , Dose-Response Relationship, Drug , Female , Injections, Intraperitoneal , Injections, Intravenous , Male , Mice , Motor Activity/drug effects , Neurotoxicity Syndromes/etiology , Neurotoxins/administration & dosage , Neurotoxins/isolation & purification , Reaction Time/drug effects , Respiratory Insufficiency/chemically induced , Salivation/drug effects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Thermosensing/drug effects , Time FactorsABSTRACT
Acetylcholine-binding protein (AChBP) is a water-soluble protein released from molluscan glial cells and modulates ACh-mediated synaptic transmission. Acetylcholine-binding protein (AChBP) is a water-soluble homolog of the ligand-binding domain of nicotinic receptors and other members of the pharmaceutically important family of pentameric ligand-gated ion channels (LGICs), GABAA, GABAC, 5-HT3 serotonin, and glycine receptors. The crystal structure of AChBP from Lymnaea stagnalis has become an established model for the extracellular domain of the pentameric LGICs, and homology models have been generated to analyze receptor-ligand interactions. AChBP has pharmacological properties similar to the homomeric alpha7 subtype of nicotinic ACh receptors (nAChRs), with relatively weak affinity for ACh and a 10-fold higher affinity for nicotine.
Subject(s)
Carrier Proteins/physiology , Lymnaea/metabolism , Animals , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Conotoxins/chemistry , Mollusk Venoms/chemistry , Protein Conformation , SnailsABSTRACT
Short- and long-chain alpha-neurotoxins from snake venoms are potent blockers of nicotinic acetylcholine receptors (nAChRs). Short alpha-neurotoxins consist of 60-62 amino acid residues and include 4 disulfide bridges, whereas long alpha-neurotoxins have 66-75 residues and 5 disulfides. The spatial structure of these toxins is built by three loops, I-III "fingers," confined by four disulfide bridges; the fifth disulfide of long-chain alpha-neurotoxins is situated close to the tip of central loop II. An accurate knowledge of the mode of alpha-neurotoxin-nAChR interaction is important for rational design of new nAChR agonists and antagonists for medical purposes. Ideas on the topography of toxin-nAChR complexes were based until recently on nAChR interactions with selectively labeled alpha-neurotoxins, mutations in toxins, nAChR, or both. Recently, crystal structures have been solved for the Torpedo marmorata nAChR (4A[Unwin, 2005]) and for the acetylcholine-binding protein (AChBP) complexed with mollusk alpha-conotoxin (2.4 A[Celie et al., 2005]) or alpha-cobratoxin, long-chain alpha-neurotoxin (4 A [Bourne et al., 2005]). However, there were no angstrom-resolution models for complexes of short-chain alpha-neurotoxins. Here, we report the model of the Torpedo californica nAChR extracellular domain complexed to a short-chain alpha-neurotoxin II (NTII) from Naja oxiana cobra venom.
Subject(s)
Neurotoxins/pharmacokinetics , Receptors, Nicotinic/metabolism , Animals , Elapid Venoms/pharmacokinetics , Elapid Venoms/toxicity , Models, Biological , Receptors, Nicotinic/drug effects , TorpedoABSTRACT
Alpha-conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of alpha-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into alpha-conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in alpha-conotoxins GI and SI, or D12K substitution in alpha-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent alpha-conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in alpha-conotoxin [A10L]PnIA, a blocker of neuronal alpha7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine-binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica. We found that [A10L,D14K]PnIA also distinguishes two alpha7-like anion-selective nAChR subtypes present on identified neurons of L. stagnalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to alpha-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C-terminus of alpha-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle-type alpha-conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling.
Subject(s)
Conotoxins/metabolism , Receptors, Nicotinic/metabolism , Torpedo/metabolism , Amino Acid Sequence , Animals , Chloride Channels/drug effects , Circular Dichroism , Conotoxins/chemical synthesis , Conotoxins/chemistry , Models, Molecular , Molecular Sequence Data , Protein Subunits , Receptors, Nicotinic/chemistry , Structure-Activity RelationshipABSTRACT
Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup alpha-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 A of alpha-Ctx PnIA (A10L D14K), a potent blocker of the alpha(7)-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. Alpha-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.
