Structure-Function Elucidation of a New α-Conotoxin, Lo1a, from Conus longurionis

α-Conotoxins are small peptide toxins that are found in the venom of marine snails, Conus, and extremely potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs are cholinergic receptors that form ligand-gated ion channels in the plasma membranes of certain neurons and on the postsynaptic side of the neuromuscular junction. As nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies like epilepsy, myasthenic syndromes, schizophrenia, Parkinson’s and Alzheimer’s diseases. In order to expand the knowledge about cone snail toxins, we examined the venom of an unknown Conus species, Conus longurionis. This study covers the isolation of an 18-amino acid peptide from the venom of the marine snail Conus longurionis that is active on nAChRs. As far as we are aware, this is the first characterization of a conotoxin from this species. The peptide, named α-conotoxin Lo1a, was characterized by electrophysiological screening against several types of cloned nicotinic receptors expressed in Xenopus laevis oocytes. The toxin, member of the α4/7 family, blocks the response to acetylcholine in oocytes expressing α7 nAChRs, an important channel target in brain disorders, with an IC50 of 3.24 ± 0.7 µM. Also α3β4 was found to be blocked with a percentage of 40% at 10 µM. Furthermore, Lo1a shows a high selectivity for neuronal versus muscle subtype nAChRs. The three-dimensional solution structure of the α-conotoxin Lo1a was determined by NMR spectroscopy. The NMR structure reveals that the peptide backbone adopts a compact “W-shaped” conformation having two loops that are reinforced by two disulfide bonds (Cys3-Cys9 and Cys4-Cys17). Moreover, we designed two mutants, Lo1a-ΔD and Lo1a-RRR, in which an Asp is deleted or an Arg-tail replaces the Asp at the C-terminus respectively. These mutant toxins not only block α7 with a lower IC50, but also surprisingly, shift the selectivity from the neuronal subtype nAChRs towards the muscle subtype nAChRs. In summary, our findings provide insight into the assembly, structure and activity of the α-contoxin Lo1a that targets preferably α7-nAChR. Consequently, our study contributes to the discovery of new ligands to affect brain disorders including schizophrenia, Alzheimer’s disease, traumatic brain injury and ADHD. ispartof: Structure-function elucidation of a new α-conotoxin, Lo1a, from Conus longurionis vol:289 issue:14 pages:9573-9583 ispartof: location:Paris status: published