Characterization of a Novel α-Conotoxin from Conus textile That Selectively Targets α6/α3β2β3 Nicotinic Acetylcholine Receptors

Abstract

α6β2 Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons in the CNS are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine addiction and Parkinson disease. However, recent studies indicate that the α6 subunit can also associate with the β4 subunit to form α6β4 nAChRs that are difficult to pharmacologically distinguish from α6β2, α3β4, and α3β2 subtypes. The current study characterized a novel 16-amino acid α-conotoxin (α-CTx) TxIB from Conus textile whose sequence is GCCSDPPCRNKHPDLC-amide as deduced from gene cloning. The peptide and an analog with an additional C-terminal glycine were chemically synthesized and tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes. α-CTx TxIB blocked α6/α3β2β3 nAChR with an IC(50) of 28 nm. In contrast, the peptide showed little or no block of other tested subtypes at concentrations up to 10 μm. The three-dimensional solution structure of α-CTx TxIB was determined using NMR spectroscopy. α-CTx TxIB represents a uniquely selective ligand for probing the structure and function of α6β2 nAChRs.

Expression of cloned α6* nicotinic acetylcholine receptors

“…Subsequently, various a-conotoxins and their mutants were developed with equal or better selectivity for a6 versus a3 than that of a-CtxMII, and used to identify a6* subtypes and their physiological importance in aminergic neurons (Dowell et al, 2003;McIntosh et al, 2004;Azam et al, 2005Azam et al, , 2010Luo et al, 2013). Via analyzing sequences interacting with AChRs, a mutant form of a-CtxMII[E11A] was developed that exhibited more than 50 fold higher binding affinity for a6b2* AChRs than a3b2* AChRs .…”

Optimal Cleavage and Oxidative Folding of α-Conotoxin TxIB as a Therapeutic Candidate Peptide

“…For one-step oxidation, all cysteine residues were trityl protected. For two-step oxidation, Cys residues were protected in pairs with either S -trityl on Cys1 and Cys3 or S-acetamidomethyl on Cys2 and Cys4, as described previously [15]. The crude peptide was cleaved from the resin and precipitated with four different Fmoc cleavage cocktails at 3 different cleavage times, respectively (Table 1).…”

“…Our laboratory has recently identified α-CTx TxIB as a highly selective blocker of α6/α3β2β3 nAChRs, with little or no activity against other receptor subtypes [15]. α6β2* nAChRs are potential targets for the treatment of several neuropsychiatric diseases, including addiction and Parkinson’s disease.…”

Expression in Escherichia coli of fusion protein comprising α‐conotoxin TxIB and preservation of selectivity to nicotinic acetylcholine receptors in the purified product

“…Many conotoxins showed specific functions on ligand-gated ion channel receptors, such as acetylcholine, norepinephrine, and G-protein-coupled receptors, which are very important in human neurological diseases. [10] It contains four cysteines (Cys), which form two couples of disulfide bonds with Cys (I-III) and Cys (II-IV) connectivities. [7] Although there are about 500-700 species of cone snails, each species of which contains ~2,000 kinds of conopeptides in the venom, only less than 0.1% of them have been discovered and named in a systematic nomenclature.…”

“…[7] Although there are about 500-700 species of cone snails, each species of which contains ~2,000 kinds of conopeptides in the venom, only less than 0.1% of them have been discovered and named in a systematic nomenclature. [10] So it can be used as a new tool to distinguish α6β2* subtype from α6β4 and other nAChR subtypes. [10] It contains four cysteines (Cys), which form two couples of disulfide bonds with Cys (I-III) and Cys (II-IV) connectivities.…”