Characterization of a T-superfamily conotoxin TxVC from Conus textile that selectively targets neuronal nAChR subtypes

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Highlights

  • TxVC is the first T-superfamily conotoxin specifically inhibiting nAChRs.

  • TxVC inhibits nAChRs subtype α4β2 more potently than α3β2.

  • The C terminus of TxVC forms a helical structure.

  • Ile6 and Leu14 of TxVC are the functional hydrophobic pharmacophores.

Abstract

T-superfamily conotoxins have a typical cysteine pattern of “CC–CC”, and are known to mainly target calcium or sodium ion channels. Recently, we screened the targets of a series of T-superfamily conotoxins and found that a new T-superfamily conotoxin TxVC (KPCCSIHDNSCCGL-NH2) from the venom of Conus textile. It selectively targeted the neuronal nicotinic acetylcholine receptor (nAChR) subtypes α4β2 and α3β2, with IC50 values of 343.4 and 1047.2 nM, respectively, but did not exhibit obvious pharmacological effects on voltage-gated potassium, sodium or calcium channel in DRG cells, the BK channels expressed in HEK293 cells, or the Kv channels in LβT2 cells. The changes in the inhibitory activities of its Ala mutants, the NMR structure, and molecular simulation results based on other conotoxins targeting nAChR α4β2, all demonstrated that the residues Ile6 and Leu14 were the main hydrophobic pharmacophores. To our best knowledge, this is the first T-superfamily conotoxin that inhibits neuronal nAChRs and possesses high binding affinity to α4β2. This finding will expand the knowledge of the targets of T-superfamily conotoxins and the motif information could help the design of new nAChR inhibitors.

Introduction

Conotoxins are secreted by Cone snails to subdue preys and defend against predators. A large number of conotoxin genes and proteins have been identified and categorized into various superfamilies (A, B, C, D, E, I, M, O, P, S, T, et al.), according to the number of cysteine residues, the arrangement of the disulfide bonds and the consensus signal sequences [1]. These conotoxins specifically target various ion channels such as Na+, K+, and Ca2+ channels and membrane receptors such as nAChR, 5-HT3R, NMDAR and G-protein-coupled receptors [2]. Some conotoxins are neuropharmacological reagents, and several have entered clinical trials. One conotoxin was even approved by FDA for pain treatment [3].

T-superfamily conotoxins typically contain CC–CC– cysteine pattern with a disulfide bond connectivity of I–III, II–IV, or CC–C–C pattern with a disulfide bond connectivity of I–IV, II–III. They selectively interact with Na+ channels, presynaptic Ca2+ channels or G protein-coupled presynaptic receptors in general [4], [5]. However, we isolated a T-superfamily conotoxin TxVC (KPCCSIHDNSCCGL-NH2) from C. textile years ago, determined its peptide sequence by Edman method [6], investigated its disulfide bond configuration using the synthesized peptide, and then surprisingly found that this TxVC selectively interacted with neuronal nicotinic acetylcholine receptor (nAChR) subtype α4β2 and α3β2 without obvious pharmacological effects on voltage-gated K+, Na+ and Ca2+ ion channels in DRG cells. In order to probe its functional residues, its Ala mutants were generated and the inhibitory activities of those mutants were studied. The NMR structure of TxVC and molecular simulations based on other conotoxins targeting nAChR α4β2 revealed the key functional residues. To our best knowledge, this was the first T-superfamily conotoxin that was found to inhibit neuronal nAChRs particularly with high binding affinity to the α4β2 subtype. Our findings therefore expanded the knowledge of the T-superfamily conotoxin targets and this novel motif could be used to design new nAChR inhibitors.

Section snippets

Peptide synthesis

TxVC and its mutants were synthesized using the method described previously [7]. Briefly, the peptide was synthesized and then cleaved from Rink resin with the cleavage solution [trifluoroacetic acid (TFA), 8.8 ml/water, 0.5 ml/DTT, 0.5 g/Triisopropylsilane, 0.2 ml]. The released peptides were oxidized in 0.1 M NH4HCO3 at room temperature, pH 8.0–8.2. The folded products were then purified using semi-preparative RP-HPLC. The final products were assessed using analytical reversed-phase HPLC. The

Chemical identity of the synthesized TxVC and its analogues

Native TxVC was isolated from the crude venom of C. textile by RP-HPLC [6]. Synthesized TxVC and its analogues were assessed using analytical reversed-phase HPLC, and the results showed that their purities were all above 98%. The purified TxVC and the native TxVC had the same retention time as shown in Fig. 1A. All the peptides had the same expected molecular weights, which were determined using Ultraflex III TOF/TOF mass spectrometry (Bruker).

Disulfide bridge pattern of TxVC was I–III, II–IV

HPLC results of the one-step and two-step folding

Discussion

About 200 T-superfamily conotoxins have been deposited in the ConoServer database and a few are pharmacologically important [11]. T-superfamily conotoxins with a CC–CC cysteine pattern generally target voltage-sensitive Na+ channel, presynaptic Ca2+ channel, or G protein-coupled presynaptic receptors. For example, ε-TxIX selectively reduces neurotransmitter release by reducing the presynaptic influx of Ca2+ [4], [12], and Lt5d potently inhibits TTX-sensitive sodium currents in adult rat dorsal

Conflict of interest statement

The authors hereby declare that there are no conflicts of interest related to this study.

Acknowledgments

This work was supported by grants from the National Natural Sciences Foundation of China (No. 81173035) and the National Basic Research Program of China (No. 2010CB529802).

References (23)

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1

Shuo Wang and Tianpeng Du contributed equally to this work.

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