| Cone snails are genus of predatory marine gastropod.Around 700 species of cone snails are existed in the world.They could be divided into 3 groups dependent upon their choice of prey:fish,worms,or mollusks.When hunting,cone snails inject their venom into the prey through radular tooth,which could make the prey immobilized at once.Conotoxins(CTxs)are various of bioactive peptides isolated from the venom of cone snails.They could be divided into 26 superfamilies according to their highly conserved signal sequence,including A,B1,B2,B3,C,D,E,F,G,H,11,12,13,J,K,L,M,N,01,02,03,P,S,T,V,Y.Besides,they also could be classified into several pharmacological families based on their distinct targets,such as α,μ,ω,δ,χ,σ,λ,κ,γ,etc.a-Conotoxins(α-CTxs)specifically targeting different nicotinic acetylcholine receptors(nAChRs)subtypes are consisting of 12-20 amino acids and two couples of disulfide-bonds,which are among the ealiest discovered and most studied CTxs.As a-CTxs have many advantages,such as their high potency,stable conformation and high specificity etc,they are usually used as probes for the relationship research between ligands and nAChRs,as well as the structure-activity study of nAChRs,even further develped as potential probes or medication for the related disease states.nAChRs functioned as transmembrane pentamers are important ligand-gated ion channels that are widely distributed in the central and peripheral nervous system,and involved in many diseases including pain,nicotine or drug addiction,Parkinson’s disease,schizophrenia,depression,Alzheimer’s disease,and breast and lung cancers.There are 17 homologous nA ChR subunits(α1-10,β1-4,ε,δ,γ)identified so far.To further investigate the molecular mechanism of a-CTxs targeting distinct nAChR subtypes and structure-function relationship of a-CTxs,as well as develop novel efficient ligands,we designed a series of modification on a-CTxs TxID,Tx1B and RegIIA.All mutants were synthesized artificially,and their potency was evaluated by electrophysiological approaches.The following research was carried out.(1)The systematic modification and structure-activity research of a-CTxs TxID.TxID is a novel a4/6-CTx identified from C.textile,which is among the most potent antagonist of a3P4 nAChR with an IC50 of 3.6 nM.However,it also exhibits inhibition on a6/α3β4 nAChR with an IC50 of 33.9 nM.So the native TxID only had about 10-fold greater potency for α3β4 versus α6/α3β4 nAChRs,which was not enough for distinguishing between these two subtypes.Antagonists of α3β4 nAChR also could inhibit α6/α3β4 nAChR generally.The results of alanine scanning of Tx1D showed that[S9A]TxED had about 50-fold greater potency for α3β4 versus a6/α3β4 nAChRs,which suggested a vital role of residues at position 9 on the selectivity of TxID.In order to develop an effective molecular probe for the research of α3β4 nAChR and related disease states,the critical Ser9 of TxID was systematically substituted with a series of amino acid residues with different characteristics to further improve the specificity of TxID on α3β4 nAChR.TxID and its mutants were synthesized artificially,while their inhibitory activities were analyzed by two-electrode voltage clamp electrophysiology on different nAChR subtypes expressed on Xenopus laevis oocytes.As the results showed,the mutant[S9K]TxID only exhibited inhibition on α3β4 nAChR with an IC50 of 6.9 nM,while have no obvious inhibition on other tested nAChR subtypes.It could be the most potent and selective antagonist of α3β4 nAChR identified so far.The three-dimensional solution structure of[S9K]TxED was determined using NMR methods to compare the structure difference relative to the native TxID.Molecular models of the complexes between the native TxID and α3β3 or α6β4 nAChRs were also generated by molecular docking,to rationalize the interaction between ligands and receptors.(2)The study of species specificity of rat and human α7 nicotinic acetylcholine receptors towards different a-CTxs and a-cobratoxin,α7 nAChR is an important transmembrane protein functioned as homomeric pentamers(composed of five α7 subunits).When compared the rat/human species specificity of α7 nAChR towards a series of a-CTxs and a-cobratoxin,we found that these ligands showed different potency on rat and human α7 nAChRs,among which a-CTxs[K11A]TxIB and[H5D]RegIIA are>50-fold more sensitive towards rα7 nAChR versus ha7 nAChR.As the sequences of the extracellular N-terminal ligand-binding domain of ra7 and ha7 nAChRs are highly conserved and from 208 amino acid residues only 10 are different,each of the latter in ra7 nAChR was substituted for the corresponding residue from ha7 nAChR.Electrophysiological measurements showed that the key amino acid residue responsible for this difference is at the position 185 in al subunit(Arg in ha7,Lys in ra7),which was explained by molecular modeling.These results could be a basis for successful design of peptide analogs with improved affinity to the α7 nAChR.(3)Quantity production of a-CTx TxIB by expression in Escherichia coli(E.coli).TxIB is a novel α4/7-CTx identified from C.textile,which is a specific antagonist of α6/3β2β3 nAChR with an ICs0 of 28 nM.However,the quantity of native a-CTxs extracted from cone snail is limited,and chemical synthesis of a-CTxs is expensive.For quantity production of TxIB with low cost on the premise of not changing its potency,the gene of TxIB was synthesized and ligated with the vector pET-31b(+),which was transformed into E.coli strain BLR(DE3)pLysS for expression.After purified by Ni-NTA affinity chromatography column and cleaved by cyanogen bromide(CNBr),recombinant a-conotoxin TxIB(rTxIB)was released and purified by reverse-phase high-performance liquid chromatography(RP-HPLC).Pharmacological activity of rTxIB was assessed by electrophysiological approaches.The results indicated that it had the same selectivity and similar potency as the native TxIB did,which may provide an alternative method to obtain a large amount of small polypeptide drugs on the premise of not changing their potency.Above all,using the combined method of molecular biology,mutation of ligands or receptors,molecular docking and two-electrode voltage clamp electrophysiology technique,our study made a deeper insight of the interaction between α-CTxs and nAChRs,as well as the expression of α-CTxs.These results could help in the design of peptide analogs with higher potency on receptors,also be useful for future structure/function studies and clinical applications. |
