Nicotinic acetylcholine receptors(nAChRs)are widely distributed ligand gated ion channels throughout the peripheral and central nervous systems of mammals.There are 16 different n AChR subunits,α1-α7,α9,α10 andβ1...Nicotinic acetylcholine receptors(nAChRs)are widely distributed ligand gated ion channels throughout the peripheral and central nervous systems of mammals.There are 16 different n AChR subunits,α1-α7,α9,α10 andβ1-β4,as well asγ,δ,andε,which assemble into pentamers to form different nAChR subtypes with distinct pharmacological properties in mammals.Among themα6β2*(*designates other possible subunit),α3β4 andα4β2 nAChR subtypes are potential therapeutic targets for the treatment of addiction.However,various n AChR subtypes are very difficult to pharmacologically distinguish from each other.Theα6*n AChRs are expressed by dopaminergic neurons in the central nervous system,which modulate the release of dopamine and are believed to be important in mediating tobacco,morphine,cocaine and ethanol addiction.Theα3β4 nAChRs present in the medial habenula with important role in influencing nicotine addiction.Blockage ofα3β4 nAChRs in the medial habenula decreased the dose of nicotine that rodents would self-administer.Thus,new antagonists ofα6β2*orα3β4 nA ChR subtypes are of considerable interest,which would give strategies to selectively modulateα6β2*orα3β4 nA ChR function.We characterized anα-conotoxin(α-CTx)TxIB with 16 amino acids and anα-CTx TxID with 15 amino acids from Conus textile.The sequence of TxIB is GCCSDPPCRNKHPDLCamide.The sequence of TxID is GCCSHPVCSAMSPIC with C-terminal amidation too.Both peptides with aⅠ-ⅢandⅡ-Ⅳdisulfide con-nectivity were chemically synthesized.The residues between Cys-Ⅱand Cys-Ⅲand Cys-Ⅲand Cys-Ⅳofα-CTx are commonly referred to as loops 1 and 2,respectively.The number of residues in each of these loops is used to further classify theα-CTx.So TxIB is classified as a 4/7α-CTx,whereas theα-CTx TxIB has a 4/6 spacing.Both peptides were tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes.Theα-CTx TxIB blockedα6/α3β2β3 nAChR with an IC50 of 28 nmol·L^(-1),which showed little or no block of all the other tested subtypes at concentrations up to 10μmol·L^(-1).TxIB blockingα6/α3β2β3 nAChR is rapidly reversed after toxin washout.The ability ofα-CTx TxIB to discriminate betweenα6/α3β2β3 and the other nAChR receptors is unique.There are no small molecules have this selectivity profile.Previously describedα-CTx that potently blockα6/α3β2β3 nA ChR s also block eitherα6/α3β4 nAChRs,α3β2 nAChRs and(or)other nAChRs subtypes.TxID was the very potentα3β4 nAChR antagonists blocking ratα3β4 n AChRs with an IC-50 of 12.5 nmol·L1.However,TxID also blocked the closely relatedα6/α3β4 with an IC50 of 94 nmol·L^(-1).In fact,the expression profile ofα3β4 nAChRs andα6/α3β4 nAChRs overlap in a variety of tissues.So TxI D can′t differentiateα3β4 nA ChR fromα6/α3β4 nA ChR effectively.To distinguish between these two close subtypes,positional-scanning mutagenesis of TxID was performed to identify critical residues that confer potency forα3β4 nAChRs,and hope to obtain more selective mutant to discriminate between these two close subtypes.The effects of 15 analogues and TxID were tested on bothα3β4 andα6/α3β4 nAChRs.An analogue,ie[S9 A]TxID had46-fold greater potency forα3β4 versusα6/α3β4 nAChRs,which showed significantly improved selectivity forα3β4 versusα6/α3β4 nAChRs.Both TxI D and[S9 A]TxI D had little activity on other nA ChR subtypes.The three-dimensional solution structures of TxIB,TxID and[S9 A]TxID were determined using NMR spectroscopy.α-CTx TxI B,TxID and[S9 A]TxID represent uniquely selective ligand for probing the structure and function ofα6β2*andα3β4 nA ChR s respectively.It is known about20%people have used drugs recreationally resulting in a substance use disorder finally.Therefore,structural insights derived from these ligands may facilitate the development of novel therapeutics for addiction involvingα6β2*andα3β4 nA ChR s.展开更多
文摘Nicotinic acetylcholine receptors(nAChRs)are widely distributed ligand gated ion channels throughout the peripheral and central nervous systems of mammals.There are 16 different n AChR subunits,α1-α7,α9,α10 andβ1-β4,as well asγ,δ,andε,which assemble into pentamers to form different nAChR subtypes with distinct pharmacological properties in mammals.Among themα6β2*(*designates other possible subunit),α3β4 andα4β2 nAChR subtypes are potential therapeutic targets for the treatment of addiction.However,various n AChR subtypes are very difficult to pharmacologically distinguish from each other.Theα6*n AChRs are expressed by dopaminergic neurons in the central nervous system,which modulate the release of dopamine and are believed to be important in mediating tobacco,morphine,cocaine and ethanol addiction.Theα3β4 nAChRs present in the medial habenula with important role in influencing nicotine addiction.Blockage ofα3β4 nAChRs in the medial habenula decreased the dose of nicotine that rodents would self-administer.Thus,new antagonists ofα6β2*orα3β4 nA ChR subtypes are of considerable interest,which would give strategies to selectively modulateα6β2*orα3β4 nA ChR function.We characterized anα-conotoxin(α-CTx)TxIB with 16 amino acids and anα-CTx TxID with 15 amino acids from Conus textile.The sequence of TxIB is GCCSDPPCRNKHPDLCamide.The sequence of TxID is GCCSHPVCSAMSPIC with C-terminal amidation too.Both peptides with aⅠ-ⅢandⅡ-Ⅳdisulfide con-nectivity were chemically synthesized.The residues between Cys-Ⅱand Cys-Ⅲand Cys-Ⅲand Cys-Ⅳofα-CTx are commonly referred to as loops 1 and 2,respectively.The number of residues in each of these loops is used to further classify theα-CTx.So TxIB is classified as a 4/7α-CTx,whereas theα-CTx TxIB has a 4/6 spacing.Both peptides were tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes.Theα-CTx TxIB blockedα6/α3β2β3 nAChR with an IC50 of 28 nmol·L^(-1),which showed little or no block of all the other tested subtypes at concentrations up to 10μmol·L^(-1).TxIB blockingα6/α3β2β3 nAChR is rapidly reversed after toxin washout.The ability ofα-CTx TxIB to discriminate betweenα6/α3β2β3 and the other nAChR receptors is unique.There are no small molecules have this selectivity profile.Previously describedα-CTx that potently blockα6/α3β2β3 nA ChR s also block eitherα6/α3β4 nAChRs,α3β2 nAChRs and(or)other nAChRs subtypes.TxID was the very potentα3β4 nAChR antagonists blocking ratα3β4 n AChRs with an IC-50 of 12.5 nmol·L1.However,TxID also blocked the closely relatedα6/α3β4 with an IC50 of 94 nmol·L^(-1).In fact,the expression profile ofα3β4 nAChRs andα6/α3β4 nAChRs overlap in a variety of tissues.So TxI D can′t differentiateα3β4 nA ChR fromα6/α3β4 nA ChR effectively.To distinguish between these two close subtypes,positional-scanning mutagenesis of TxID was performed to identify critical residues that confer potency forα3β4 nAChRs,and hope to obtain more selective mutant to discriminate between these two close subtypes.The effects of 15 analogues and TxID were tested on bothα3β4 andα6/α3β4 nAChRs.An analogue,ie[S9 A]TxID had46-fold greater potency forα3β4 versusα6/α3β4 nAChRs,which showed significantly improved selectivity forα3β4 versusα6/α3β4 nAChRs.Both TxI D and[S9 A]TxI D had little activity on other nA ChR subtypes.The three-dimensional solution structures of TxIB,TxID and[S9 A]TxID were determined using NMR spectroscopy.α-CTx TxI B,TxID and[S9 A]TxID represent uniquely selective ligand for probing the structure and function ofα6β2*andα3β4 nA ChR s respectively.It is known about20%people have used drugs recreationally resulting in a substance use disorder finally.Therefore,structural insights derived from these ligands may facilitate the development of novel therapeutics for addiction involvingα6β2*andα3β4 nA ChR s.
