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 and ...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 and β1-β4,as well as γ,δ,and ε,which assemble into pentamers to form different nAChR subtypes with distinct pharmacological properties in mammals.Among them α6β2*(*designates other possible subunit),α3β4 and α4β2 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.The α6* 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.The α3β4 nAChRs present in the medial habenula with important role in influencing nicotine addiction.Blockage of α3β4 nAChRs in the medial habenula decreased the dose of nicotine that rodents would self-administer.Thus,new antagonists of α6β2* or α3β4 nA ChR subtypes are of considerable interest,which would give strategies to selectively modulate α6β2* or α3β4 nA ChR function.We characterized an α-conotoxin(α-CTx)TxIB with 16 amino acids and an α-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 a Ⅰ-Ⅲ and Ⅱ-Ⅳ disulfide con-nectivity were chemically synthesized.The residues between Cys-Ⅱ and Cys-Ⅲ and Cys-Ⅲand Cys-Ⅳ of α-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 the α-CTx.So TxIB is classified as a 4/7α-CTx,whereas the α-CTx TxIB has a 4/6 spacing.Both peptides were tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes.The α-CTx TxIB blocked α6/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 10 μmol·L^(-1).TxIB blocking α6/α3β2β3 nAChR is rapidly reversed after toxin washout.The ability ofα-CTx TxIB to discriminate between α6/α3β2β3 and the other nAChR receptors is unique.There are no small molecules have this selectivity profile.Previously described α-CTx that potently blockα6/α3β2β3 nA ChR s also block either α6/α3β4 nAChRs,α3β2 nAChRs and(or) other nAChRs subtypes.TxID was the very potent α3β4 nAChR antagonists blocking rat α3β4 n AChRs with an IC-50 of 12.5 nmol·L1.However,TxID also blocked the closely related α6/α3β4 with an IC50 of 94 nmol·L^(-1).In fact,the expression profile ofα3β4 nAChRs and α6/α3β4 nAChRs overlap in a variety of tissues.So TxI D can′t differentiate α3β4 nA ChR from α6/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 for α3β4 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 both α3β4 and α6/α3β4 nAChRs.An analogue,ie [S9 A]TxID had46-fold greater potency for α3β4 versus α6/α3β4 nAChRs,which showed significantly improved selectivity for α3β4 versus α6/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 of α6β2*and α3β4 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 involving α6β2* and α3β4 nA ChR s.展开更多
The voltage-gated sodium channel(Na v)is widely present in mammals and can generate cell action potentials,which are related to many diseases.Theμ-Conotoxins(μ-CTx)isolated from the venom of cone snails can specific...The voltage-gated sodium channel(Na v)is widely present in mammals and can generate cell action potentials,which are related to many diseases.Theμ-Conotoxins(μ-CTx)isolated from the venom of cone snails can specifically block the voltage-gated sodium channel;it can be widely used as a necessary probe to distinguish the Na v channel subtypes.In this study,the effects of eightμ-CTx on different Na v channel isoforms were reviewed,and sequence alignment and protein homologous modeling were used to predict their biological activities,and the structure-activity relationship betweenμ-CTx and mutagenesis strategies.展开更多
ω-Conotoxins inhibit N-type voltage-gated calcium(Ca_(v)2.2)channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index.Here,we synthesized and characterized a novelω-conotoxin,Bu8...ω-Conotoxins inhibit N-type voltage-gated calcium(Ca_(v)2.2)channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index.Here,we synthesized and characterized a novelω-conotoxin,Bu8 from Conus bullatus,which consists of 25 amino acid residues and three disulfide bridges.Bu8 selectively and potently inhibits depolarization-activated Ba^(2+ )currents mediated by rat Ca_(v)2.2 expressed in HEK293 T cells(IC_(50)=89 nmol/L).Bu8 is two-fold more potent thanω-conotoxin MVIIA,aω-conotoxin currently used for the treatment of severe chronic pain.It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish.Its lower side effects may be attributed to its faster binding rate and higher recovery ratios.The NMR structure demonstrates that Bu8 contains a small irregular tripleβ-strand.The structure-activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of Ca_(v)2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA.This study characterizes a novel,more potentω-conotoxin and provides new insights for designing Ca_(v)2.2 antagonists.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 and β1-β4,as well as γ,δ,and ε,which assemble into pentamers to form different nAChR subtypes with distinct pharmacological properties in mammals.Among them α6β2*(*designates other possible subunit),α3β4 and α4β2 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.The α6* 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.The α3β4 nAChRs present in the medial habenula with important role in influencing nicotine addiction.Blockage of α3β4 nAChRs in the medial habenula decreased the dose of nicotine that rodents would self-administer.Thus,new antagonists of α6β2* or α3β4 nA ChR subtypes are of considerable interest,which would give strategies to selectively modulate α6β2* or α3β4 nA ChR function.We characterized an α-conotoxin(α-CTx)TxIB with 16 amino acids and an α-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 a Ⅰ-Ⅲ and Ⅱ-Ⅳ disulfide con-nectivity were chemically synthesized.The residues between Cys-Ⅱ and Cys-Ⅲ and Cys-Ⅲand Cys-Ⅳ of α-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 the α-CTx.So TxIB is classified as a 4/7α-CTx,whereas the α-CTx TxIB has a 4/6 spacing.Both peptides were tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes.The α-CTx TxIB blocked α6/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 10 μmol·L^(-1).TxIB blocking α6/α3β2β3 nAChR is rapidly reversed after toxin washout.The ability ofα-CTx TxIB to discriminate between α6/α3β2β3 and the other nAChR receptors is unique.There are no small molecules have this selectivity profile.Previously described α-CTx that potently blockα6/α3β2β3 nA ChR s also block either α6/α3β4 nAChRs,α3β2 nAChRs and(or) other nAChRs subtypes.TxID was the very potent α3β4 nAChR antagonists blocking rat α3β4 n AChRs with an IC-50 of 12.5 nmol·L1.However,TxID also blocked the closely related α6/α3β4 with an IC50 of 94 nmol·L^(-1).In fact,the expression profile ofα3β4 nAChRs and α6/α3β4 nAChRs overlap in a variety of tissues.So TxI D can′t differentiate α3β4 nA ChR from α6/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 for α3β4 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 both α3β4 and α6/α3β4 nAChRs.An analogue,ie [S9 A]TxID had46-fold greater potency for α3β4 versus α6/α3β4 nAChRs,which showed significantly improved selectivity for α3β4 versus α6/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 of α6β2*and α3β4 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 involving α6β2* and α3β4 nA ChR s.
基金Supported by Special Scientific Research Project of the Hainan Academician Innovation Platform,China(YSPTZX202132)Hainan Provincial Natural Science Foundation,China(820RC636)Hainan Provincial Key Point Research and Invention Program,China(ZDYF2022SHFZ309).
文摘The voltage-gated sodium channel(Na v)is widely present in mammals and can generate cell action potentials,which are related to many diseases.Theμ-Conotoxins(μ-CTx)isolated from the venom of cone snails can specifically block the voltage-gated sodium channel;it can be widely used as a necessary probe to distinguish the Na v channel subtypes.In this study,the effects of eightμ-CTx on different Na v channel isoforms were reviewed,and sequence alignment and protein homologous modeling were used to predict their biological activities,and the structure-activity relationship betweenμ-CTx and mutagenesis strategies.
基金supported by the National Natural Science Foundation of China(grant number 81473192)the National Basic Research Program of China(grant number 2010CB529802)
文摘ω-Conotoxins inhibit N-type voltage-gated calcium(Ca_(v)2.2)channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index.Here,we synthesized and characterized a novelω-conotoxin,Bu8 from Conus bullatus,which consists of 25 amino acid residues and three disulfide bridges.Bu8 selectively and potently inhibits depolarization-activated Ba^(2+ )currents mediated by rat Ca_(v)2.2 expressed in HEK293 T cells(IC_(50)=89 nmol/L).Bu8 is two-fold more potent thanω-conotoxin MVIIA,aω-conotoxin currently used for the treatment of severe chronic pain.It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish.Its lower side effects may be attributed to its faster binding rate and higher recovery ratios.The NMR structure demonstrates that Bu8 contains a small irregular tripleβ-strand.The structure-activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of Ca_(v)2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA.This study characterizes a novel,more potentω-conotoxin and provides new insights for designing Ca_(v)2.2 antagonists.
