Antagonistic Activity of ��-Conotoxin TxIB Isomers on Rat and Human ��6/��3��2��3 Nicotinic Acetylcholine Receptors
ZHANGSUN Dong-ting, WU Yong, ZHU Xiao-peng, LUO Su-lan*
Key Laboratory of Tropical Biological Resources, Ministry of Education; Key Lab for Marine Drugs of Haikou; Pharmacy Department of Ocean College; Hainan University, Haikou 570228, China
Abstract��OBJECTIVE To investigate antagonistic activities of three isomers of ��-conotoxin TxIB on rat and human ��6/��3��2��3 nicotinic acetylcholine receptors (nAChRs). METHODS Three disulfide bond isomers were synthesized using Fmoc chemistry, which were identified by ultra performance liquid chromatography (UPLC)and confirmed by MALDI-TOF mass spectrometry. Rat and human ��6/��3��2��3 nAChRs were expressed in oocytes of Xenopus laevis, which were used to test the antagonistic abilities of the 3 isomers. RESULTS The three isomers of ��-conotoxin TxIB were synthesized successfully.The retention time of each isomer of ��-conotoxin TxIB was different each other significantly. The observed molecular masses of three isomers were the same, which were consistent with their theoretical molecular mass.Their hydrophilicity orders were globular > ribbon> bead. Both rat and human ��6/��3��2��3 nAChRs were expressed in oocytes well. Inhibition of three isomers of ��-conotoxin TxIB on rat and human ��6/��3��2��3 nAChRs were evaluated respectively. Among the three isomers of TxIB, the activity of the globular isomer was the most potent one, which had almost same activity at rat and human ��6/��3��2��3 nAChRs with corresponding IC50 of 28.2 and 32.0 nmol��L-1respectively. However, the other two isomers, ribbon and bead isomers displayed little antagonistic effect on both rat and human ��6/��3��2��3 nAChRs only with an IC50 of ��10 ��mol��L-1. CONCLUSION The synthesized globular isomer of ��-conotoxin TxIB in this work has a high selectivity and potent antagonistic activity on rat and human ��6/��3��2��3 nAChRs, which would be helpful for its new drug development.
DAVIS J, JONES A, LEWIS R J. Remarkable inter- and intra-species complexity of conotoxins revealed by LC/MS[J]. Peptides, 2009, 30(7):1222-1227.
[2]
LAVERGNE V, HARLIWONG I, JONES A, et al. Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks[J]. Proc Natl Acad Sci USA, 2015, 112(29):3782-3791.
[3]
KAAS Q, YU R, JIN A H, et al. ConoServer: updated content, knowledge, and discovery tools in the conopeptide database[J]. Nucleic Acids Res, 2012, 40(database issue):325-330.
[4]
LEWIS R J, DUTERTRE S, VETTER I, et al. Conus venom peptide pharmacology[J]. Pharmacol Rev, 2012, 64(2):259-298.
[5]
SANFORD M. Intrathecal ziconotide: a review of its use in patients with chronic pain refractory to other systemic or intrathecal analgesics[J]. CNS Drugs, 2013, 27(11):989-1002.
[6]
GOTTI C, CLEMENTI F. Neuronal nicotinic receptors: from structure to pathology[J]. Prog Neurobiol, 2004, 74(6):363-396.
[7]
LEBBE E K, PEIGNEUR S, MAITI M, et al. Discovery of a new subclass of alpha-conotoxins in the venom of Conus australis[J]. Toxicon, 2014, 91:145-154.
[8]
LUO S, ZHANGSUN D, WU Y, et al. Characterization of a novel alpha-conotoxin from conus textile that selectively targets alpha6/alpha3beta2beta3 nicotinic acetylcholine receptors[J]. J Biol Chem, 2013, 288(2):894-902.
[9]
MCINTOSH J M, AZAM L, STAHELI S, et al. Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors[J]. Mol Pharmacol, 2004, 65(4):944-952.
[10]
FANG L C, SHEN L Z, YU J P, et al. Optimization of receptors expressed on Xenopus laevis oocytes [J]. Biotechno Bull(���＼��ͨ��), 2013, 29(6):167-171.
[11]
HALAI R, CLARK R J, NEVIN S T, et al. Scanning mutagenesis of alpha-conotoxin Vc1. 1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor[J]. J Biol Chem, 2009, 284(30):20275-20284.
[12]
VINCLER M, WITTENAUER S, PARKER R, et al. Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors[J]. Proc Natl Acad Sci USA, 2006, 103(47):17880-17884.
[13]
AZAM L, MCINTOSH J M. Molecular basis for the differential sensitivity of rat and human alpha9alpha10 nAChRs to alpha-conotoxin RgIA[J]. J Neurochem, 2012, 122(6):1137-1144.
YANG K C, JIN G Z, WU J. Mysterious alpha6-containing nAChRs: function, pharmacology, and pathophysiology[J]. Acta Pharmacol Sin, 2009, 30(6):740-751.
[16]
KLINK R, DE KERCHOVE D'EXAERDE A, ZOLI M, et al. Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei[J]. J Neurosci, 2001, 21(5):1452-1463.
[17]
AZAM L, WINZER-SERHAN U H, CHEN Y, et al. Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons[J]. J Comp Neurol, 2002, 444(3):260-274.
[18]
CHAMPTIAUX N, GOTTI C, CORDERO-ERAUSQUIN M, et al. Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice[J]. J Neurosci, 2003, 23(21):7820-7829.
[19]
PONS S, FATTORE L, COSSU G, et al. Crucial role of alpha4 and alpha6 nicotinic acetylcholine receptor subunits from ventral tegmental area in systemic nicotine self-administration[J]. J Neurosci, 2008, 28(47):12318-12327.
[20]
LE NOVERE N, ZOLI M, CHANGEUX J P. Neuronal nicotinic receptor alpha 6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain[J]. Eur J Neurosci, 1996, 8(11):2428-2439.
[21]
VAILATI S, HANKE W, BEJAN A, et al. Functional alpha6-containing nicotinic receptors are present in chick retina[J]. Mol Pharmacol, 1999, 56(1):11-19.
[22]
CHAMPTIAUX N, HAN Z Y, BESSIS A, et al. Distribution and pharmacology of alpha 6-containing nicotinic acetylcholine receptors analyzed with mutant mice[J]. J Neurosci, 2002, 22(4):1208-1217.
[23]
QUIK M, POLONSKAYA Y, KULAK J M, et al. Vulnerability of 125I-alpha-conotoxin MII binding sites to nigrostriatal damage in monkey[J]. J Neurosci, 2001, 21(15):5494-5500.
[24]
QUIK M, BORDIA T, FORNO L, et al. Loss of alpha-conotoxinMII- and A85380-sensitive nicotinic receptors in Parkinson's disease striatum[J]. J Neurochem, 2004, 88(3):668-679.
[25]
JACKSON K J, MCINTOSH J M, BRUNZELL D H, et al. The role of alpha6-containing nicotinic acetylcholine receptors in nicotine reward and with drawal[J]. J Pharmacol Exp Ther, 2009, 331(2):547-554.
[26]
SANJAKDAR S S, MALDOON P P, MARKS M J, et al. Differential roles of alpha6beta2* and alpha4beta2* neuronal nicotinic receptors in nicotine-and cocaine-conditioned reward in mice[J]. Neuropsychopharmacology, 2015, 40(2):350-360.
[27]
SCHILATY N D, HEDGES D M, JANG E Y, et al. Acute ethanol inhibits dopamine release in the nucleus accumbens via alpha6 nicotinic acetylcholine receptors[J]. J Pharmacol Exp Ther, 2014, 349(3):559-567.
2017, Vol. 52 
