Isolation and characterization of α-conotoxin LsIA with potent activity at nicotinic acetylcholine receptors.
A new α-conotoxin LsIA was isolated from the crude venom of Conus limpusi using assay-guided RP-HPLC fractionation. Synthetic LsIA was a potent antagonist of α3β2, α3α5β2 and α7 nAChRs, with half-maximal inhibitory concentrations of 10, 31 and 10 nM, respectively. The structure of LsIA determined by NMR spectroscopy comprised a characteristic disulfide bond-stabilized α-helical structure and disordered N-terminal region. Potency reductions of up to 9-fold were observed for N-terminally truncated analogues of LsIA at α7 and α3β2 nAChRs, whereas C-terminal carboxylation enhanced potency 3-fold at α3β2 nAChRs but reduced potency 3-fold at α7 nAChRs. This study gives further insight into α-conotoxin pharmacology and the molecular basis of nAChR selectivity, highlighting the influence of N-terminal residues and C-terminal amidation on conotoxin pharmacology.
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia.
Marco C
MC
Inserra
Shiva N
SN
Kompella
Irina
I
Vetter
Andreas
A
Brust
Norelle L
NL
Daly
Hartmut
H
Cuny
David J
DJ
Craik
Paul F
PF
Alewood
David J
DJ
Adams
Richard J
RJ
Lewis
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D056148:Chromatography, Reverse-Phase; D020916:Conotoxins / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D052078:Conus Snail / Q000737:chemistry*; D005456:Fluorescent Dyes; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D018733:Nicotinic Antagonists / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000378:metabolism; D017433:Protein Structure, Secondary; D021281:Quantitative Structure-Activity Relationship; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013379:Substrate Specificity
D020916:Conotoxins; D005456:Fluorescent Dyes; D018733:Nicotinic Antagonists; D020033:Protein Isoforms; D011978:Receptors, Nicotinic; D002118:Calcium
1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea; 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; ACh; AChBP; AFU; CCD; CYANA; Conus limpusi; DIPEA; DMSO; DQF-COSY; EDTA; Electrophysiology; FBS; FLIPR; G protein-coupled receptor; GPCR; HBTU; IC(50); N,N-diisopropylethylamine; NMR; NMR structure; NOESY; Nicotinic acetylcholine receptor; PNU-12596; RP-HPLC; RPMI; Rosewell Park Memorial Institute; TFA; TIPS; TOCSY; acetylcholine; acetylcholine binding protein; arbitrary fluorescent units; charge coupled device; combined assignment and dynamics algorithm for NMR applications; dimethylsulfoxide; double quantum filtered correlation spectroscopy; ethylenediaminetetraacetic acid; fluorometric imaging plate reader; foetal bovine serum; half-maximal inhibitory concentration; nAChR; nicotinic acetylcholine receptor; nuclear magnetic resonance; nuclear overhauser effect spectroscopy; reverse phase-high performance liquid chromatography; total correlation spectroscopy; trifluoroacetic acid; triisopropylsilyl; α-Conotoxin