title: Key residues in the nicotinic acetylcholine receptor β2 subunit contribute to α-conotoxin LvIA binding.
abstract: α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val(111) is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr(59) and Phe(119) of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.
authors.affiliation: From the Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, Hainan University, Haikou, Hainan 570228, China.
authors.forename: Dongting
authors.initials: D
authors.lastname: Zhangsun
authors.affiliation: From the Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, Hainan University, Haikou, Hainan 570228, China.
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Zhu
authors.affiliation: From the Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, Hainan University, Haikou, Hainan 570228, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: From the Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, Hainan University, Haikou, Hainan 570228, China.
authors.forename: Yuanyan
authors.initials: Y
authors.lastname: Hu
authors.affiliation: the Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072 Australia, and.
authors.forename: Quentin
authors.initials: Q
authors.lastname: Kaas
authors.affiliation: the Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072 Australia, and.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.affiliation: the George E. Wahlen Veterans Affairs Medical Center and Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah 84112.
authors.forename: J Michael
authors.initials: JM
authors.lastname: McIntosh
authors.affiliation: From the Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, Hainan University, Haikou, Hainan 570228, China, luosulan2003@163.com.
authors.forename: Sulan
authors.initials: S
authors.lastname: Luo
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D020916:Conotoxins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D052078:Conus Snail / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D008564:Membrane Potentials / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
chemical_list: D000596:Amino Acids; D020916:Conotoxins; D010455:Peptides; D021122:Protein Subunits; D011978:Receptors, Nicotinic; C419362:nicotinic receptor alpha3beta2
keywords: Docking; Mutant α3β2 Subtype.; Receptor; Receptor Structure-Function; Receptor-interacting Protein (RIP); Toxin; α-Conotoxin LvIA; α3β2 nAChR; β subunit contribution