title: Positional scanning mutagenesis of α-conotoxin PeIA identifies critical residues that confer potency and selectivity for α6/α3β2β3 and α3β2 nicotinic acetylcholine receptors.
abstract: The nicotinic acetylcholine receptor (nAChR) subtype α6β2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The α6 subunit is closely related to the α3 subunit, and this presents a problem in designing ligands that discriminate between α6β2* and α3β2* nAChRs. We used positional scanning mutagenesis of α-conotoxin PeIA, which targets both α6β2* and α3β2*, in combination with mutagenesis of the α6 and α3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed α6/α3β2β3 and α3β2 receptors. Mutagenesis of PeIA revealed that Asn(11) was located in an important position that interacts with the α6 and α3 subunits. Substitution of Asn(11) with a positively charged amino acid essentially abolished the activity of PeIA for α3β2 but not for α6/α3β2β3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on α6/α3β2β3 than α3β2 receptors. Analogs with an N11R substitution were then used to show a critical interaction between the 11th position of PeIA and Glu(152) of the α6 subunit and Lys(152) of the α3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target α6β2* nAChRs.
authors.affiliation: the Departments of Biology and.
authors.forename: Arik J
authors.initials: AJ
authors.lastname: Hone
authors.affiliation: the Departments of Biology and.
authors.forename: Miguel
authors.initials: M
authors.lastname: Ruiz
authors.affiliation: the Departments of Biology and.
authors.forename: Mick'l
authors.initials: M
authors.lastname: Scadden
authors.affiliation: the Departments of Biology and.
authors.forename: Sean
authors.initials: S
authors.lastname: Christensen
authors.affiliation: the Departments of Biology and.
authors.forename: Joanna
authors.initials: J
authors.lastname: Gajewiak
authors.affiliation: the Departments of Biology and.
authors.forename: Layla
authors.initials: L
authors.lastname: Azam
authors.affiliation: the Departments of Biology and; From the Interdepartmental Program in Neuroscience and; Psychiatry, University of Utah, Salt Lake City, Utah 84132 and; the George E. Wahlen Veterans Affairs Medical Center, Salt Lake City, Utah 84148. Electronic address: mcintosh.mike@gmail.com.
authors.forename: J Michael
authors.initials: JM
authors.lastname: McIntosh
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002121:Calcium Channel Blockers / Q000737:chemistry* / Q000378:metabolism; D020916:Conotoxins / Q000737:chemistry* / Q000378:metabolism; D016296:Mutagenesis; D020125:Mutation, Missense; D011485:Protein Binding; D021122:Protein Subunits; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D002121:Calcium Channel Blockers; D020916:Conotoxins; D021122:Protein Subunits; D011978:Receptors, Nicotinic
keywords: Electrophysiology; Neurotoxin; Neurotransmitter Receptors; Nicotinic Acetylcholine Receptors; Oocyte; alpha-Conotoxin; alpha3beta2; alpha6beta2