title: Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to alpha-bungarotoxin.
abstract: We have investigated the molecular determinants responsible for alpha-bungarotoxin (alphaBgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous alpha subunits, one highly sensitive to alphaBgtx block (alpha1) and the other, alphaBgtx-insensitive (alpha3). By replacing rat alpha3 residues 184-191 with the corresponding region from the Torpedo alpha1 subunit, we introduced a cluster of five alpha1 residues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-191) into the alpha3 subunit. Functional activity and alphaBgtx sensitivity were assessed following co-expression in Xenopus oocytes of the chimeric alpha3 subunit (alpha3/alpha1[5]) with either rat beta2 or beta4 subunits. Agonist-evoked responses of alpha3/alpha1[5]-containing receptors were blocked by alphaBgtx with nanomolar affinity (IC(50) values: 41 nM for alpha3/alpha1[5]beta2 and 19 nM for alpha3/alpha1[5]beta4). Furthermore, receptors containing the single point mutation alpha3K189Y acquire significant sensitivity to alphaBgtx block (IC(50) values: 186 nM for alpha3K189Ybeta2 and 179 nM for alpha3K189Ybeta4). Another alpha3 chimeric subunit, alpha3/alpha7[6], similar to alpha3/alpha1[5] but incorporating the corresponding residues from the alphaBgtx-sensitive alpha7 subunit, also conferred potent alphaBgtx sensitivity to chimeric receptors when co-expressed with the beta4 subunit (IC(50) value = 31 nM). Our findings demonstrate that the residues between positions 184 and 191 of the alphaBgtx-sensitive subunits alpha1 and alpha7 play a critical functional role in the interaction of alphaBgtx with nicotinic acetylcholine receptors sensitive to this toxin.
authors.affiliation: Department of Molecular Pharmacology, Physiology, and Biotechnology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Levandoski
authors.forename: Y
authors.initials: Y
authors.lastname: Lin
authors.forename: L
authors.initials: L
authors.lastname: Moise
authors.forename: J T
authors.initials: JT
authors.lastname: McLaughlin
authors.forename: E
authors.initials: E
authors.lastname: Cooper
authors.forename: E
authors.initials: E
authors.lastname: Hawrot
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
chemical_list: D000596:Amino Acids; D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins