title: Docking of mu-conotoxin GIIIA in the sodium channel outer vestibule.
abstract: mu-Conotoxin GIIIA (mu-CTX) is a high-affinity ligand for the outer vestibule of selected isoforms of the voltage-gated Na(+) channel. The detailed bases for the toxin's high affinity binding and isoform selectivity are unclear. The outer vestibule is lined by four pore-forming (P) loops, each with an acidic residue near the mouth of the vestibule. mu-CTX has seven positively charged residues that may interact with these acidic P-loop residues. Using pair-wise alanine replacement of charged toxin and channel residues, in conjunction with double mutant cycle analysis, we determined coupling energies for specific interactions between each P-loop acidic residue and selected toxin residues to systematically establish quantitative restraints on the toxin orientation in the outer vestibule. Xenopus oocytes were injected with the mutant or native Na(+) channel mRNA, and currents measured by two-electrode voltage clamp. Mutant cycle analysis revealed novel, strong, toxin-channel interactions between K9/E403, K11/D1241, K11/D1532, and R19/D1532. Experimentally determined coupling energies for interacting residue pairs provided restraints for molecular dynamics simulations of mu-CTX docking. Our simulations suggest a refined orientation of the toxin in the pore, with toxin basic side-chains playing key roles in high-affinity binding. This modeling also provides a set of testable predictions for toxin-channel interactions, hitherto not described, that may contribute to high-affinity binding and channel isoform selectivity.
authors.affiliation: Department of Medicine, Providence Veterans Affairs Medical Center/Brown University, Providence, Rhode Island 02904, USA. Gaurav_Choudhary@brown.edu
authors.forename: Gaurav
authors.initials: G
authors.lastname: Choudhary
authors.forename: Marcela P
authors.initials: MP
authors.lastname: Aliste
authors.forename: D Peter
authors.initials: DP
authors.lastname: Tieleman
authors.forename: Robert J
authors.initials: RJ
authors.lastname: French
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Dudley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003198:Computer Simulation; D020916:Conotoxins / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012908:Snails; D015222:Sodium Channels / Q000378:metabolism*; D014981:Xenopus
chemical_list: D020916:Conotoxins; D020033:Protein Isoforms; D012333:RNA, Messenger; D015222:Sodium Channels; C046101:conotoxin GIII