Single-residue alteration in alpha-conotoxin PnIA switches its nAChR subtype selectivity

Biochemistry. 1999 Nov 2;38(44):14542-8. doi: 10.1021/bi991252j.

Abstract

alpha-Conotoxins are disulfide-rich peptides that are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Despite their small size, different alpha-conotoxins are able to discriminate among different subtypes of mammalian nAChRs. In this report, the activity of two peptides from the venom of Conus pennaceus, alpha-conotoxins PnIA and PnIB, are examined. Although the toxins differ in only two residues, PnIA preferentially blocks alpha3beta2 nAChRs, whereas PnIB prefers the alpha7 subtype. Point mutation chimeras of these alpha-conotoxins were synthesized and their activities assessed on Xenopus oocytes expressing specific nAChRs. Change of a single residue, Ala10 to Leu, in PnIA (to form PnIA [A10L]) converts the parent peptide from alpha3beta2-preferring to alpha7-preferring; furthermore, PnIA [A10L] blocks the alpha7 receptor with an IC(50) (12.6 nM) that is lower than that of either parent peptide. Kinetic analysis indicates that differences in affinity among the analogues are primarily due to differences in off-rate, with PnIA [A10L]'s interaction with alpha7 having the smallest off-rate (k(off) = 0.17 min(-)(1)). Thermodynamic analysis indicates that Leu10 enhances the peptide's interaction with alpha7, but not alpha3beta2, receptors, whereas Ser11 (in PnIA [N11S]) reduces its affinity for both alpha7 and alpha3beta2 nAChRs.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Binding Sites
  • Binding, Competitive
  • Brain / drug effects
  • Brain / metabolism
  • Cholinergic Antagonists / chemistry*
  • Cholinergic Antagonists / toxicity*
  • Conotoxins*
  • Female
  • In Vitro Techniques
  • Kinetics
  • Male
  • Models, Molecular
  • Molecular Sequence Data
  • Mollusk Venoms / chemistry*
  • Mollusk Venoms / genetics
  • Mollusk Venoms / toxicity*
  • Mutagenesis, Site-Directed
  • Oligopeptides / chemistry*
  • Oligopeptides / genetics
  • Oligopeptides / toxicity*
  • Oocytes / drug effects
  • Oocytes / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, Cholinergic / classification
  • Receptors, Cholinergic / drug effects
  • Receptors, Cholinergic / metabolism
  • Thermodynamics
  • Xenopus

Substances

  • Cholinergic Antagonists
  • Conotoxins
  • Mollusk Venoms
  • Oligopeptides
  • Receptors, Cholinergic
  • alpha-conotoxin PnIA