Single amino acid substitutions in kappa-conotoxin PVIIA disrupt interaction with the shaker K+ channel.
kappa-Conotoxin PVIIA (kappa-PVIIA), a 27-amino acid peptide with three disulfide cross-links, isolated from the venom of Conus purpurascens, is the first conopeptide shown to inhibit the Shaker K(+) channel (Terlau, H., Shon, K., Grilley, M., Stocker, M., Stühmer, W., and Olivera, B. M. (1996) Nature 381, 148-151). Recently, two groups independently determined the solution structure for kappa-PVIIA using NMR; although the structures reported were similar, two mutually exclusive models for the interaction of the peptide with the Shaker channel were proposed. We carried out a structure/function analysis of kappa-PVIIA, with alanine substitutions for all amino acids postulated to be key residues by both groups. Our data are consistent with the critical dyad model developed by Ménez and co-workers (Dauplais, M., Lecoq, A., Song, J. , Cotton, J., Jamin, N., Gilquin, B., Roumestand, C., Vita, C., de Medeiros, C., Rowan, E. G., Harvey, A. L., and Ménez, A. (1997) J. Biol. Chem. 272, 4802-4809) for polypeptide antagonists of K(+) channels. In the case of kappa-PVIIA, Lys(7) and Phe(9) are essential for activity as predicted by Savarin et al. (Savarin, P., Guenneugues, M., Gilquin, B., Lamthanh, H., Gasparini, S., Zinn-Justin, S., and Ménez, A. (1998) Biochemistry 37, 5407-5416); these workers also correctly predicted an important role for Lys(25). Thus, although kappa-conotoxin PVIIA has no obvious sequence homology to polypeptide toxins from other venomous animals that interact with voltage-gated K(+) channels, there may be convergent functional features in diverse K(+) channel polypeptide antagonists.
Jacobsen
R B
RB
Departments of Biology and Medicinal Chemistry, University of Utah, Salt Lake City, Utah, 84112, USA.
Koch
E D
ED
Lange-Malecki
B
B
Stocker
M
M
Verhey
J
J
Van Wagoner
R M
RM
Vyazovkina
A
A
Olivera
B M
BM
Terlau
H
H
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D020916:Conotoxins / Q000737:chemistry* / Q000494:pharmacology*; D004220:Disulfides; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D017354:Point Mutation; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D012908:Snails
D020916:Conotoxins; D004220:Disulfides; D010455:Peptides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D011994:Recombinant Proteins; C108818:kappa-conotoxin PVIIA; D000409:Alanine