Abstract
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.
Highlights
§ These authors contributed to this work. ʈ To whom correspondence should be addressed: Dept. of Biology, University of Utah, 257 S. 1400 E. 201, Salt Lake City, UT 84112-0840
Perfamily, Conus peptides specific for various subtypes of calcium channels [9] and sodium channels [10] have been described. -Conotoxin PVIIA from the venom of the Eastern Pacific fish-hunting species Conus purpurascens was the first conopeptide described that targets voltage-gated potassium channels [1, 11]
The role of -conotoxin PVIIA in prey capture has been established [1]; this peptide is key to the very rapid immobilization of fish prey by C. purpurascens. This cone species uses the following two groups of toxins to affect different physiological programs in the prey after venom injection: a very rapid excitotoxic shock and a slower onset but irreversible neuromuscular block. -Conotoxin PVIIA plays a role in the initial excitotoxic shock response to venom injection; in combination with ␦-conotoxin PVIA, an immediate tetanic-like immobilization is elicited upon injection into fish
Summary
Solid-phase Peptide Synthesis—The protected peptide resin was built using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as described previously [11] with side chains protected as follows: pentamethylchromasulfonyl (Arg), t-butyl (Hyp, Asp, and Ser), S-trityl (Asn, Gln, His, and Cys), and t-butoxycarbonyl (Lys). The pattern of mixed folded isomers obtained for all analogs was very similar to wild-type -PVIIA. The earliest eluting major peak was collected and purified, and in each case, presumed to have the same disulfide connectivity as native PVIIA; one substitution for each of the three loci (Arg2–Ala, Lys7–Ala, and Phe9–Met) was analyzed by NMR and CD to confirm directly that the disulfide connectivity was native. The Leu12–Ala analog in particular showed an unusual pattern of isomers following folding and needs to be further investigated. CRNA coding for Shaker H4 was injected into oocytes, and the cells were incubated 1–7 days to allow expression of the protein. To estimate the IC50 value of -conotoxin PVIIA and the corresponding analogs, whole cell currents of oocytes expressing either wild-type. Data shown represent mean Ϯ S.E. calculated from IC50 values independently determined for each cell (see “Experimental Procedures”); the number of independent experimental trails are specified in the table. n.d., not done
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