A disulfide tether stabilizes the block of sodium channels by the conotoxin µO§-GVIIJ.

A cone snail venom peptide, µO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. µO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVß2 or rNaVß4, but not that of rNaVß1 or rNaVß3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVß- and NaVα-subunits are present in native neurons.

I(1)-superfamily conotoxins and prediction of single D-amino acid occurrence.

The considerable diversity of Conus peptides in the I(1)-superfamily provides a rare opportunity to define parameters important for the post-translational l- to d-isomerization of amino acids. This subtlest of post-translational modifications is not readily detectable by most techniques, and it would be a considerable advance if one could predict its potential occurrence purely from gene sequences. We previously described three I(1)-conotoxins, iota-RXIA (formerly designated r11a), r11b and r11c, each containing a d-amino acid at the third position from the C-terminus. In this work, we investigated two novel I(1)-superfamily members, r11d and ar11a, which we show have only l-amino acids. Based on these observations and an analysis of cDNA sequences of other group members, we suggest that there is a rule to predict d-amino acids in I(1)-superfamily peptides. Two factors are important: the residue to be modified should be three amino acids from the C-terminus of the precursor sequence, and it should be in a suitable sequence context. We apply the rule to other members of the I(1)-superfamily, to determine a priori which are probably modified.

Characterization of D-amino-acid-containing excitatory conotoxins and redefinition of the I-conotoxin superfamily.

Post-translational isomerization of l-amino acids to d-amino acids is a subtle modification, not detectable by standard techniques such as Edman sequencing or MS. Accurate predictions require more sequences of modified polypeptides. A 46-amino-acid-long conotoxin, r11a, belonging to the I-superfamily was previously shown to have a d-Phe residue at position 44. In this report, we characterize two related peptides, r11b and r11c, with d-Phe and d-Leu, respectively, at the homologous position. Electrophysiological tests show that all three peptides induce repetitive activity in frog motor nerve, and epimerization of the single amino acid at the third position from the C-terminus attenuates the potency of r11a and r11b, but not that of r11c. Furthermore, r11c (but neither r11a nor r11b) also acts on skeletal muscle. We identified more cDNA clones encoding conopeptide precursors with Cys patterns similar to r11a/b/c. Although the predicted mature toxins have the same cysteine patterns, they belong to two different gene superfamilies. A potential correlation between the identity of the gene superfamily to which the I-conotoxin belongs and the presence or absence of a d-amino acid in the primary sequence is discussed. The great diversity of I-conopeptide sequences provides a rare opportunity for defining parameters that may be important for this most stealthy of all post-translational modifications. Our results indicate that neither the chemical nature of the side chain nor the precise vicinal sequence around the modified residue seem to be critical, but there may be favored loci for isomerization to a d-amino acid.