1. We have cloned a cDNA encoding a Phe:-Met-Arg-Phe-:NH2 (FMRFamide)-gated Na+ channel from nervous tissue of the pond snail Helisoma trivolvis (HtFaNaC) and expressed the channel in Xenopus oocytes. The deduced amin...
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1. We have cloned a cDNA encoding a Phe:-Met-Arg-Phe-:NH2 (FMRFamide)-gated Na+ channel from nervous tissue of the pond snail Helisoma trivolvis (HtFaNaC) and expressed the channel in Xenopus oocytes. The deduced amino acid sequence of the protein expressed by HtFaNaC is 65 % identical to that of the FMRFamide-gated channel cloned fr om Helix aspersa (HaFaNaC). 2. HtFaNaC expressed in oocytes was less sensitive to FMRFamide (EC50 = 70 mu M) than HaFaNaC (EC50 = 2 mu M). The two had a similar selectivity for Naf. The amplitude of the FMRFamide response of HtFaNsC was increased by reducing the extracellular concentration of divalent cations. 3. The conductance of the two channels was similar, but the mean open time of unitary events was shorter for expressed HtFaNaC compared to expressed HaFaNaC. Each channel was susceptible to peptide block by high agonist concentrations. 4. In marked contrast to HaFaNaC and other amiloride-sensitive Na+ channels, amiloride, and the related drugs benzamil and 5- (N-ethyl-N-isopropyl)-amiloride (EIPA), enhanced the FMRFamide response in oocytes expressing HtFaNaC cRNA. The potentiating effects of EIPA and benzamil were greater than those of amiloride. Unitary current analysis showed that with such drugs, there was channel blockade as well as an increased probability of channel opening. 5. The similar permeability of the oocyte-expressed HtFaNaC and the Helisoma neuronal channel, and the susceptibility of both to agonist blockade and blockade by divalent cations, suggest that the channels are the same. However, neuronal channels were less susceptible to enhancement by amiloride analogues and in some patches were more sensitive to FMRFamide than expressed HtFaNaC.
We report the discovery and initial characterization of the T-superfamily of conotoxins. Eight different T-superfamily peptides from five Conus species were identified; they share a consensus signal sequence, and a co...
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We report the discovery and initial characterization of the T-superfamily of conotoxins. Eight different T-superfamily peptides from five Conus species were identified; they share a consensus signal sequence, and a conserved arrangement of cysteine residues (- -CC- -CC-). T-superfamily peptides were found expressed in venom ducts of all major feeding types of Conus; the results suggest that the T-superfamily will be a large and diverse group of peptides, widely distributed in the 500 different Conus species. These peptides are likely to be functionally diverse; although the peptides are small (11-17 amino acids), their sequences are strikingly divergent, with different peptides of the superfamily exhibiting varying extents of post-translational modification. Of the three peptides tested for in vivo biological activity, only one was active on mice but all three had effects on fish. The peptides that have been extensively characterized are as follows: p5a, GCCPKQMRCCTL*; tx5a, γCCγDGW+CCT§AAO; and au5a, FCCPFIRYCCW (where γ = γ-carboxyglutamate, W^+ = bromotryptophan, O = hydroxyproline, T§ = glycosylated threonine, and * = COOH-terminal amidation). We also demonstrate that the precursor of tx5a contains a functional γ-carboxylation recognition signal in the -1 to -20 propeptide region, consistent with the presence of γ-carboxyglutamate residues in this peptide.
To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the Venom of the marine snail Conus textile, the cDNA encoding this peptide was cloned from a C. textile venom duc...
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To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the Venom of the marine snail Conus textile, the cDNA encoding this peptide was cloned from a C. textile venom duct library. The cDNA of the precursor form of epsilon-TxTX encodes a 67 amino acid precursor peptide, including an N-terminal prepro-region, the mature peptide, and four residues posttranslationally cleaved from the C-terminus. To determine the role of the propeptide in gamma-carboxylation, peptides were designed and synthesized based on the propeptide sequence of the Gla-containing conotoxin epsilon-TxIX and used in assays with the vitamin K-dependent gamma-glutamyl carboxylase from C. textile venom ducts. The mature acarboxy peptide epsilon-TxIX was a high K-M substrate for the gamma-carboxylase. Synthetic peptides based on the precursor epsilon-TxIX were low K-M substrates (5 mu M) if the peptides included at least 12 residues of propeptide sequence, from -12 to -1. Leucine-19, leucine-16, asparagine-13, leucine-12, leucine-8 and leucine-4 contribute to the interaction of the pro-conotoxin with carboxylase since their replacement by aspartic acid increased the K-M of the substrate peptide. Although the Conus propeptide and the propeptides of the mammalian vitamin K-dependent proteins show no obvious sequence homology, synthetic peptides based upon the structure of pro-epsilon-TxIX were intermediate K-M substrates for the bovine carboxylase. The propeptide of epsilon-TxIX contains significant alpha-helix, as estimated by measurement of the circular dichroism spectra, but the region of the propeptide that plays the dominant role in directing carboxylation does not contain evidence of helical structure. These results indicate that the gamma-carboxylation recognition site is defined by hydrophobic residues in the propeptide of this conotoxin precursor.
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