Abstract
We describe the properties of a family of 22-amino acid peptides, the mu-conotoxins, which are useful probes for investigating voltage-dependent sodium channels of excitable tissues. The mu-conotoxins are present in the venom of the piscivorous marine snail, Conus geographus L. We have purified seven homologs of the mu-conotoxin set and determined their amino acid sequences, as follows, where Hyp = trans-4-hydroxyproline. GIIIA R.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2 [Pro6]GIIIA R.D.C.C. T.P.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2 [Pro7]GIIIA R.D.C.C.T.Hyp.P.K.K.C.K.D.R.Q.C.R.Hyp.Q.R.C.C.A-NH2 GIIIB R.D.C.C.T.Hyp.Hyp.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2 [Pro6]GIIIB R.D.C.C.T.P.Hyp.R.K.C.K.D.R.R. C.K.Hyp.M.K.C.C.A-NH2 [Pro7]GIIIB R.D.C.C.T.Hyp.P.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2 GIIIC R.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.R.C.K.Hyp.L.K.C.C.A-NH2. Using the major peptide (GIIIA) in electrophysiological studies on nerve-muscle preparations and in single channel studies using planar lipid bilayers, we have established that the toxin blocks muscle sodium channels, while having no discernible effect on nerve or brain sodium channels. In bilayers the blocking kinetics of GIIIA were derived by statistical analysis of discrete transitions between blocked and unblocked states of batrachotoxin-activated sodium channels from rat muscle. The kinetics conform to a single-site, reversible binding equilibrium with a voltage-dependent binding constant. The measured value of the equilibrium KD for GIIIA is 100 nM at OmV, decreasing e-fold/34 mV of hyperpolarization. This voltage dependence of blocking is similar to that of tetrodotoxin and saxitoxin as measured by the same technique. The tissue specificity and kinetic characteristics suggest that the mu-conotoxins may serve as useful ligands to distinguish sodium channel subtypes in different tissues.
Highlights
EXPERIMENTAL PROCEDURESC. geographus venom was extracted [17], and soluble proteins and peptides were fractionated on a column of Sephadex G-25 [18] as previously described
Yield Net pmol Cys" Lys' Hyp LeuAla "Analyzed as PTH-cysteic acid, which extracts poorly from the sequencer cup.Analyzed as 6-N-acetyl-PTH-lysine,which elutes just after PTHAla on HPLC. 'indicating a blocked a-carboxyl
We provide definitive biochemical and electrophysiological evidence that one toxin in this series ( p conotoxin GIIIA) does directly block muscle Na channels
Summary
C. geographus venom was extracted [17], and soluble proteins and peptides were fractionated on a column of Sephadex G-25 [18] as previously described. The p-conotoxins were found in the second peak This material was fractionated by reversed-phase HPLC' using a Supelco semiprep column (1.25 X 25 cm; 5-pm particle size; C18, not end-capped; 25 "C) ata flow rate of ml/min. HPLC on the VYDAC column using conditions similar to those described above It was digested for 3 hat 37 "C with clostripain (50 pl of a 1 mg/ml solution in 0.025 M potassium phosphate, pH 7.5, containing 0.2 mM calcium acetate and 2.5 mM dithiothreitol) [21]. The digest was diluted with 200 pl of water and applied directly to the sequencer cup, which contained precycled Polybrene For this sample we used a 0.25 M Quadrol program with separate benzene and ethyl acetate washes
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