Channel Blocking Peptides Research Articles

Structural determinants of scorpion toxin affinity: the charybdotoxin (alpha-KTX) family of K(+)-channel blocking peptides.

The structure-activity relationships for the α-K family of K+ channel blockers from scorpion venoms demonstrate the critical contribution of site-directed mutagenesis, electrophysiology, together with three-dimensional information. Additions to the current database of toxin structures should enable us to make increasingly accurate and detailed predictions about the selectivity of channel-toxin interactions from the molecular structure data. Amino acid sequences, alone, cannot provide this information because the sequences provide little information about the location, in 3-dimensional space, of key side-chains relative to the backbone and to each other. In cases when the 3-dimensional structures are known, the use of these α-K toxins has provided a large amount of information about the structure of the outer vestibule of cloned channels. More recently the toxins have been useful in classifying the types of K+ channels that are present in several native preparations.

A unified nomenclature for short-chain peptides isolated from scorpion venoms: α-KTx molecular subfamilies

Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families of peptides have been purified from scorpion venom. In this article, the classification of K +-channel-blocking peptides belonging to family 2 peptides and comprising 30–40 amino acids linked by three or four disulfide bridges, will be discussed. Evidence is provided for the existence of 12 molecular subfamilies, named α-KTx1–12, containing 49 different peptides. Because of the pharmacological divergence of these peptides, the principle of classification was based on a primary sequence alignment, combined with maximum parsimony and Neighbour-Joining analysis.

Molecular cloning and sequencing of two ‘short chain’ and two ‘long chain’ K + channel-blocking peptides from the Chinese scorpion Buthus martensii Karsch

Five full-length cDNAs encoding the precursors of two ‘short chain’ scorpion non-toxic peptides active on Ca 2+-activated K + channels (BmP02 and BmP03) and two novel putative long chain K + channel-blocking peptides (named BmTXKβ and BmTXKβ2) were first isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). BmTXKβ2 showed a high similarity with AaTXKβ, while BmTXKβ was completely different in the deduced primary structure from the long chain and short chain scorpion toxins already characterized. Thus, BmTXKβ expands the scorpion long chain K + channel-blocking peptide family. Although little sequence similarity exists between the above two short and two long peptides, they are similar at the positions of six cysteines, suggesting that they should all share a similar scaffold composed of an α-helix and a three-stranded β-sheet.

Competition for binding between veratridine and KIFMK: an open channel blocking peptide of the RIIA sodium channel.

Veratridine, an alkaloid isolated from the rhizome of V. album, binds and slows the inactivation of the brain sodium channels. The synthetic pentapeptide KIFMK causes a voltage- and use-dependent open-channel block of the RIIA (rat brain type IIA) sodium channel (Eaholtz, Scheuer & Catterall, 1994). Our studies on the RIIA sodium channel expressed in CHO cells reveal that the fraction of veratridine modified sodium channels decreases linearly with increasing KIFMK concentration. However, the time constant for dissociation of veratridine from the channel remains unchanged in the presence of a high concentration of KIFMK, as opposed to that in the presence of QX314 where the dissociation appears to be more complex. These data are consistent with mutually exclusive binding of the open channel blocking peptide and veratridine to the brain sodium channel.

Shaker pore structure as predicted by annealed atomic simulation using symmetry and novel geometric restraints

Recent studies making use of channel-blocking peptides as molecular calipers have revealed the architecture of the pore-forming region of Shaker-type potassium channels. Here we show that the low-resolution, experimentally derived geometric information can be incorporated as restraints within the context of an annealed molecular dynamics simulation to predict an atomic structure for the channel pore which, by virtue of restraints, conforms to the experimental evidence. The simulation is reminiscent of the computational method employed by nuclear magnetic resonance (NMR) spectroscopists to resolve solution structures of biological macromolecules, but in lieu of restraints conventionally derived from NMR spectra, novel restraints are developed that include side-chain orientation of amino acid residues and assumed symmetry of protein subunits. The method presented here offers the possibility of expanding cooperation between simulation and experiment in developing structural models, especially for systems such as ion channels whose three-dimensional structures may not be amenable to determination by direct methods at the present time.

Synthesis and expression of the gene coding for noxiustoxin, a K + channel-blocking peptide from the venom of the scorpion Centruroides noxius

A set of six synthetic overlapping oligonucleotides coding for noxiustoxin were coupled into a continuous DNA fragment by means of recursive polymerase chain reaction. The polymerase chain reaction product was digested with SalI and HindIII, ligated into the E. coli vector pCSP105 and expressed as a fusion protein. The fusion protein was purified and digested with trypsin and the hydrolysis products were separated by high-performance liquid chromatography. Approximately 1.3 mg of recombinant noxiustoxin per liter of culture was obtained. Amino acid analysis and N-terminal amino acid sequence of the recombinant noxiustoxin confirmed the nucleotide sequence of the cloned DNA. Binding experiments using rat brain synaptosomal membranes revealed that recombinant noxiustoxin displaced bound radioactive native NTX with a similar efficiency to cold native noxiustoxin.

A novel structural class of K+-channel blocking toxin from the scorpion Pandinus imperator.

A novel peptide was purified and characterized from the venom of the scorpion Pandinus imperator. Analysis of its primary structure reveals that it belongs to a new structural class of K+-channel blocking peptide, composed of only 35 amino acids, but cross-linked by four disulphide bridges. It is 40, 43 and 46% identical to noxiustoxin, margatoxin and toxin 1 of Centruroides limpidus respectively. However, it is less similar (26 to 37% identity) to toxins from scorpions of the geni Leiurus, Androctonus and Buthus. The disulphide pairing was determined by sequencing heterodimers produced by mild enzymic hydrolysis. They are formed between Cys-4-Cys-25, Cys-10-Cys-30, Cys-14-Cys-32 and Cys-20-Cys-35. Three-dimensional modelling, using the parameters determined for charybdotoxin, showed that is it possible to accommodate the four disulphide bridges in the same general structure of the other K+-channel blocking peptides. The new peptide (Pil) blocks Shaker B K+ channels reversibly. It also displaces the binding of a known K+-channel blocker, [125I]noxiustoxin, from rat brain synaptosomal membranes with an IC50 of about 10 nM.

Structural and functional characterization of noxiustoxin, a K + channel blocking-peptide from the venom of the scorpion Centruroides noxius

Structural and functional characterization of noxiustoxin, a K + channel blocking-peptide from the venom of the scorpion Centruroides noxius

A new cysteine framework in sodium channel blocking conotoxins.

Two novel sodium channel blocking peptides from the venom of the molluscivorous snail Conus pennaceus, muPnIVA and muPnIVB, are described. Elucidation of their amino acid sequences was complicated by a previously undescribed anomalous product of reduction and pyridylethylation, which occurs on N-terminal cysteine residues and gives a PTH derivative eluting at the same position as PTH-Trp in reverse-phase chromatography. The amino acid sequences of the toxins were determined by a combination of Edman degradation and mass-spectrometric techniques as CCKYGWTCLLGCSPCGC (PnIVA) and CCKYGWTCWLGCSPCGC (PnIVB). These toxins block sodium channels in molluscan neurons, but have no effect on sodium currents in bovine chromaffin cells or in rat brain synaptosomes. Although there is only one amino acid difference in the two sequences, PnIVB is approximately 6 times more potent than PnIVA in blockade of the sodium current in Lymnaea neurons. The PnIV sequences reveal a new cysteine residue framework for conotoxins (CC-----C---C--C-C). Strikingly, the only charged residue in PnIVA/B is Lys3. Iodination reaction experiments on the adjacent Tyr4 suggest that this region of the peptide must be solvent exposed and essential for activity. These structurally novel mu-conotoxins target a sodium channel subtype with low affinity for tetrodotoxin and therefore provide new probes for functional studies on sodium channels.

Characterization of a potassium channel toxin from the Caribbean sea anemone Stichodactyla helianthus

A peptide toxin, ShK, that blocks voltage-dependent potassium channels was isolated from the whole body extract of the Caribbean sea anemone Stichodactyla helianthus. It competes with dendrotoxin I and α-dendrotoxin for binding to synaptosomal membranes of rat brain, facilitates acetylcholine release at an avian neuromuscular junction and suppresses K + currents in rat dorsal root ganglion neurones in culture. Its amino acid sequence is R 1SCIDTIPKS 10RCTAFQCKHS 20MKYRLSFCRK 30-TCGTC 35. There is no homology with other K + channel-blocking peptides, except for BgK from the sea anemone Bunodosoma granulifera. ShK and BgK appear to be in a different structural class from other toxins affecting K + channels.

Mapping function to structure in a channel-blocking peptide: electrostatic mutants of charybdotoxin.

Electrostatic interactions between charybdotoxin (CTX), a specific peptide pore blocker of K+ channels, and a Ca(2+)-activated K+ channel were investigated with a genetically manipulable recombinant CTX. Point mutations at certain charged residues showed only small effects on the binding affinity of the toxin molecule: Lys11, Glu12, Arg19, His21, Lys31, and Lys32. Replacement by Gln at Arg25, Lys27, or Lys34 strongly decreased the affinity of the toxin. These affinity changes were mainly due to large increases of toxin dissociation rates without much effect on association rates, as if close-range interactions between the toxin and its receptor site of the channel were disrupted. We also found that the neutralization of Lys27 to Gln removed the toxin's characteristic voltage dependence in dissociation rate. Mutation and functional mapping of charged residues revealed a molecular surface of CTX which makes direct contact with the extracellular mouth of the K+ channel.

K+ Channel blocking peptides isolated from the venom of scorpions

K+ Channel blocking peptides isolated from the venom of scorpions

Potassium channel blocking peptides from sea anemones: D. Mebs, 1 I. Claus, 1 M. Deist, 2 H. Repp, 2 J. Beise 2 and F. Dreyer ( 1Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt, F.R.G.; 2Rudolf-Buchheim Institute of Pharmacology, University of Giessen, F.R.G.)

Potassium channel blocking peptides from sea anemones: D. Mebs, 1 I. Claus, 1 M. Deist, 2 H. Repp, 2 J. Beise 2 and F. Dreyer ( 1Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt, F.R.G.; 2Rudolf-Buchheim Institute of Pharmacology, University of Giessen, F.R.G.)