ABSTRACT
Animal venoms are rich sources of ligands for studying ion channels and other pharmacological targets. Proteomic analyses of the soluble venom from the Mexican scorpion Vaejovis mexicanus smithi showed that it contains more than 200 different components. Among them, a 36-residue peptide with a molecular mass of 3864 Da (named Vm24) was shown to be a potent blocker of Kv1.3 of human lymphocytes (K(d) â¼ 3 pM). The three-dimensional solution structure of Vm24 was determined by nuclear magnetic resonance, showing the peptide folds into a distorted cystine-stabilized α/ß motif consisting of a single-turn α-helix and a three-stranded antiparallel ß-sheet, stabilized by four disulfide bridges. The disulfide pairs are formed between Cys6 and Cys26, Cys12 and Cys31, Cys16 and Cys33, and Cys21 and Cys36. Sequence analyses identified Vm24 as the first example of a new subfamily of α-type K(+) channel blockers (systematic number α-KTx 23.1). Comparison with other Kv1.3 blockers isolated from scorpions suggests a number of structural features that could explain the remarkable affinity and specificity of Vm24 toward Kv1.3 channels of lymphocytes.
Subject(s)
Kv1.3 Potassium Channel/antagonists & inhibitors , Potassium Channel Blockers/pharmacology , Scorpion Venoms/chemistry , Scorpion Venoms/pharmacology , T-Lymphocytes/drug effects , Amino Acid Motifs , Animals , Disulfides/chemistry , Drug Evaluation, Preclinical/methods , Humans , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Peptides/chemical synthesis , Peptides/chemistry , Peptides/pharmacology , Phylogeny , Protein Conformation , Scorpion Venoms/chemical synthesis , Scorpions/chemistryABSTRACT
A novel potassium channel blocker peptide was purified from the venom of the scorpion Centruroides suffusus suffusus by high-performance liquid chromatography and its amino acid sequence was completed by Edman degradation and mass spectrometry analysis. It contains 38 amino acid residues with a molecular weight of 4000.3Da, tightly folded by three disulfide bridges. This peptide, named Css20, was shown to block preferentially the currents of the voltage-dependent K+-channels Kv1.2 and Kv1.3. It did not affect several other ion channels tested at 10 nM concentration. Concentration-response curves of Css20 yielded an IC50 of 1.3 and 7.2 nM for Kv1.2- and Kv1.3-channels, respectively. Interestingly, despite the similar affinities for the two channels the association and dissociation rates of the toxin were much slower for Kv1.2, implying that different interactions may be involved in binding to the two channel types; an implication further supported by in silico docking analyses. Based on the primary structure of Css20, the systematic nomenclature proposed for this toxin is alpha-KTx 2.13.
Subject(s)
Kv1.2 Potassium Channel/antagonists & inhibitors , Kv1.3 Potassium Channel/antagonists & inhibitors , Potassium Channel Blockers/pharmacology , Scorpion Venoms/pharmacology , Amino Acid Sequence , Animals , COS Cells , Cell Line, Tumor , Cells, Cultured , Chlorocebus aethiops , Humans , Kv1.2 Potassium Channel/physiology , Kv1.3 Potassium Channel/physiology , Mice , Models, Molecular , Molecular Sequence Data , Potassium Channel Blockers/isolation & purification , Rats , Scorpion Venoms/chemistry , Scorpion Venoms/isolation & purification , ScorpionsABSTRACT
From the venom of the Mexican scorpion Centruroides elegans Thorell five peptides were isolated to homogeneity by chromatographic procedures and their full amino acid sequence was determined by automatic Edman degradation. They all belong to the Noxiustoxin subfamily of scorpion toxins and were given the systematic names alpha-KTx 2.8 to 2.12, with trivial names Ce1 to Ce5, respectively. They have 39 amino acid residues, except for Ce3 which has only 38, but all of them have three disulfide bridges, and have molecular weights of 4255, 4267, 4249, 4295 and 4255 atomic mass units, respectively for Ce1 to Ce5. The C-terminal residues of Ce2, Ce4 and Ce5 were found to be amidated. The electrophysiological assay (whole-cell patch-clamp) showed that out of the five peptides, Ce1 (alpha-KTx 2.8), Ce2 (alpha-KTX2.9) and Ce4 (alpha-KTx 2.11) were effective blockers of Kv1.3 channels of human T lymphocytes, whereas these peptides did not inhibit the Ca2+-activated K+ channels (IKCa1) of the same cells. The equilibrium dissociation constants of these peptides for Kv1.3 were 0.70, 0.25 and 0.98nM for Ce1, Ce2 and Ce4, respectively. Furthermore, toxins Ce1, Ce2 and Ce4 practically did not inhibit the related voltage gated Shaker K+ channels, and rKv2.1 channels of the Shab family. The high affinity blockage of Kv1.3 channels by these peptides and their selectivity for Kv1.3 over IKCa1 may have significance in the development of novel tools for suppressing the function of those T cell subsets whose proliferation critically depends on the activity of Kv1.3 channels.