Agitoxin 2

Recombinant expression of margatoxin and agitoxin-2 in Pichia pastoris: an efficient method for production of KV1.3 channel blockers.[Pubmed:23300835]

PLoS One. 2012;7(12):e52965.

The K(v)1.3 voltage-gated potassium channel regulates membrane potential and calcium signaling in human effector memory T cells that are key mediators of autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. Thus, subtype-specific K(v)1.3 blockers have potential for treatment of autoimmune diseases. Several K(v)1.3 channel blockers have been characterized from scorpion venom, all of which have an alpha/beta scaffold stabilized by 3-4 intramolecular disulfide bridges. Chemical synthesis is commonly used for producing these disulfide-rich peptides but this approach is time consuming and not cost effective for production of mutants, fusion proteins, fluorescently tagged toxins, or isotopically labelled peptides for NMR studies. Recombinant production of K(v)1.3 blockers in the cytoplasm of E. coli generally necessitates oxidative refolding of the peptides in order to form their native disulfide architecture. An alternative approach that avoids the need for refolding is expression of peptides in the periplasm of E. coli but this often produces low yields. Thus, we developed an efficient Pichia pastoris expression system for production of K(v)1.3 blockers using margatoxin (MgTx) and agitoxin-2 (AgTx2) as prototypic examples. The Pichia system enabled these toxins to be obtained in high yield (12-18 mg/L). NMR experiments revealed that the recombinant toxins adopt their native fold without the need for refolding, and electrophysiological recordings demonstrated that they are almost equipotent with the native toxins in blocking K(V)1.3 (IC(50) values of 201+/-39 pM and 97 +/- 3 pM for recombinant AgTx2 and MgTx, respectively). Furthermore, both recombinant toxins inhibited T-lymphocyte proliferation. A MgTx mutant in which the key pharmacophore residue K28 was mutated to alanine was ineffective at blocking K(V)1.3 and it failed to inhibit T-lymphocyte proliferation. Thus, the approach described here provides an efficient method of producing toxin mutants with a view to engineering K(v)1.3 blockers with therapeutic potential.

Solution structure of the potassium channel inhibitor agitoxin 2: caliper for probing channel geometry.[Pubmed:8520473]

Protein Sci. 1995 Aug;4(8):1478-89.

The structure of the potassium channel blocker Agitoxin 2 was solved by solution NMR methods. The structure consists of a triple-stranded antiparallel beta-sheet and a single helix covering one face of the beta-sheet. The cysteine side chains connecting the beta-sheet and the helix form the core of the molecule. One edge of the beta-sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, Agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface-exposed residues, Arg24, Lys27, and Arg31 of the beta-sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K+ channel. The sequential and spatial locations of Arg24 and Arg31 are not conserved among the homologous toxins. Knowledge on the details of the channel-binding sites of Agitoxin 2 formed a basis for site-directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel-toxin interactions.

Agitoxin footprinting the shaker potassium channel pore.[Pubmed:8789954]

Neuron. 1996 Feb;16(2):399-406.

In voltage-dependent K+ channels, each of the four identical subunits contributes one pore loop to the central ion selectivity unit at the interface between the subunits. The pore loop is also the target for scorpion venom peptide inhibitors. These inhibitors bind at the pore entryway between the four subunits and can assume any one of four orientations. The orientations become distinguishable only if the binding site symmetry is disrupted. We have used mutagenesis and site-directed chemical modification to alter pore loop amino acids in either one or four subunits. The effects of these alterations on inhibitor affinity define the eccentricity of amino acids in the pore entryway and imply a different secondary structure for the amino and carboxyl ends of the pore loop.

Purification and characterization of three inhibitors of voltage-dependent K+ channels from Leiurus quinquestriatus var. hebraeus venom.[Pubmed:8204618]

Biochemistry. 1994 Jun 7;33(22):6834-9.

Three new toxins from the venom of the scorpion Leiurus quinquestriatus var. hebraeus have been identified on the basis of their ability to block the Shaker K+ channel. These toxins have been purified using HPLC techniques and characterized as 38 amino acid peptides by mass spectroscopy, amino acid analysis, and sequence determination. Their chemical identity was confirmed by producing fully functional synthetic toxins using recombinant methods. These peptides are potent inhibitors of the Shaker K+ channel (Kd < 1 nM) as well as the mammalian homologues of Shaker. They are related to other previously described K+ channel toxins, but form a new subclass within the larger family of K+ channel inhibitors derived from scorpion venom. We have named these toxins agitoxin 1, 2, and 3, respectively.