Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of KV1.3 Channel Blockers

Abstract

The Kv1.3 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 Kv1.3 blockers have potential for treatment of autoimmune diseases. Several Kv1.3 channel blockers have been characterized from scorpion venom, all of which have an α/β 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 Kv1.3 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 Kv1.3 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 KV1.3 (IC50 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 KV1.3 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 Kv1.3 blockers with therapeutic potential.

Margatoxin is a non-selective inhibitor of human Kv1.3 K+ channels

“…When MgTx was isolated and its high affinity interaction with Kv1.3 was precisely characterized it was only tested on a limited number of channels excluding Kv1.1, Kv1.2 and Kv1.4 channels, those with high sequence homology to Kv1.3 (Garcia-Calvo et al, 1993). Later the authors who described MgTx and other workgroups refer to the peptide as potent blocker of Kv1.1, Kv1.2 and Kv1.3 channels (Anangi et al, 2012;Koch et al, 1997;SuarezKurtz et al, 1999;Vianna-Jorge et al, 2003) whereas the references do not describe or contain any information about the interaction of MgTx and the Kv1.1 or Kv1.2 channels. Without the precise characterization of the selectivity profile of MgTx, results from radioactively labeled MgTx binding assays, observed potassium current block or other biological effects of the peptide may not be considered as a direct proof of Kv1.3 expression.…”

Margatoxin‐bound quantum dots as a novel inhibitor of the voltage‐gated ion channel Kv1.3

“…When Lys28 is mutated to Ala, MgTx's ability to inhibit Kv1.3 is abolished (Anangi et al . ). Although we had no direct control over which basic residues were targeted during the conjugation to QDs, our electrophysiological data demonstrated that chemical conjugation through this methodology did not prevent MgTx from inhibiting Kv1.3, thus it is highly likely that our conjugation did not significantly affect the surface residues required to form favorable electrostatic interactions with the channel.…”

Binding Modes of Two Scorpion Toxins to the Voltage-Gated Potassium Channel Kv1.3 Revealed from Molecular Dynamics

“…Of all the scorpion venom peptides that have been isolated, margatoxin (MgTx) and hongotoxin (HgTx) are among the most potent for Kv1.3. Both toxins inhibit Kv1.3 with picomolar affinities [ 8 , 9 ], but they are equally potent for several other Kv channel isoforms such as Kv1.1 and Kv1.2, which are closely related to Kv1.3 [ 8 , 10 ]. For the pharmacological development of MgTx and HgTx the specificity of the toxins for Kv1.3 needs to be improved.…”