Dataset Information

Combining mKate2-Kv1.3 Channel and Atto488-Hongotoxin for the Studies of Peptide Pore Blockers on Living Eukaryotic Cells.

ABSTRACT: The voltage-gated potassium Kv1.3 channel is an essential component of vital cellular processes which is also involved in the pathogenesis of some autoimmune, neuroinflammatory and oncological diseases. Pore blockers of the Kv1.3 channel are considered as potential drugs and are used to study Kv1 channels' structure and functions. Screening and study of the blockers require the assessment of their ability to bind the channel. Expanding the variety of methods used for this, we report on the development of the fluorescent competitive binding assay for measuring affinities of pore blockers to Kv1.3 at the membrane of mammalian cells. The assay constituents are hongotoxin 1 conjugated with Atto488, fluorescent mKate2-tagged Kv1.3 channel, which was designed to improve membrane expression of the channel in mammalian cells, confocal microscopy, and a special protocol of image processing. The assay is implemented in the "mix and measure", format and allows the screening of Kv1.3 blockers, such as peptide toxins, that bind to the extracellular vestibule of the K⁺-conducting pore, and analyzing their affinity.

SUBMITTER: Orlov NA

PROVIDER: S-EPMC9780825 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Combining mKate2-Kv1.3 Channel and Atto488-Hongotoxin for the Studies of Peptide Pore Blockers on Living Eukaryotic Cells.

Orlov Nikita A NA Ignatova Anastasia A AA Kryukova Elena V EV Yakimov Sergey A SA Kirpichnikov Mikhail P MP Nekrasova Oksana V OV Feofanov Alexey V AV

Toxins 20221205 12

The voltage-gated potassium Kv1.3 channel is an essential component of vital cellular processes which is also involved in the pathogenesis of some autoimmune, neuroinflammatory and oncological diseases. Pore blockers of the Kv1.3 channel are considered as potential drugs and are used to study Kv1 channels' structure and functions. Screening and study of the blockers require the assessment of their ability to bind the channel. Expanding the variety of methods used for this, we report on the devel ...[more]

PMID: 36548755

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Project description: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 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 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.

Dataset Information

Combining mKate2-Kv1.3 Channel and Atto488-Hongotoxin for the Studies of Peptide Pore Blockers on Living Eukaryotic Cells.

Publications

Combining mKate2-Kv1.3 Channel and Atto488-Hongotoxin for the Studies of Peptide Pore Blockers on Living Eukaryotic Cells.

Similar Datasets

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