Design of Far-red Fluorescent Kv1.3 Channel for Membrane Expression in Eukaryotic Cells and Its Interactions with Hongotoxin1

Orlov, Nikita A.; Ignatova, Anastasia A.; Nekrasova, Oksana V.; Kirpichnikov, Mikhaǐl P.; Feofanov, Аlexey V.

doi:10.1017/s1431927620017936

Cited by 2 publications

(1 citation statement)

References 3 publications

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“…In the following experiments we have verified, if AgTx2-L2-GFP, His6-AgTx2-L2-GFP and GFP-L1-AgTx2 can be used as fluorescent probes of Kv1.3 channels on the membrane of eukaryotic cells. Recently, we have designed a plasmid that encoded the fluorescently labeled human Kv1.3 channel (mKate2-hKv1.3-del) with enhanced presentation on the membrane of eukaryotic cells [ 20 ]. This far-red fluorescent Kv1.3 channel was demonstrated to be able to bind specifically peptide blockers (hongotoxin 1 and its fluorescently labeled derivative) after transient expression in eukaryotic cells.…”

Section: Resultsmentioning

confidence: 99%

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

Nekrasova

Primak

Ignatova

et al. 2020

Toxins

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Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

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Section: Resultsmentioning

confidence: 99%

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

Nekrasova

Primak

Ignatova

et al. 2020

Toxins

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

Orlov

Ignatova

Kryukova

et al. 2022

Toxins

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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.

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Design of Far-red Fluorescent Kv1.3 Channel for Membrane Expression in Eukaryotic Cells and Its Interactions with Hongotoxin1

Cited by 2 publications

References 3 publications

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site

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

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