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

Oksana V Nekrasova,Valery N Novoseletsky,Alexandra L Primak,Alexey V Feofanov,Mikhail P Kirpichnikov,Olga V Geras’Kina,Anastasia A Ignatova,Ksenia S Kudryashova,Sergey A Yakimov,Alexander S Arseniev

doi:10.3390/toxins12120802

Oksana V Nekrasova, Valery N Novoseletsky + Show 8 more

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https://doi.org/10.3390/toxins12120802

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Abstract

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.

Highlights

Many of peptide toxins from animal venoms are highly specific ligands of potassium, sodium, calcium and acid-sensing channels, nicotinic acetylcholine, and NMDA receptors [1]
Interactions of green fluorescent protein (GFP)-L2-AgTx2 with the pore blocker binding sites of the Kv1.x (x = 1, 3, 6) channels were studied with the bioengineering systems based on E. coli spheroplasts that expose hybrid potassium channels KcsA-Kv1.x (x = 1, 3, 6) in their inner membrane
Data obtained with KcsA-Kv1.x (x = 1, 3, 6) for small organic compounds, peptide toxins, as well as fluorescent protein-tagged pore blockers are in agreement with the results of electrophysiological studies [12,16]

Summary

Introduction

Many of peptide toxins from animal venoms are highly specific ligands of potassium, sodium, calcium and acid-sensing channels, nicotinic acetylcholine, and NMDA receptors [1]. Genetically encoded fluorescent protein-scorpion toxin chimeras (FP-Tx), which are the polypeptide ligands of potassium voltage-gated Kv1-channels were recently developed [12]. These FP-Tx carry a fluorescent protein moiety at the N-terminus of the peptide ligand and exhibit high affinity to the target channels. The new type of fluorescent ligands is represented by two structurally different chimeras, namely green fluorescent protein (GFP) fused with peptide OSK1 (GFP-OSK1) and red fluorescent protein TagRFP fused with agitoxin 2 (RFP-AgTx2)

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