Fluorescent protein-scorpion toxin chimera is a convenient molecular tool for studies of potassium channels.

Alexey I. Kuzmenkov,Kseniya S. Kudryashova,Alexey V. Stepanov,Steve Peigneur,Jan Tytgat,Alexander A. Vassilevski,Eugene V. Grishin,Alexey V. Feofanov,Mikhail P. Kirpichnikov,Oksana V. Nekrasova

doi:10.1038/srep33314

Abstract

Ion channels play a central role in a host of physiological and pathological processes and are the second largest target for existing drugs. There is an increasing need for reliable tools to detect and visualize particular ion channels, but existing solutions suffer from a number of limitations such as high price, poor specificity, and complicated protocols. As an alternative, we produced recombinant chimeric constructs (FP-Tx) consisting of fluorescent proteins (FP) fused with potassium channel toxins from scorpion venom (Tx). In particular, we used two FP, eGFP and TagRFP, and two Tx, OSK1 and AgTx2, to create eGFP-OSK1 and RFP-AgTx2. We show that these chimeras largely retain the high affinity of natural toxins and display selectivity to particular ion channel subtypes. FP-Tx are displaced by other potassium channel blockers and can be used as an imaging tool in ion channel ligand screening setups. We believe FP-Tx chimeras represent a new efficient molecular tool for neurobiology.

Highlights

Previous alternatives to channel labeling were to use either chemically modified toxins or antibodies. Both possibilities suffered from serious drawbacks, such as complicated synthesis and high price, and poor selectivity and need to work with fixed samples
We rationalize that our Fluorescent protein-scorpion toxin (FP-Tx) tools are easy to use and can be produced just by the recombinant technique, avoiding any chemical modification
The binding is specific and reversible: FP-Tx are displaced from the complexes by pore blockers of KV including OSK1, AgTx2, and TEA

Summary

Introduction

Such hybrids bind pore blockers of KV, and the dissociation constants of these complexes are in good agreement with the constants estimated from electrophysiological measurements on native KV channels. EGFP-OSK1 and RFP-AgTx2 bind to KcsA-Kv1.1 with dissociation constants (mean ±S.E., n = 3) of 3.2 ± 1.1 and 0.4 ± 0.1 nM, respectively.

Results

Conclusion