GFP-Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers.

Kristina R Denisova,Elena A Kryukova,Sergey A Yakimov,Oksana V Nekrasova,Mikhail P Kirpichnikov,Dmitry A Dolgikh,Nikita A Orlov,Alexey V Feofanov

doi:10.3390/ijms23031724

Abstract

Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the structure and functions of the voltage-gated potassium Kv1.3 channel, which is considered as a pharmacological target in the treatment of autoimmune and neurological disorders. We present Kv1.3 fluorescent ligand, GFP–MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3. Expression of the fluorescent ligand in E. coli cells resulted in correctly folded and functionally active GFP–MgTx with a yield of 30 mg per 1 L of culture. Complex of GFP–MgTx with the Kv1.3 binding site is reported to have the dissociation constant of 11 ± 2 nM. GFP–MgTx as a component of an analytical system based on the hybrid KcsA–Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3. GFP–MgTx can be used in screening and pre-selection of Kv1.3 channel blockers as potential drug candidates.

Highlights

Voltage-gated potassium channel Kv1.3 is involved in a variety of physiological processes ranging from regulating action potential threshold to migration, proliferation and hormone secretion
We present Kv1.3 fluorescent ligand, GFP–MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3
GFP–MgTx as a component of an analytical system based on the hybrid KcsA–Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3

Summary

Introduction

Voltage-gated potassium channel Kv1.3 is involved in a variety of physiological processes ranging from regulating action potential threshold to migration, proliferation and hormone secretion. Peptide toxins from scorpion venoms, which are distinguished by their high affinity and selectivity for the target potassium channels, are commonly used to study Kv1.3 channel activity. These peptides are Kv1.3 channel blockers, which interact with the external vestibule of the channel occluding the ion-conducting pathway of the pore domain [2]. Inhibiting Kv1.3 channels by peptide blockers results in pronounced physiological effects, providing an opportunity to design peptide-based drugs with high selectivity and low off-target effects [4]. A demand for peptide blockers with biased selectivity towards the target channel guides the ongoing studies of peptide toxins from the natural sources, as well as further characterization of their binding activities

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Conclusion