A Fluorescent Protein-Tagged Scorpion Toxin with Picomolar Affinity for the Kv1.3 K <b> ⁺ </b> Channel

The fusion of fluorophores and peptide pore blockers generates valuable molecular tools to identify cells that express the voltage-gated potassium channel Kv1.3. Changes in the Kv1.3 expression are associated with numerous autoimmune and neurological disorders. In this study, Vm24, a potent and selective Kv1.3 inhibitor peptide isolated from scorpion venom, was genetically linked to mCherry, a red fluorescent protein characterized by high photostability, maturation, and tagging tolerance. The recombinant fusion protein (mCherry-Vm24) was expressed utilizing the <i>Pichia pastoris</i> expression system. Following protein purification using affinity chromatography and size-exclusion HPLC, the production of the correct fusion-peptide was confirmed using native PAGE, mass spectrometry, spectrophotometry, and electrophysiology. mCherry-Vm24 showed ∼90-fold lower, albeit still picomolar (<i>K</i> _d = 280 pM), affinity for Kv1.3 as compared the native Vm24, but retained its high Kv1.3 selectivity and practically irreversible binding to Kv1.3. Confocal microscopy and flow cytometry reported that CHO cells expressing hKv1.3 channels were specifically labeled with mCherry-Vm24. Biodistribution of the fusion protein in mice showed the highest fluorescence signal in the kidneys and a specific increase of the mCherry-Vm24 signal in the spleen over nonconjugated mCherry. These unique properties make the mCherry-Vm24 construct suitable for visualization of Kv1.3 both in vitro and in vivo.