Oral 9th Venoms to Drugs 2026

Properties of the mCherry fluorescence protein-tagged scorpion toxin Vm24 with picomolar affinity for the Kv1.3 K+ channel (145701)

Jesus Borrego 1 , Muhammad Umair Naseem 1 , Amna S Al Olaimi 1 , Eva Korpos 2 , Arpad Szoor 1 , Gyula Jr. Batta 3 , Ildiko Godeny 4 , Gyorgy Panyi 1
  1. University of Debrecen, Dept. of Biophysics and Cell Biology, Debrecen, HUNGARY, Hungary
  2. HUN-REN-UD Cell Biology and Signaling Research Group, Debrecen, Hungary
  3. University of Debrecen, Debrecen, Hungary
  4. Pharmion Ltd, Debrecen, Hungary

Kv1.3 is a voltage-gated K+ channel belonging to the Shaker-related subfamily. Kv1.3 is highly expressed in macrophages, microglia, and effector memory cells (TEM cells), suggesting that Kv1.3 plays a crucial role in immune and inflammatory responses to several human diseases, where the expression of this channel is significantly elevated. The aim of the study was to generate a fusion of red fluorescence protein and a peptide blocker of Kv1.3 to identify cells that express Kv1.3.

Vm24, a potent (Kd = 3 pM) and selective Kv1.3 inhibitor peptide isolated from the scorpion Vaejovis mexicanus smithi venom, with an almost irreversible binding to the channel, was genetically linked to mCherry, characterized by high photostability, maturation and tagging tolerance. The recombinant protein was expressed utilizing the Pichia pastoris expression system, and its potential as a visualization tool for the Kv1.3 channel was evaluated using confocal microscopy, flow cytometry and ex vivo imaging of selected organs.

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 (Kd=280 pM), affinity for Kv1.3 as compared the native Vm24, but retained its high Kv1.3 selectivity against Kv1.1, Kv1.2 and KCa3.1, 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: pre-incubation of the Kv1.3-transfected CHO with excess of wild-type Vm24 prevented the subsequent labeling of the cells 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 non-conjugated mCherry. These unique properties make the mCherry-Vm24 construct suitable for visualization of Kv1.3 both in vitro and in vivo.