N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site
| Autor: | Alexandra L Primak, Alexey V. Feofanov, V. N. Novoseletsky, S.A. Yakimov, Mikhail P. Kirpichnikov, Ksenia S Kudryashova, Oksana V. Nekrasova, Olga Geraskina, Alexander S. Arseniev, Anastasia A. Ignatova |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
agitoxin
Charybdotoxin Health Toxicology and Mutagenesis Green Fluorescent Proteins Kaliotoxin lcsh:Medicine Scorpion Venoms Peptide selective ligand Toxicology GFP complex mixtures Agitoxin Article Protein Structure Secondary Green fluorescent protein 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Potassium Channel Blockers Humans Amino Acid Sequence Binding site 030304 developmental biology chemistry.chemical_classification 0303 health sciences Binding Sites Kv1.3 Potassium Channel lcsh:R Fluorescence Potassium channel HEK293 Cells chemistry Biophysics 030217 neurology & neurosurgery potassium channel |
| Zdroj: | Toxins Toxins, Vol 12, Iss 802, p 802 (2020) Volume 12 Issue 12 |
| ISSN: | 2072-6651 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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