Recombinant Kv Channels at the Membrane of Escherichia coli Bind Specifically Agitoxin2

• Published in: Journal of neuroimmune pharmacology Vol. 4; no. 1; pp. 83 - 91
• Main Authors: Nekrasova, Oksana V., Ignatova, Anastasia A., Nazarova, Anna I., Feofanov, Alexey V., Korolkova, Yuliya V., Boldyreva, Elena F., Tagvei, Anna I., Grishin, Eugene V., Arseniev, Alexander S., Kirpichnikov, Mikhail P.
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.03.2009; Springer Nature B.V
• Subjects: Animals; Binding sites; Biomedical and Life Sciences; Biomedicine; Cell Biology; Cell Membrane - metabolism; Chromatography, High Pressure Liquid; Cloning, Molecular; DNA Primers; DNA, Bacterial - genetics; E coli; Escherichia coli; Escherichia coli - metabolism; Immunology; Ligands; Microscopy, Confocal; Neurosciences; Original Article; Pharmacology/Toxicology; Plasmids - genetics; Potassium Channels, Voltage-Gated - genetics; Potassium Channels, Voltage-Gated - metabolism; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Scorpion Venoms - metabolism; Spheroplasts - metabolism; Streptomyces lividans - genetics; Virology; agitoxin 2; fluorescence; membrane protein; cell-based; confocal; Kv channel; ligand binding
• ISSN: 1557-1890; 1557-1904
• DOI: 10.1007/s11481-008-9116-4

Potassium voltage-gated channels (Kv) are considered as molecular targets in a number of serious neuronal, immune, and cardiac disorders. Search for efficient low-molecular weight modulators of Kv channel function provides a basis for the development of an appropriate therapy for various Kv-mediated diseases. We report here on a new bacterial cell-based system, which is suitable for study of interactions between ligands and ligand-binding sites of eukaryotic Kv1.3 and Kv1.1 channels. To create this system, high-level expression of KcsA-Kv1.3 and KcsA-Kv1.1 hybrid proteins (ligand-binding sites of Kv1.3 or Kv1.1 fused with prokaryotic KcsA potassium channel) was achieved in the plasma membrane of Escherichia coli . An efficient procedure of E. coli conversion to intact spheroplasts was developed. We demonstrate that fluorescently labeled agitoxin 2 binds specifically to high-affinity and lower-affinity sites of KcsA-Kv1.3 and KcsA-Kv1.1, respectively, at the membrane of spheroplasts. Number of binding sites per cell is estimated to be (1.0 ± 0.6) ×10 5 and (0.3 ± 0.2) ×10 5 for KcsA-Kv1.3- and KcsA-Kv1.1-presenting cells, respectively, that allows reliable detection of ligand–receptor interactions by confocal laser scanning microscopy. This bacterial cell-based system is intended for screening of ligands to membrane-embedded pharmaceutical targets.