Recombinant Kv Channels at the Membrane of Escherichia coli Bind Specifically Agitoxin2
| Autor: | Anna I. Tagvei, E. F. Boldyreva, Oksana V. Nekrasova, Anna Nazarova, Alexey V. Feofanov, Mikhail P. Kirpichnikov, Yuliya V. Korolkova, Eugene V. Grishin, Alexander S. Arseniev, Anastasia A. Ignatova |
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| Rok vydání: | 2008 |
| Předmět: |
DNA
Bacterial Immunology Neuroscience (miscellaneous) KcsA potassium channel Scorpion Venoms Spheroplasts Plasma protein binding Biology medicine.disease_cause complex mixtures Agitoxin Cell membrane Escherichia coli medicine Animals Immunology and Allergy natural sciences Cloning Molecular Binding site Chromatography High Pressure Liquid DNA Primers Pharmacology Microscopy Confocal Reverse Transcriptase Polymerase Chain Reaction Cell Membrane Spheroplast Cell biology medicine.anatomical_structure nervous system Membrane protein Potassium Channels Voltage-Gated Streptomyces lividans Plasmids Protein Binding |
| Zdroj: | Journal of Neuroimmune Pharmacology. 4:83-91 |
| ISSN: | 1557-1904 1557-1890 |
| Popis: | 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) x 10(5) and (0.3 +/- 0.2) x 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. |
| Databáze: | OpenAIRE |
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