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

Abstract

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.

Variability of Potassium Channel Blockers in Mesobuthus eupeus Scorpion Venom with Focus on Kv1.1

“…Kv1 channels were studied using flow cytometry (60), single molecule fluorescent microscopy (61), and the analysis of membrane potential changes using fluorescent dyes and fluorescence readers (62). Recently, a convenient fluorescent screening system based on KcsA-Kv1.3 hybrid receptors was developed and applied successfully to screen Orthochirus scrobiculosus venom for peptide toxins, which specifically target the pore region of Kv1.3 (36,38). This system, which represents an efficient, robust, and rapid binding assay, is a powerful alternative to radioligand and patch clamp techniques.…”

“…KcsA-Kv1.1 is readily expressed in E. coli and forms tetramers in the plasma membrane with the outer pore vestibule of the channel oriented extracellularly. Spheroplasts prepared from E. coli cells with membrane-embedded KcsA-Kv1.1 bind specifically fluorescently labeled agitoxin-2 (R-AgTx2) (38). Therefore, RAgTx2 and spheroplasts bearing KcsA-Kv1.1 can be used as components of a fluorescent system for investigation of Kv1.1 ligands just as it was realized recently for the KcsA-Kv1.3 system (36).…”

“…coli BL21(DE3) cells that express KcsA-Kv1.1 proteins in the inner membrane were obtained as described earlier (37,38). Cell cultivation and preparation of spheroplasts were performed as described elsewhere (36).…”

Studying of Membrane Localization of Recombinant Potassium Channels in E.coli

“…For this, we used a procedure for binding KcsA-Kv1.3 to a fluorescently labeled peptide toxin - agitoxin2, on the entire surface of E. coli cells [26]. Agitoxin (AgTx2), a 38-a.a. peptide from the venom of a scorpion, Leiurus quinquestriatus, is an effective blocker of the Kv1.3 channel [27].…”

Nanobiology for the Pharmacology of Cellular Ion Channels

“…The authors present the recent data about domain organization of eukaryotic potassium voltage-gated ion channels and discuss the interaction between the domains and the corresponding conformational changes upon activation of the channel. This theme is continued in a study by Nekrasova et al (2008), which reports on a new bacterial cell-based system, suitable for study of interactions between ligands and ligand-binding sites of eukaryotic Kv1.3 and Kv1.1 channels. This new system may be useful for screening of ligands to membrane-embedded pharmaceutical targets.…”