Quantitative Confocal Microscopy Analysis as a Basis for Search and Study of Potassium Kv1.x Channel Blockers

摘要

Artificial KcsA-Kv1.x (x= 1, 3) receptors were recently designed by transferring the ligand-binding site from human Kv1.x voltage-gated potassium channels into corresponding domain of the bacterial KscA channel. We found that KcsA-Kv1.x receptors expressed in E. coli cells are embedded into cell membrane and bind ligands when the cells are transformed to spheroplasts. We supposed that E. coli spheroplasts with membrane-embedded KcsA-Kv1.x and fluorescently labeled ligand agitoxin-2 (R-AgTx2) can be used as elements of an advanced analytical system for search and study of Kv1-channel blockers. To realize this idea, special procedures were developed for measurement and quantitative treatment of fluorescence signals obtained from spheroplast membrane using confocal laser scanning microscopy (CLSM). The worked out analytical "mix and read"systems supported by quantitative CLSM analysis were demonstrated to be reliable alternative to radioligand and electrophysiology techniques in the search and study of selective Kv1.x channel blockers of high scientific and medical importance.