Screening of receptor antagonists using agonist-activated patch clamp detection in chemical separations.
We present a capillary electrophoresis-patch clamp detection system optimized for screening of antagonists and inhibitors of ligand-gated ion channels. In this system, highly selective receptor agonists are delivered through the electrophoresis capillary to the cell surface where they continuously activate a receptor, resulting in increased steady-state transmembrane currents. Thus, receptor selection and biosensor functionality is simply achieved by selection of an appropriate agonist. The antagonists are fractionated in the same electrophoresis capillary and inhibit the agonist-evoked response, resulting in transiently decreased steady-state transmembrane currents. Specifically, a mixture containing 6-cyano-7-nitroquinoxaline-2,3-dione, that reversibly blocks alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate receptors, and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, a broad-spectrum glutamate receptor antagonist, were separated and detected by kainate-activated patch-clamped interneurons freshly dissociated from rat brain olfactory bulb. In addition, Mg2+ that reversibly blocks the N-methyl-D-aspartate receptor in a voltage-dependent way was detected using the same cell detector system when activated by N-methyl-D-aspartate and the co-agonist glycine. The presented method offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor system of interest.
Department of Anatomy and Cell Biology, Göteborg University, Sweden.
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Jardemark
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Farre
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Jacobson
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Zare
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Orwar
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000032:analysis*; D000818:Animals; D001667:Binding, Competitive; D019075:Electrophoresis, Capillary / Q000295:instrumentation; D018690:Excitatory Amino Acid Agonists / Q000032:analysis / Q000302:isolation & purification; D018691:Excitatory Amino Acid Antagonists / Q000032:analysis*; D005998:Glycine / Q000494:pharmacology; D007473:Ion Channels / Q000037:antagonists & inhibitors*; D008433:Mathematics; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism; D009830:Olfactory Bulb / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011810:Quinoxalines / Q000032:analysis*; D051381:Rats; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors*
D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D007473:Ion Channels; D011810:Quinoxalines; D011956:Receptors, Cell Surface; D016202:N-Methylaspartate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; C057665:6,7-dichloro-3-hydroxy-2-quinoxalinecarboxylic acid; D005998:Glycine