SCREENING OF RECEPTOR ANTAGONISTS USING AGONIST-ACTIVATED PATCH-CLAMPDETECTION IN CHEMICAL SEPARATIONS

We present a capillary electrophoresis-patch clamp detection system op timized for screening of antagonists and inhibitors of ligand-gated io n channels. In this system, highly selective receptor agonists are del ivered 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 f unctionality is simply achieved by selection of an appropriate agonist . The antagonists are fractionated in the same electrophoresis capilla ry and inhibit the agonist-evoked response, resulting in transiently d ecreased steady-state transmembrane currents, Specifically, a mixture containing 6-cyano-7-nitroquinoxaline-2,3-dione, that reversibly block s pha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate recep tors, and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, a broad-sp ectrum glutamate receptor antagonist, were separated and detected by k ainate-activated patch-clamped interneurons freshly dissociated from r at 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-aspar tate and the co-agonist glycine, The presented method offers new possi bilities for drug screening and for identifying endogenous receptor an tagonists and to determine their mode of action on any ionotropic rece ptor system of interest.