INTERACTIONS OF THE DYE EVANS-BLUE AND GYKI-52466, A 2,3-BENZODIAZEPINE, WITH LPHA-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLEPROPIONIC ACID RECEPTORS IN CULTURED RAT CORTICAL-NEURONS - ELECTROPHYSIOLOGICAL EVIDENCE FOR AT LEAST 2 DIFFERENT BINDING-SITES FOR NONCOMPETITIVE ANTAGONISTS

The effects of the dye Evans Blue and GYKI 52466, a 2,3-benzodiazepine , on lpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/ka inate receptors in primary cultured rat cortical neurons were investig ated using the patch-clamp technique. Evans Blue and GYKI 52466 reduce d the currents induced by the application of 100 mu M kainate with IC5 0 values of 10.6 +/- 1.4 mu M and 12.1 +/- 0.4 mu M, respectively. In contrast to the similar potencies of the two compounds, their kinetics of block were quite different with those of Evans Blue being more com plex. The on-, as well as the off-reaction of the block by GYKI 52466 could be described by single exponential functions, whereas two differ ent time-constants for binding and one time-constant for the unbinding of Evans Blue were found. The block of AMPA receptors by Evans Blue w as not completely reversible under the experimental conditions applied in this study. GYKI 52466 was not able to augment the recovery after inhibiting AMPA receptors with Evans Blue. Moreover, preapplication of a high concentration of GYKI 52466 did not prevent the inhibition of AMPA receptors by Evans Blue. We therefore conclude that GYKI 52466 an d Evans Blue bind to two different sites at AMPA receptors in primary cultured cortical neurons.