BENZODIAZEPINE MODULATION OF RECOMBINANT ALPHA-1-BETA-3-GAMMA-2 GABA(A) RECEPTOR FUNCTION EFFICACY DETERMINATION USING THE CYTOSENSOR MICROPHYSIOMETER

gamma-Aminobutyric acid (GABA) dose dependently increased extracellula r acidification rate in Ltk(-) cells stably expressing human recombina nt alpha 1 beta 3 gamma 2 GABAA receptors but had no effect in non-tra nsfected controls. Cells seeded at 1 x 10(5) cells/cup, with 4-5 days induction, had basal acidification rates of 105 +/- 2 mu Vs(-1) at 37 degrees C (mean +/- standard error of mean, n = 37). GABA responses ha d a characteristic time-course with an initial alkalinisation followed by a peak of acidification, which was optimized by increasing agonist exposure from 15 s to 25-30 s. The maximum concentration of GABA test ed (100 mu M) produced a 40 +/- 2% increase over basal acidification r ate (n = 3), with an EC50 of 15.5 mu M and a Hill slope of 1.5. Respon ses were specifically antagonized by bicuculline and could be modulate d by benzodiazepine Ligands with varying efficacies. Full benzodiazepi ne agonists flunitrazepam(1 mu M) and zolpidem (10 mu M) significantly potentiated the response to 10 mu M GABA by 124 +/- 15% (rt = 7) and 117 +/- 23% (n = 3), respectively. The partial agonist bretazenil (100 nM) produced a 45 +/- 13% (n = 3) potentiation whilst the inverse ago nist DMCM (10 mu M) (methyl ,7-dimethoxy-4-ethyl-beta-carboline-3-carb oxylate) inhibited the response to 20 mu M GABA by 53 +/- 5%. The micr ophysiometer offers an alternative functional measure for GABA, recept ors with the sensitivity to measure subtle modulatory effects of benzo diazepine site ligands and to determine their relative efficacy. (C) 1 998 Elsevier Science B.V. All rights reserved.