REGULATION OF ALLOSTERIC COUPLING AND FUNCTION OF STABLY EXPRESSED GAMMA-AMINOBUTYRIC-ACID (GABA)(A) RECEPTORS BY CHRONIC TREATMENT WITH GABA(A) AND BENZODIAZEPINE AGONISTS

Chronic treatments with drugs that stimulate or potentiate gamma-aminobutyric acid (GABA)(A) receptor function cause uncoupling of allosteric sites and downregulation of the GABA(A) receptors expressed in neurons. To study these effects on receptors having a defined subunit composition, we treated stably transfected mouse Ltk(-) cells (PA3 cells) with drugs known to uncouple GABA(A) receptors. Because dexamethasone controls the expression of bovine alpha-1, beta-1 and gamma 2L GABA(A) receptor subunit genes in PA3 cells, this expression system provides a way to study receptors in the absence of neuronal subunit gene promoters. We assayed binding of [H-3]flunitrazepam to measure allosteric coupling and uptake of Cl-36(-) to measure GABA(A) receptor function. Chronic (4 day) treatment of PA3 cells with muscimol, GABA or flunitrazepam reduced the GABA enhancement of binding of [H-3]flunitrazepam to PA3 cells. Chronic flurazepam or muscimol treatments also caused downregulation of benzodiazepine potentiation of muscimol-stimulated Cl-36(-) uptake. Chronic treatment with muscimol did not affect levels of subunit mRNAS and alpha 1- or beta 1-subunit protein of GABA(A) receptors and chronic flunitrazepam did not affect subunit mRNAs or all protein. We conclude that chronic drug treatments regulate allosteric coupling and function of GABA(A) receptors in stably transfected cells and this regulation cannot be attributed to decreases in the expression of receptor subunits or to expression of subunits other than alpha 1, beta 1 or gamma 2L.