DIFFERENTIAL RESPONSES OF EXPRESSED RECOMBINANT HUMAN GAMMA-AMINOBUTYRIC ACIDA RECEPTORS TO NEUROSTEROIDS

Neuroactive steroids, in particular 3-alpha-hydroxypregnanes, are allosteric modulators of the gamma-aminobutyric acid(A) (GABA(A)) receptor. Regionally selective expression of receptor subunit subtypes may account for differential responsiveness of tissues to GABAergic inhibition and neurosteroid modulatory effects. The effect of 5-alpha-pregnan-3-alpha-ol-20-one (epiallopregnanolone) on heterotropic cooperativity on the GABA(A) receptor complex has been studied in three subtypes of expressed recombinant human receptors and in rat brain and spinal cord. Steroid potentiation of [H-3]flunitrazepam binding was greatest for the alpha-3-beta-1-gamma-2 receptor complex, whereas alpha-1-beta-1-gamma-2 and alpha-2-beta-1-gamma-2 complexes showed < 100% enhancement in binding. Previous studies suggest that the spinal cord is devoid of alpha-1, whereas cerebellum is rich in alpha-1 subunits. Correspondingly, a differential enhancement of [H-3]flunitrazepam binding in spinal cord (51%) versus cerebellum (28%) was also observed. The structure of neuroactive steroids is important in determining the extent of neuromodulatory activity. The 5-beta-pregnanes, 5-beta-pregnan-3-alpha-ol-20-one (epipregnanolone) and 5-beta-pregnan-3-alpha,21-diol-20-one (5-beta-tetrahydrodeoxycorticosterone), were both less potent than their corresponding 5-alpha derivatives. A 3-alpha-hydroxyl group is essential for neuromodulatory activity in the expressed receptors, as demonstrated by the observation that 5-alpha-pregnan-3-beta-ol-20-one (allopregnanolone) and 4-pregnen-3,20-dione (progesterone) were both inactive. The ability to screen synthetic molecules using expressed human receptors that selectively contain individual subunit subtype combinations may prove to be a powerful tool in the development of therapeutic agents that act as allosteric modulators of the GABA(A) receptor and other neurotransmitter receptors as well.