ROXY-3-BETA-TRIFLUOROMETHYL-5-ALPHA-PREGNAN-20-ONE (CO-2-1970) - A PARTIAL AGONIST AT THE NEUROACTIVE STEROID SITE OF THE GAMMA-AMINOBUTYRIC-ACID(A) RECEPTOR

Neuroactive steroids bind to a unique site on the gamma-aminobutyric a cid, (GABA(A) receptor complex and allosterically modulate the binding of convulsant ([S-35]t-butylbicyclophosphorothionate, [S-35]TBPS), GA BA ([H-3]muscimol), and benzodiazepine ([H-3]flunitrazepam) site ligan ds. In rat cortical membranes, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) is a full agonist at the steroid site, inhibiting 96% of specific [S-35]TBPS binding and enhancing [H-3]flunitrazepam an d [H-3]muscimol binding 95% and 69% above control levels, respectively . In contrast, the synthetic steroid 3 alpha-hydroxy-3 beta-trifluorom ethyl-5 alpha-pregnan-20-one (Co 2-1970) has limited efficacy for modu lating the binding of [S-35]TBPS (44% inhibition), [H-3]flunitrazepam (41% enhancement), and [H-3]muscimol (<10% enhancement). In competitio n experiments, Co 2-1970 (10 mu M) reduced the apparent potency of 3 a lpha,5 alpha-P by 7-17-fold for modulating the binding of these radiol igands in rat cortical membranes, suggesting that it has partial agoni st properties. Because cortical membranes contain a heterogeneous popu lation of receptors, Co 2-1970 was examined in recombinant GABA(A) rec eptors stably expressed in human embryonic kidney 293 cells. Co 2-1970 inhibited [S-35]TBPS binding with limited efficacy (39-65% inhibition ) in the five receptor combinations examined and, at 10 mu M, reduced the apparent potency of 3 alpha,5 alpha-P 57-fold for inhibiting [S-35 ]TBPS binding to alpha 1 beta 1 gamma 2L receptors. To verify these fi ndings functionally, the effects of 3 alpha,5 alpha-P and Co 2-1970 we re examined electrophysiologically in Xenopus oocytes expressing alpha 1 beta 1 gamma 2L receptors. Co 2-1970 showed limited efficacy potent iation of GABA-evoked chloride currents relative to 3 alpha,5 alpha-P (28% and 86% of the GABA maximum current, respectively). Moreover, Co 2-1970 produced a concentration-dependent antagonism of the 3 alpha,5 alpha-P-induced potentiation that was associated with a reduction in t he apparent affinity of 3 alpha,5 alpha-P (11-fold at 10 mu M Co 2-197 0). Taken together, these data indicate that Co 2-1970 is a partial ag onist at the neuroactive steroid site associated with GABA(A) receptor s.