3α-hydroxy-3β-trifluoromethyl-5α-pregnan-20-one (Co 2-1970): A partial agonist at the neuroactive steroid site of the γ-Aminobutyric acid_A receptor

Neuroactive steroids bind to a unique site on the γ-aminobutyric acid_A (GABA_A) receptor complex and allosterically modulate the binding of convulsant ([³⁵S]t-butylbicyclophosphorothionate, [³⁵S]TBPS), GABA ([³H]muscimol), and benzodiazepine ([³H]flunitrazepam) site ligands. In rat cortical membranes, 3α-hydroxy-5α-pregnan-20-one (3α,5α-P) is a full agonist at the steroid site, inhibiting 96% of specific [³⁵S]TBPS binding and enhancing [³H]flunitrazepam and [³H]muscimol binding 95% and 69% above control levels, respectively. In contrast, the synthetic steroid 3α-hydroxy-3β-trifluoromethyl-5α-pregnan-20-one (Co 2-1970) has limited efficacy for modulating the binding of [³⁵S]TBPS (44% inhibition), [³H]flunitrazepam (41% enhancement), and [³H]muscimol (<10% enhancement). In competition experiments, Co 2-1970 (10 μM) reduced the apparent potency of 3α,5α-P by 7-17-fold for modulating the binding of these radioligands in rat cortical membranes, suggesting that it has partial agonist properties. Because cortical membranes contain a heterogeneous population of receptors, Co 2-1970 was examined in recombinant GABA_A receptors stably expressed in human embryonic kidney 293 cells. Co 2-1970 inhibited [³⁵S]TBPS binding with limited efficacy (39-65% inhibition) in the five receptor combinations examined and, at 10 μM, reduced the apparent potency of 3α,5α-P 57-fold for inhibiting [³⁵S]TBPS binding to α1β1γ2L receptors. To verify these findings functionally, the effects of 3α,5α-P and Co 2-1970 were examined electrophysiologically in Xenopus oocytes expressing α1β1γ2L receptors. Co 2-1970 showed limited efficacy potentiation of GABA-evoked chloride currents relative to 3α,5α-P (28% and 86% of the GABA maximum current, respectively). Moreover, Co 2-1970 produced a concentration-dependent antagonism of the 3α,5α-P-induced potentiation that was associated with a reduction in the apparent affinity of 3α,5α-P (11-fold at 10 μM Co 2-1970). Taken together, these data indicate that Co 2-1970 is a partial agonist at the neuroactive steroid site associated with GABA_A receptors.