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

Jon E. Hawkinson, John A. Drewe, Catherine L. Kimbrough, Jie Sheng Chen, Derk J. Hogenkamp, Nancy C. Lan, Kelvin W. Gee, Ke Zhong Shen, Edward R. Whittemore, Richard M. Woodward

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Abstract

Neuroactive steroids bind to a unique site on the γ-aminobutyric acidA (GABAA) 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 GABAA 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 GABAA receptors.

Original languageEnglish (US)
Pages (from-to)897-906
Number of pages10
JournalMolecular pharmacology
Volume49
Issue number5
StatePublished - May 1 1996

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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    Hawkinson, J. E., Drewe, J. A., Kimbrough, C. L., Chen, J. S., Hogenkamp, D. J., Lan, N. C., Gee, K. W., Shen, K. Z., Whittemore, E. R., & Woodward, R. M. (1996). 3α-hydroxy-3β-trifluoromethyl-5α-pregnan-20-one (Co 2-1970): A partial agonist at the neuroactive steroid site of the γ-Aminobutyric acidA receptor. Molecular pharmacology, 49(5), 897-906.