Abstract

This study reports the actions of enantiomer pairs of anesthetic steroids 3α5αP/ent-3α5αP and 3α5βP/ent-3α5βP as modulators of γ-aminobutyric acid (GABA)_A receptors and as anesthetics. The enantiomers of structurally related 17-carbonitrile analogs also are examined. These studies were aimed at 1) determining whether the steroid recognition site could distinguish between molecules differing in shape, but not other physical properties (enantioselectivity); 2) providing further insight into the structure-activity relationships of anesthetic steroids; and 3) determining whether modulation of GABA_A receptor function correlates with anesthetic potency for anesthetic steroid enantiomers. Stereoselective actions of the compounds were evaluated in four different bioassays: 1) noncompetitive displacement of [³⁵S]t-butylbicyclophosphorothionate from the picrotoxin site of GABA_A receptors present in rat brain membrane preparations; 2) modulation of GABA currents in cultured rat hippocampal neurons; 3) loss of righting reflex in tadpoles; and 4) loss of righting reflex in mice. The data indicate that 5α-reduced steroids, but not 5β-reduced steroids, show a high degree of enantioselectivity/enantiospecificity in their actions as modulators of GABA_A receptors and as anesthetics. For all compounds studied, the effects on GABA_A receptor function closely tracked with anesthetic effects. These data show that the anesthetic steroid recognition site is capable of distinguishing enantiomers, suggesting a protein-binding site of specific dimensions and shape. The results are compatible either with a structural model of the binding site that can accommodate 3α5αP, 3α5βP, andent-3α5βP, but not ent-3α5αP, or with two different binding sites for steroid anesthetics.