A synthetic 18-norsteroid distinguishes between two neuroactive steroid binding sites on GABA_A receptors

In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [³⁵S]t-butylbicyclophosphorothionate in a biphasic manner, with enhancement of binding at low concentrations (site NS1) and inhibition at higher concentrations (site NS2). In the current study, compound (3 α,5 β,17 β)-3-hydroxy-18-norandrostane-17- carbonitrile (3α5β-18-norACN), an 18-norsteroid, is shown to be a full agonist at site NS1 and a weak partial agonist at site NS2 in both rat brain membranes and heterologously expressed GABA_A receptors. 3α5β-18-norACN also inhibits the action of a full neurosteroid agonist, (3α,5α,17β)-3-hydroxy-17-carbonitrile (3α5αACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5β-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABA_A receptors show that 3α5α-18-norACN potently and efficaciously potentiates the GABA currents elicited by low concentrations of GABA but that it has low efficacy as a direct activator of GABA_A receptors. 3α5α-18-norACN also inhibits direct activation of GABA_A receptors by 3α5αACN. 3α5α-18-norACN also produces loss of righting reflex in tadpoles and mice, indicating that action at NS1 is sufficient to mediate the sedative effects of neurosteroids. These data provide insight into the pharmacophore required for neurosteroid efficacy at the NS2 site and may prove useful in the development of selective agonists and antagonists for neurosteroid sites on the GABA_A receptor.