A Synthetic 18-Norsteroid Distinguishes between Two Neuroactive Steroid Binding Sites on GABA(A) Receptors

Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Journal of Pharmacology and Experimental Therapeutics (Impact Factor: 3.97). 05/2010; 333(2):404-13. DOI: 10.1124/jpet.109.164079
Source: PubMed


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 (3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile (3alpha5beta-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 GABAA receptors. 3alpha5beta-18-norACN also inhibits the action of a full neurosteroid agonist, (3alpha,5alpha,17beta)-3-hydroxy-17-carbonitrile (3alpha5alphaACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5beta-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABAA receptors show that 3alpha5beta-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 GABAA receptors. 3alpha5beta-18-norACN also inhibits direct activation of GABAA receptors by 3alpha5alphaACN. 3alpha5beta-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 GABAA receptor.

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    • "[ 35 S]TBPS Binding. [ 35 S]TBPS binding assays were performed by using previously described methods (Evers et al., 2010). Aliquots of membranes (final protein concentration, 20 g/ml) were resuspended in 100 mM KCl, 10 mM potassium phosphate, pH 7.5, containing 1 to 2 nM [ 35 S]TBPS (60–100 Ci/mmol; PerkinElmer Life and Analytical Sciences, Waltham, MA) and 5-l aliquots of 6-AziP in dimethylsulfoxide solution (final steroid concentrations, 3 nM–30 M), in a total assay volume of 1 ml. "
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    ABSTRACT: Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5β)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-β3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-β3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-β3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the β3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3β,5β)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-β3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole β3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.
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    • "Nanomolar enhancement and micromolar displacement have been observed for GABA agonists, barbiturates and neurosteroids on cerebrocortical [ 35 S]t-butylbicyclophosphorothionate binding to GABA A receptors (Evers et al., 2010; Maksay and Simonyi, 1986). Nanomolar enhancement and micromolar displacement by neuroactive steroids have been correlated with potentiation and direct activation, respectively (Evers et al., 2010). Binding kinetics of convulsant radioligands has been used to characterize the in vitro efficacy and potency of allosteric modulators of GABA A receptors (Maksay, 1996). "
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