Neuroactive steroids modulate the function of ␥-aminobutyric acid, type A (GABA A ) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3␣,5-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA A receptors as evidenced by its ability to inhibit binding of [ 35 S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA A receptor subunits (␣ 1  2 ␥ 2 ). Certain endogenous pregnane steroids and their structural analogues are potent anesthetics in vertebrates (1, 2). These neuroactive steroids produce a rapid and reversible depression of the central nervous system indicating that their actions, unlike those of other steroid hormones, are not mediated by transcriptional regulation. In the 1980s it was demonstrated that neuroactive steroids could modulate the function of ␥-aminobutyric acid (GABA), 1 type A (GABA A ) receptors in the central nervous system (3-5). Low concentrations of the steroids potentiate the actions of GABA whereas higher concentrations directly open the GABA A receptor ion channel (6, 7). These observations led to the hypothesis that neuroactive steroids produce anesthesia by activating GABA A receptors and thus enhancing inhibitory synaptic transmission. The strong correlation between the ability of various neuroactive steroid analogues to modulate GABA A receptors and their ability to produce anesthesia strongly supports this hypothesis (8).The mechanism by which neuroactive steroids modulate GABA A receptor function remains unclear. In previous work we have tested the enantiomers of allopregnanolone (9) and pregnanolone (10) for their abilities to produce anesthesia and to modulate GABA A receptor function. These studies showed that both steroid anesthesia and steroid modulation of GABA A receptor function are highly enantioselective, particularly in the case of allopregnanolone. This indicates that neuroactive steroids most likely act via binding to specific recognition sites on the GABA A receptor protein complex, because the enantiomeric pairs have identical physical properties but mirror image shapes. Potentiation of GABA action by neuroactive steroids does not require any specific GABA A subunit (11) although the absence of the ␦ subunit does decrease the sensitivity of the receptor to neuroactive steroids (12). Radioligand binding studies and electrophysiological studies indicate that the putative neuroactive steroid binding sites are not identical to or overlapping with the identified binding sites for benzodiazepines (13), GABA (13) or picrotoxin (3) or to the putative binding site for barbiturates (13,14).A variety of anesthetic agents, including propofol, etomidate, benzodiazepines, and the halogenated alkanes and ethers, have also been shown to modulate GABA A receptor f...
In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [35 S]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␣,5,17)-3-hydroxy-18-norandrostane-17-carbonitrile (3␣5-18-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. 3␣5-18-norACN also inhibits the action of a full neurosteroid agonist, (3␣,5␣,17)-3-hydroxy-17-carbonitrile (3␣5␣ACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABA A receptors show that 3␣5-18-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. 3␣5-18-norACN also inhibits direct activation of GABA A receptors by 3␣5␣ACN. 3␣5-18-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.
Neuroactive steroids modulate the function of ␥-aminobutyric acid type A (GABA A ) receptors in brain; this is the presumed basis of their action as anesthetics. In a previous study using the neuroactive steroid analog, (3␣,5)-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity-labeling reagent, we showed that voltage-dependent anion channel-1 (VDAC-1) was the predominant protein labeled in brain. Antisera to VDAC-1 were shown to coimmunoprecipitate GABA A receptors, suggesting a functional relationship between steroid binding to VDAC-1 and modulation of GABA A receptor function. This study examines the contribution of steroid binding to VDAC proteins to modulation of GABA A receptor function and anesthesia. Photolabeling of 35-kDa protein with [ 3 H]6-AziP was reduced 85% in brain membranes prepared from VDAC-1-deficient mice but was unaffected by deficiency of VDAC-3. The photolabeled 35-kDa protein in membranes from VDAC-1-deficient mice was identified by two-dimensional electrophoresis and electrospray ionization-tandem mass spectrometry as VDAC-2. The absence of VDAC-1 or VDAC-3 had no effect on the ability of neuroactive steroids to modulate GABA A receptor function as evidenced by radioligand ([ 35 S] t-butylbicyclophosphorothionate) binding or by electrophysiological studies. Electrophysiological studies also showed that neuroactive steroids modulate GABA A receptor function normally in VDAC-2-deficient fibroblasts transfected with ␣ 1  2 ␥ 2 GABA A receptor subunits. Finally, the neuroactive steroid pregnanolone [(3␣,5)-3-hydroxypregnan-20-one] produced anesthesia (loss of righting reflex) in VDAC-1-and VDAC-3-deficient mice, and there was no difference in the recovery time between the VDAC-deficient mice and wild-type controls. These data indicate that neuroactive steroid binding to VDAC-1, -2, or -3 is unlikely to mediate GABA A receptor modulation or anesthesia.
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