Inhibitory Actions of Potentiating Neuroactive Steroids in the Human α1β3γ2L γ-Aminobutyric Acid Type A Receptor.

The γ-aminobutyric acid type A (GABA _A ) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3 β -hydroxy steroids inhibit, while 3 α -hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the α 1 β 3γ2L GABA _A receptor by the endogenous neurosteroid 3 α -hydroxy-5 β -pregnan-20-one (3 α 5 β P) and the synthetic neuroactive steroid 3 α -hydroxy-5 α -androstane-17 β -carbonitrile (ACN). The receptors were expressed in Xenopus oocytes. All experiments were done using two-electrode voltage-clamp electrophysiology. In the presence of low concentrations of GABA, 3 α 5 β P and ACN potentiate the GABA _A receptor. To reveal inhibition, we conducted the experiments on receptors activated by the combination of a saturating concentration of GABA and propofol to fully activate the receptors and mask potentiation, or on mutant receptors in which potentiation is ablated. Under these conditions, both steroids inhibited the receptor with IC ₅₀ s of ∼13 μ M and maximal inhibitory effects of 70-90%. Receptor inhibition by 3 α 5 β P was sensitive to substitution of the α 1 transmembrane domain (TM) 2-2' residue, previously shown to ablate inhibition by pregnenolone sulfate. However, results of coapplication studies and the apparent lack of state dependence suggest that pregnenolone sulfate and 3 α 5 β P inhibit the GABA _A receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the α 1 and β 3 subunits statistically significantly, albeit weakly and incompletely, reduced inhibition by 3 α 5 β P and ACN. SIGNIFICANCE STATEMENT: The heteromeric GABA _A receptor is inhibited by sulfated steroids and 3 β -hydroxy steroids, while 3 α -hydroxy steroids are considered to potentiate the receptor. We show here that 3 α -hydroxy steroids have inhibitory effects on the α 1 β 3γ2L receptor, which are observed in specific experimental settings and are expected to manifest under different physiological conditions.
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