TY - JOUR
T1 - Inhibitory Actions of Potentiating Neuroactive Steroids in the Human a1b3g2L g-Aminobutyric Acid Type A Receptor
AU - Pierce, Spencer R.
AU - Germann, Allison L.
AU - Covey, Douglas F.
AU - Evers, Alex S.
AU - Steinbach, Joe Henry
AU - Akk, Gustav
N1 - Publisher Copyright:
© 2024 by The American Society for Pharmacology and Experimental Therapeutics.
PY - 2024/11/1
Y1 - 2024/11/1
N2 - The g-aminobutyric acid type A (GABAA) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3b-hydroxy steroids inhibit, while 3a-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the a1b3g2L GABAA receptor by the endogenous neurosteroid 3a-hydroxy-5b-pregnan-20-one (3a5bP) and the synthetic neuroactive steroid 3a-hydroxy-5a-androstane-17b-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, 3a5bP and ACN potentiate the GABAA 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 IC50s of 13 lM and maximal inhibitory effects of 70-90%. Receptor inhibition by 3a5bP was sensitive to substitution of the a1 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 3a5bP inhibit the GABAA receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the a1 and b3 subunits statistically significantly, albeit weakly and incompletely, reduced inhibition by 3a5bP and ACN.
AB - The g-aminobutyric acid type A (GABAA) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3b-hydroxy steroids inhibit, while 3a-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the a1b3g2L GABAA receptor by the endogenous neurosteroid 3a-hydroxy-5b-pregnan-20-one (3a5bP) and the synthetic neuroactive steroid 3a-hydroxy-5a-androstane-17b-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, 3a5bP and ACN potentiate the GABAA 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 IC50s of 13 lM and maximal inhibitory effects of 70-90%. Receptor inhibition by 3a5bP was sensitive to substitution of the a1 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 3a5bP inhibit the GABAA receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the a1 and b3 subunits statistically significantly, albeit weakly and incompletely, reduced inhibition by 3a5bP and ACN.
UR - http://www.scopus.com/inward/record.url?scp=85206959191&partnerID=8YFLogxK
U2 - 10.1124/molpharm.124.000960
DO - 10.1124/molpharm.124.000960
M3 - Article
C2 - 39214710
AN - SCOPUS:85206959191
SN - 0026-895X
VL - 106
SP - 264
EP - 277
JO - Molecular pharmacology
JF - Molecular pharmacology
IS - 5
ER -