Multiple Functional Neurosteroid Binding Sites on GABA_AReceptors

Abstract

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While GABA A receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill-defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middledown mass spectrometry, we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α 1 β 3 GABA A receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α 1 and β 3 subunits, adjacent to the extracellular domains. An intersubunit site in the interface between the β 3 (+) and α 1 (-) subunits of the GABA A receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABA A receptors.Electrophysiological studies based on these predictions indicate that both the α 1 intrasubunit and β 3 -α 1 intersubunit sites are critical for neurosteroid action. 5 circumvent challenges associated with mass spectrometric identification (predominantly neutral loss) and quantification of neurosteroid-peptide adducts 35 . Using these approaches we have identified three clusters of neurosteroid photolabeled residues on the human  1  3 GABA A receptor. Computational docking studies, guided by the photolabeling data, were used to describe three binding sites and the orientation of the neurosteroids within each site. The docking studies were also used to predict critical residues to test the contribution of each of these sites to neurosteroid modulation of GABA A currents. Site-directed mutagenesis of these sites and electrophysiological studies indicate that at least two of three structurally distinct sites contribute to allosteric modulation of GABA currents. Results Development and characterization of allopregnanolone-analogue photolabeling reagentsAllopregnanolone (3α-hydroxy-5α-pregnan-20-one) is a potent, endogenous positive allosteric modulator of GABA A receptors (figure 1a). We synthesized three photolabeling analogues of allopregnanolone in which photolabeling moieties were placed at various positions around the steroid backbone. KK123 has a 6-diazirine photolabeling group on the C5-C6-C7 edge of the sterol, which is a likely binding interface with α-helices 36 and minimally perturbs neurosteroid structure 37 . KK123 is, however, an aliphatic diazirine and, as such, may preferentially label nucleophilic amino acids 38 . The two other reagents, KK202 and KK200 incorporate a trifluoromethylphenyl-diazirine (TPD) group at either the 3-or 17carbon. These were designed to sample th...

Inhibitable photolabeling by neurosteroid diazirine analog in the β3-Subunit of human hetereopentameric type A GABA receptors

“…Multiple steroid photolabels 5-11, incorporating photoreactive groups at various positions on the steroid backbone, have been tested in GABA A Rs. 17,21 However, to date no tritiated steroid photolabels have proven useful in such protection studies at steroid binding sites. Previously, we developed tritiated allopregnanolone analogs 12 and 13 containing photoreactive substituents at the 11-position, which were potent anesthetics and GABA A R modulators, but their photoincorporation in GABA A Rs was not inhibited by other steroid modulators.…”

“…We hypothesized that the upper edge of the steroid framework, where our C-11 photolabels were located, projected out of the binding site, perhaps facing towards the lipids. 22 Consequently, in this work, we placed the photoreactive residues on the opposite edge of the steroid (C-6), where previous studies on the β3 homomer had been successful, 17,21 and on the steroid sidechain (C17 of the framework). Here, we report the synthesis of three neurosteroid analogs equipped with diazirines located at C-6 or C-21 (14)(15)(16) that act as positive allosteric modulators of heteropentameric α1β3 and α1β3γ2L receptors.…”

Enhancement of muscimol binding and gating by allosteric modulators of the GABAA receptor: relating occupancy to state functions

“…Construct design and cell culture were performed as described previously (Chen et al, 2019). Briefly, the human α 1 and β 3 subunits were subcloned into pcDNA3 for molecular manipulations and cRNA synthesis.…”

“…The [ 3 H]muscimol binding assays were performed using a previously described method (Chen et al, 2019). To perform [ 3 H]muscimol binding isotherms, HEK cell membrane proteins (50 μg/ml final concentration) were incubated with 0.3 nM-1 μM [ 3 H]muscimol (30 Ci/mmol; PerkinElmer, Boston, MA), neurosteroid and binding buffer (10 mM potassium phosphate, 100 mM potassium chloride, pH 7.5) in a total volume of 1 ml.…”

Intrasubunit and intersubunit steroid binding sites independently and additively mediate α1β2γ2L GABAA receptor potentiation by the endogenous neurosteroid allopregnanolone

“…Functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the membrane-spanning domains of the heteromeric GABA A receptor (Chen et al, 2019;Hosie et al, 2006;Laverty et al, 2017;Miller et al, 2017;Ziemba et al, 2018). The sites are located at the β-α subunit interface (dubbed "Site I") and within the α ("Site II") and β subunits ("Site III") (Fig.…”