Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors

Abstract

Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analyses, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABAAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M3 (β3Pro-415, β3Leu-417, and β3Thr-418) and M4 (β3Arg-309) helices located at the base of a pocket in the β+–α– subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABAAR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABAAR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABAAR β+–α– subunit interface steroid binding site and identify several steroid PAMs that act via other sites.

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization

“…In contrast, KK148, KK150 and 3α5αP but not 3β5αP prevented labeling of β 3 G308 in the intersubunit site ( Figure 4C ), indicating that 3β5αP does not bind to the intersubunit site. Similarly, 3β5αP did not prevent labeling of the intersubunit site by a similar NS-analogue photolabeling reagent in detergent-solubilized GABA A Rs ( Jayakar et al, 2020 ).…”

Druggable Lipid Binding Sites in Pentameric Ligand-Gated Ion Channels and Transient Receptor Potential Channels

“…In pLGICs, the inner leaflet of the intersubunit groove also forms a hydrophobic pocket for allosteric modulators, especially neurosteroids. 3α-hydroxy-pregnane neurosteroids such as THDOC and the neurosteroid-analog anesthetic, alphaxalone, potentiate the GABA A R by binding to this site at the α1/β3 interface (Laverty et al, 2017;Miller et al, 2017;Chen et al, , 2019Sugasawa et al, 2019Sugasawa et al, , 2020Jayakar et al, 2020). The site is formed by residues that are generally conserved in pLGICs, including W245 and Q241 (this glutamine is conserved among α isoforms; Hosie et al, 2009) in M1 of α1, and L297, F301 and Y304 in M3 of β3.…”

Synthetic neuroactive steroids as new sedatives and anaesthetics: Back to the future

“…Molecular studies have determined that the binding site for neuroactive steroids is independent from that of benzodiazepines and barbiturates 66,67,78 . Neuroactive steroid efficacy is dependent on a the combination of different receptor subtypes, 79 but a likely binding spot for neuroactive steroids is at the α/β interface at the level of the plasma membrane 80‐85 …”

“…66,67,78 Neuroactive steroid efficacy is dependent on a the combination of different receptor subtypes, 79 but a likely binding spot for neuroactive steroids is at the α/β interface at the level of the plasma membrane. [80][81][82][83][84][85] Importantly, different GABA A receptor subunits contribute to anaesthesia differently. For example, global knockout of the α4 subunit did not impact duration of loss of righting reflex in mice after exposure to etomidate or isoflurane, but it affected the amnestic component of anaesthesia as measured by contextual fear conditioning.…”