Structural basis of neurosteroid anesthetic action on GABAA receptors

Abstract

Type A γ-aminobutyric acid receptors (GABA_ARs) are inhibitory pentameric ligand-gated ion channels in the brain. Many anesthetics and neurosteroids act through binding to the GABA_AR transmembrane domain (TMD), but the structural basis of their actions is not well understood and no resting-state GABA_AR structure has been determined. Here, we report crystal structures of apo and the neurosteroid anesthetic alphaxalone-bound desensitized chimeric α1GABA_AR (ELIC-α1GABA_AR). The chimera retains the functional and pharmacological properties of GABA_ARs, including potentiation, activation and desensitization by alphaxalone. The apo-state structure reveals an unconventional activation gate at the intracellular end of the pore. The desensitized structure illustrates molecular determinants for alphaxalone binding to an inter-subunit TMD site. These structures suggest a plausible signaling pathway from alphaxalone binding at the bottom of the TMD to the channel gate in the pore-lining TM2 through the TM1-TM2 linker. The study provides a framework to discover new GABA_AR modulators with therapeutic potential.

Fig. 1

Construction and function of the ELIC-α1GABA_AR chimera. a Schematic representation of the ELIC-α1GABA_AR chimera, constructed by fusing the ELIC extracellular domain (yellow) ending at residue R199 to the α1GABA_AR transmembrane domain (orange) beginning at residue K222. See more details in the supporting information (Supplementary Fig. 1). b The ELIC agonist propylamine (PPA) activates the α1GABA_AR chimera expressed in Xenopus oocytes in a concentration dependent manner (EC₅₀ = 19.8 ± 1.5 μM, n = 4). c The neurosteroid alphaxalone potentiates the current of oocytes expressing the α1GABA_AR chimera: (left) representative potentiation trace by 0.1 μM alphaxalone; (right) concentration-response potentiation curve with EC₅₀ = 45.5 ± 10.7 nM (n = 6). d Representative traces showing that 3 or 1 μM alphaxalone activates and then quickly desensitizes the α1GABA_AR chimera (current disappears during alphaxalone application as marked by arrows). e Picrotoxin, a known GABA_AR blocker, inhibits the α1GABA_AR chimera. Note the quick desensitization by 10 μM PPA. Error bars in b and c represent SEM. Scale bars in c, d, and e represent 30 s (horizontal) and 10 nA (vertical)

Fig. 2

Crystal structures of the ELIC-α1GABA_AR chimera. a Side (left) and bottom (right) views of the α1GABA_AR chimera in complex with alphaxalone. Alphaxalone binding to the inter-subunit sites in the TMD is indicated by the F_O–F_C omit electron density map contoured at 4 σ (green mesh). b Side view of the apo α1GABA_AR chimera. One of the five subunits is covered with the 2F_O–F_C electron density map contoured at 1 σ (blue mesh). A zoom-in view of the interfacial region shows the representative residue contacts at the interface between the TM2–TM3 loop and the Cys-loop or β1–β2 linker in the ECD: F119-A285 (3.1 Å), F116-Y282 (2.4 Å), T28-K279 (4.5 Å)

Fig. 3

Crystal structures of the pore of the ELIC-α1GABA_AR chimera. a Pore lining residues in the TM2 helices of the apo α1GABA_AR chimera covered by the 2F_O–F_C electron density map contoured at 1 σ (blue mesh). b Overlaid structures of the pore-lining TM2 helices from apo (orange) and alphaxalone-bound (cyan) α1GABA_AR chimeras. Blue and red dots define apo α1GABA_AR chimera pore radii greater or less than the radius of a hydrated Cl⁻ ion (3.2 Å), respectively. c Comparison of the pore radii of the α1GABA_AR chimera in the apo (orange) and desensitized states (cyan) to the desensitized β3GABA_AR (green), the desensitized GLIC-α1GABA_AR chimera (blue), the desensitized α5GABA_AR chimera (brown), and resting ELIC (yellow)

Fig. 4

Alphaxalone binding mode in the α1GABA_AR chimera. a 2D chemical structure of alphaxalone with rings and carbon atoms labeled according to the IUPAC standard for steroids. b Crystal structure of alphaxalone (cyan) bound to a pocket lined by residues in the transmembrane domain from the principal (yellow) and complementary (white) subunits of the α1GABA_AR chimera. Alphaxalone is surrounded by the 2F_O–F_C electron density map contoured at 1 σ (blue mesh). Three residues in close contact with alphaxalone are highlighted. Functional validation of the alphaxalone-binding site was performed by c activation of Xenopus oocytes expressing WT (solid circles), T306A (open circles), Q242L (solid squares) and W246L (open squares) α1GABA_AR chimeras by propylamine (PPA) with EC₅₀ = 20 ± 1, 23 ± 2, 39 ± 3, and 3300 ± 300 μM, respectively; d alphaxalone (0.1 μM) potentiation at the EC₁₀ concentration of PPA; e alphaxalone (3 μM) activation normalized to EC₁₀₀ PPA activation for each construct. Error bars represent SEM (n ≥ 3 oocytes). Statistical significance was assessed by one-way ANOVA followed by Fisher’s LSD post-hoc test. Asterisks indicate statistical difference from WT at p < 0.001 (***) and p < 0.05 (*)

Fig. 5

Alphaxalone interactions with nearby residues in molecular dynamics (MD) simulations. a A representative snapshot from MD simulations shows alphaxalone contacts with T306, Q242, and W246. Distances between alphaxalone and the residues are measured as marked by the dash lines. b Histograms of distances between alphaxalone and W246 atoms (1 and 2) shown in a. c Histograms of distances between alphaxalone and Q242 or T306 atoms (3 and 4, respectively) shown in a. Data in b and c are from three replicate 50-ns simulations, where snapshots were collected every 100-ps for analysis. Distances were measured for each of the five alphaxalone molecules per replicate simulation (500 snapshots × 3 replicates × 5 alphaxalone = 7500 distances in total)

Fig. 6

Alphaxalone-induced structural changes at the bottom of the TMD. a Bottom view of overlaid TM1-TM2 structures of the apo (orange) and alphaxalone-bound (cyan) ELIC-α1GABA_AR. b Side view of overlaid structures of apo (principal subunit - gold; complementary subunit - orange) and alphaxalone-bound (principal subunit - blue; complementary subunit - cyan) ELIC-α1GABA_AR. For clarity, only TM2 and TM3 are shown in the principal subunit and only TM1 and TM2 are shown in the complementary subunit. The arrow highlights structural perturbations originating from the alphaxalone binding site near W246 through the TM1–TM2 linker to the pore-lining residues P253 (−2′) and V257 (2′). c The 2F_O-F_C electron density maps (blue mesh, contoured at 1 σ) covering TM1–TM2 in the apo (left) and alphaxalone-bound (right) ELIC-α1GABA_AR. The sidechains for residues W246 to V257 (2′) are highlighted

Fig. 7

Structural changes in the ECD due to different TMDs. a Top view of crystal structures of apo ELIC-α1GABA_AR (orange) and apo ELIC (green), aligned along all ECD residues. b Displacements of Cα atoms of equivalent residues in apo ELIC-α1GABA_AR and apo ELIC in the pre-TM1 and the TM2–TM3 loop are labeled. Both the pre-TM1 and TM2–TM3 loop regions may affect the ECD. c Structural changes in the ECD are measured by displacements of the highlighted residues in several key regions