Chen Q , Wells MM , Arjunan P , Tillman TS , Cohen AE , Xu Y , Tang P .

P41 GM103393 NIGMS NIH HHS , R01 GM056257 NIGMS NIH HHS , R01GM056257 U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS), TG-MCB050030N National Science Foundation (NSF), T32 EB009403 NIBIB NIH HHS

	Fig. 1. Construction and function of the ELIC-α1GABAAR chimera. a Schematic representation of the ELIC-α1GABAAR chimera, constructed by fusing the ELIC extracellular domain (yellow) ending at residue R199 to the α1GABAAR 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 α1GABAAR 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 α1GABAAR 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 α1GABAAR chimera (current disappears during alphaxalone application as marked by arrows). e Picrotoxin, a known GABAAR blocker, inhibits the α1GABAAR 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-α1GABAAR chimera. a Side (left) and bottom (right) views of the α1GABAAR chimera in complex with alphaxalone. Alphaxalone binding to the inter-subunit sites in the TMD is indicated by the FO–FC omit electron density map contoured at 4 σ (green mesh). b Side view of the apo α1GABAAR chimera. One of the five subunits is covered with the 2FO–FC 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-α1GABAAR chimera. a Pore lining residues in the TM2 helices of the apo α1GABAAR chimera covered by the 2FO–FC 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) α1GABAAR chimeras. Blue and red dots define apo α1GABAAR 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 α1GABAAR chimera in the apo (orange) and desensitized states (cyan) to the desensitized β3GABAAR (green), the desensitized GLIC-α1GABAAR chimera (blue), the desensitized α5GABAAR chimera (brown), and resting ELIC (yellow)
	Fig. 4. Alphaxalone binding mode in the α1GABAAR 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 α1GABAAR chimera. Alphaxalone is surrounded by the 2FO–FC 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) α1GABAAR 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-α1GABAAR. b Side view of overlaid structures of apo (principal subunit - gold; complementary subunit - orange) and alphaxalone-bound (principal subunit - blue; complementary subunit - cyan) ELIC-α1GABAAR. 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 2FO-FC electron density maps (blue mesh, contoured at 1 σ) covering TM1–TM2 in the apo (left) and alphaxalone-bound (right) ELIC-α1GABAAR. 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-α1GABAAR (orange) and apo ELIC (green), aligned along all ECD residues. b Displacements of Cα atoms of equivalent residues in apo ELIC-α1GABAAR 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

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