Type A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of inhibitory neurotransmission in the human brain. Endogenous neurosteroids interact with GABAARs to regulate acute and chronic anxiety and are potent sedative, analgesic, anticonvulsant and anesthetic agents. Their mode of binding and mechanism of receptor potentiation, however, remain unknown. Here we report crystal structures of a chimeric GABAAR construct in apo and pregnanolone-bound states. The neurosteroid-binding site is mechanically coupled to the helices lining the ion channel pore and modulates the desensitization-gate conformation. We demonstrate that the equivalent site is responsible for physiological, heteromeric GABAAR potentiation and explain the contrasting modulatory properties of 3a versus 3b neurosteroid epimers. These results illustrate how peripheral lipid ligands can regulate the desensitization gate of GABAARs, a process of broad relevance to pentameric ligand-gated ion channels.
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Protein Data Bank
Protein Data Bank
We thank staff at Diamond Light Source beamlines I03 and I04 for assistance at the synchrotron, K. Harlos and T. Walter for technical support with crystallization, J. Kammonen and staff at Pfizer for very kind time and assistance with electrophysiology, E. Beke for technical assistance during nanobody discovery, Y. Zhao for technical support with tissue culture, members of STRUBI for helpful discussions and G. Sutton and T. Malinauskas for feedback regarding the manuscript. This work was supported by the UK Biotechnology and Biological Sciences Research Council grant BB/M024709/1 (A.R.A. and P.S.M.), the UK Medical Research Council grants MR/L009609/1 and MC_UP_1201/15 (A.R.A.), the Human Frontier Science Program grant RGP0065/2014 (A.R.A.) and the Wellcome Trust studentships 105247/Z/14/Z (S.S.) and 084655/Z/08/Z (S.M.). We also thank INSTRUCT, part of the European Strategy Forum on Research Infrastructures and the Research Foundation-Flanders (FWO) for their nanobody discovery support. Further support from the Wellcome Trust Core Award 090532/Z/09/Z is acknowledged.
Integrated supplementary information
Cartoon representation of the α5TMD pentamer, complexed with Nb25 (green) and pregnanolone (ball-and-stick and spacefilling models, carbon atoms in blue, oxygen atoms in red), followed by zoom-in on one neurosteroid potentiation site. Subunits each comprise the human GABAA β3 ECD (light/dark blue) and α5 TMD (M1–M4 -helices, red/pink). N-linked glycans are shown as spheres coloured by atom type (carbon atoms in orange, oxygen atoms in red). The overview structure and the inter-subunit location of pregnanolone molecules are highlighted by oscillations (0–16 s). The zoom-in on the neurosteroid potentiation site (17–27 s) shows pregnanolone bound to an inter-subunit site between M3 residues on the principal face of one subunit (red) and M1 residues on the complementary face of the next (pink).
Overview of the structural transition between the TMDs of globally superposed apo (orange/light orange) and pregnanolonebound(red/pink) α5TMD. A close-up view of one neurosteroid site (0–8 s) reveals the reshaping taking place upon pregnanolone (balland-stick representation, carbon atoms in blue, oxygen atoms in red) binding. The upper portion of the site contracts between principal face Ile305 and complementary face Gln245, whereas the lower portion of the site expands between Ile305 and complementary face Trp249. This creates torque on the lower part of the M1 helix twisting it sideways and outwards. A top-down view of the TMDs (10–16 s) shows a sideways and outward flexing of the lower (intracellular) halves of the five subunit helical bundles through the transition from apo to pregnanolone-bound states. A top-down zoom-in view of the bottom of the pore (18–24 s) shows the flexing of the transmembrane helices, the swing of the M1–M2 linker, and the dilation of the lower ring, defined by Pro256, of the desensitisation gate (Val260 surrounds the upper ring). The M2 motions are further emphasised by a side-on view between two opposing M2 helices lining the pore (29–34 s), where residues forming the desensitisation gate are highlighted.
Neurosteroid binding induces changes in the relative positions of ECDs vs TMDs. Cartoons are coloured in light/dark blue (ECD, both α5TMD structures) and orange/light orange (apo α5TMD TMD) or ruby/pink (pregnanolone-bound α5TMD TMD); one subunit is highlighted. Pregnanolone molecules are shown in ball-and-stick and space-filling representations, carbon atoms in blue, oxygen atoms in red. The movie oscillates between global superpositions of apo and pregnanolone-bound α5TMD pentamers viewed side-on (0–6s), followed by focus on a single subunit viewed from the outside of the pentamer (7–12 s) and subsequently rotated to look into the inter-subunit principal face (14–20 s). The change in shape of the helical bundle in response to preganolone binding is visible from both orientations and when viewed looking into the inter-subunit principal face (14–20 s) reveals the flexure along M2 and the outward swing of the lower half of the TMD helical bundle. These motions induce an overall straightening of individual subunits due to a rocking motion of the ECD and TMD about their shared interface.