Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer

Abstract

Type A γ-aminobutyric acid receptors (GABA A Rs) are pentameric ligand-gated ion channels (pLGICs) and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system 1 , 2 . Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia 3 , 4 . Amongst the numerous assemblies theoretically possible, α1β2/3γ2 GABA A Rs are most prevalent in the brain 5 . The β3 subunit plays an important role in maintaining inhibitory tone and expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in a CRISPR/Cas9 derived β1-3 triple knockout 6 . To date, efforts to generate accurate structural models for heteromeric GABA A Rs have been hampered by the use of engineered receptors and the presence of detergents 7 – 9 . Significantly, some recent cryo-EM reconstructions report “collapsed” conformations 8 , 9 which disagree with the prototypical pLGIC, the Torpedo nicotinic acetylcholine receptor 10 , 11 , the large body of structural work on homologous homopentameric receptor variants 12 , and the logic of a ion channel architecture. To address this problem, here we present a high-resolution cryo-EM structure of the full-length human α1β3γ2L, a major synaptic GABA A R isoform, functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator megabody and in a desensitised conformation. Unexpectedly, each GABA A R pentamer harbours two phosphatidylinositol 4,5-bisphosphate (PIP2) molecules, whose head groups occupy positively-charged pockets in the intracellular juxtamembrane regions of α1-subunits. Beyond this level, the intracellular M3-M4 loops are largely disordered, possibly because interacting post-synaptic proteins were not included. This structure illustrates the molecular principles of heteromeric GABA A receptor organization and provides a reference framework for future mechanistic investigations of GABA-ergic signalling and pharmacology.

Mechanisms of activation and desensitization of full-length glycine receptor in membranes

“…The lipid at the extracellular end was modelled as phosphatidylcholine and the lipid at the cytosolic end was modelled as phosphatidylinositol-4,5-bisphosphate (PIP2) (Supplemental Figure 2C). The PIP2 density is in a similar location to that observed in a recent GABAAR structure 8 .…”

“…GlyRs belong to the superfamily of pentameric ligand-gated ion channels (pLGIC) and assemble as either homopentamers of α subunits (α1−α4) or heteropentamers of α subunits in combination with β subunits (β1−β2). The GlyR has a conserved pLGIC architecture [6][7][8][9][10] consisting of an extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular domain (ICD). The ECD houses neurotransmitter binding pocket and the TMD governs the machinery for selective ion permeation.…”

Competitive dewetting underlies site-specific binding of general anesthetics to GABA(A) receptors

“…If sevoflurane bound to an intersubunit site has little access to these M1 residues, the experimental results could instead be explained by the photolabel binding to the ↵ intrasubunit site rather than an intersubunit site. Most of the labeled residues in Reference (30) are on the M2 helix, and many of those face the pore in both the homology model and a very recent EM structure (14). In our view, these residues collectively suggest heavy labeling of the pore.…”

“…This structure represents the active state, and the crystallized GluCl chain posesses 33 to 39% sequence identity with the GABAA subunits studied here. Cryo-EM structures of human GABA A receptor heteromers have been released recently (11)(12)(13)(14), yet none of them is representative of the active state that is best suited to study binding of positive allosteric modulators. The systems were prepared as in references 43 and 33, by embedding the protein in a lipid bilayer composed of phosphatidylcholine (POPC), solvated and completed with 0.15 mol/L NaCl using CHARMM-GUI Membrane builder (44), with the final system containing 268 POPC molecules and about 180000 atoms.…”

Structural analysis of pathogenic missense mutations in GABRA2 and identification of a novel de novo variant in the desensitization gate

“…There is no experimentally determined structure for the human GABAAR α2 subunit, either in a homo-or hetero-pentameric complex. For the structural analysis of the previously reported and the novel variants we used the recently solved cryo-electron microscopy (cryo-EM) human hetero-pentameric GABAAR α1β3γ2 complexes in closed (PDB ID 6HUG, with resolution 3.1Å) and desensitized (PDB ID 6HUP and 6I53, with resolution 3.58Å and 3.2Å respectively) forms (28,69). These structures were used because they represent the most abundant arrangement of adult GABAAR hetero-pentamers which is two α subunits, two β subunits and one γ subunit.…”