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, 24 (11), 977-985

Crystal Structures of a GABA A-receptor Chimera Reveal New Endogenous Neurosteroid-Binding Sites

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Crystal Structures of a GABA A-receptor Chimera Reveal New Endogenous Neurosteroid-Binding Sites

Duncan Laverty et al. Nat Struct Mol Biol.

Abstract

γ-Aminobutyric acid receptors (GABAARs) are vital for controlling excitability in the brain. This is emphasized by the numerous neuropsychiatric disorders that result from receptor dysfunction. A critical component of most native GABAARs is the α subunit. Its transmembrane domain is the target for many modulators, including endogenous brain neurosteroids that impact anxiety, stress and depression, and for therapeutic drugs, such as general anesthetics. Understanding the basis for the modulation of GABAAR function requires high-resolution structures. Here we present the first atomic structures of a GABAAR chimera at 2.8-Å resolution, including those bound with potentiating and inhibitory neurosteroids. These structures define new allosteric binding sites for these modulators that are associated with the α-subunit transmembrane domain. Our findings will enable the exploitation of neurosteroids for therapeutic drug design to regulate GABAARs in neurological disorders.

Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Structure and function of the GLIC-GABAARα1 chimera.
(a) Schematic representation of the GLIC-GABAARα1 chimera. The ECD is contributed by the GLIC subunit (green, residues 1-194) and the TMD is from the GABAAR α1 subunit (blue, residues 223-428, excluding the intracellular domain between M3 and M4, which derives from GLIC, green loop). (b) Peak-scaled currents induced by applied (bar) orthosteric agonists for: α1β3 GABAAR (10 mM GABA); wild-type (WT) GLIC (protons - pH 4); and chimera constructs with and without the G258V mutation (proton – pH 4). GLIC-GABAARα1G258Vcryst was used for crystallization experiments. (c) The GABAA channel blocker picrotoxin (PTX; 1 mM) inhibits proton-activated currents (pH 4) in the chimera voltage-clamped at -60 mV. Dotted lines show the extent of steady-state current inhibition. (d) Bar-graph showing current remaining after PTX inhibition of peak and steady-state pH4 currents. Values are means ± sem (n = 4 for both, independent experiments). Note the peak currents are more profoundly inhibited by PTX compared to steady-state currents. (e) Peak-scaled proton-activated (pH 4 – 4.5) currents (VH = -60 mV) for chimeras with gain-of-desensitization mutations in the α1-subunit TMD . Increased residue side-chain volume at the -3’ Val and 2’ Gly in M2 increases the rate of desensitization.
Figure 2
Figure 2. Structure of the GABAA receptor chimera.
Crystal structure of the chimera showing side (a) and plan (b) views with the ECD from GLIC (green) and the TMD of GABAARα1 (blue). M2 helices (cyan) line the ion channel. Cholesteryl hemisuccinate molecules (orange) and the detergent acyl chains (yellow) are bound at the periphery of the TMD and shown in stick form.
Figure 3
Figure 3. Coupling at the receptor chimera ECD-TMD interface.
(a) Side-view of the receptor showing two subunits forming the principal (p, +) and complementary (c, -) inter-subunit interfaces. The dashed boxes are magnified in panels b-d. (b) Residues that interact at the coupling interface between the ECD and TMD are shown. Identified residues (in stick form) are broadly conserved across GLIC and GABAA receptor subunits, and putative H-bonding is shown by black dashed lines. (c) Residues involved in putative inter-subunit H-bonding and intra-subunit salt-bridge interactions in the upper half of the TMD are shown. The Cys residues (yellow) in M1 and M3 do not form a disulfide bridge. (d) Residues involved in putative inter-subunit H-bonding and intra-subunit salt-bridge interactions in the lower half of the TMD are shown.
Figure 4
Figure 4. Structure of the GABAAR chimera channel in a desensitized state.
(a) Plan view superimposing WT GLIC and GABA β3 subunit TMDs on GLIC-GABAARα1G258Vcryst revealing conformational changes to the TMD principally by tilting of M2 and rotation of M4. (b) Two M2 α-helices of the GLIC-GABAARα1G258Vcryst (blue) are shown with equivalent M2 helices from WT GLIC (green) and GABA β3 (red) subunits. Note the tilting of the helices to form a constriction in the lower part of the pore (box). The solvent accessible volume of the channel is represented by spheres. (c) Pore radius profiles through the channel. The ordinate directly relates to (b) for GLIC-GABAARα1G258Vcryst, GABA β3 and WT GLIC open state channels.(d) Pore constrictions formed by M2 lining residues at the level of -2’ Pro, 2’ Val (desensitization gate) and 9’ Leu (activation gate; all shown as Cα-spheres with distances in angstroms (Ǻ)). (e) Residues lining the M2-M3 interface and M1-M2 linker form the components of a desensitization gate. (f) Positive electrostatic surface potential of the chimera at the cytoplasmic portal of the ion channel. Cl- ions are represented as green spheres and omit style map is calculated when ions were excluded from the refinement (contoured at 2σ, orange).
Figure 5
Figure 5. Interfacial subunit binding site for the neurosteroid THDOC.
(a) Chemical structure of THDOC in 2D and 3D. (b) Membrane currents for GLIC-GABAARα1G258Vcryst (expressed in Xenopus oocytes) activated by protons (EC10-15) in the absence and presence of THDOC revealing profound potentiation. (c) Proton and THDOC concentration-response curves for the chimera. Normalized plots represent fits to mean ± sem data points with the Hill equation for potentiation (blue) by THDOC of the pH6 (EC10) current (= 100 %), or direct activation (red) of the chimera by THDOC. EC50 value for potentiation is 1.23 ± 0.09 µM (n = 4), and for direct activation, 2.30 ± 0.09 µM (n = 3). (d) For GLIC-GABAARα1G258Vcryst, THDOC (green sticks) binds across each subunit-subunit interface (box) in the pentamer. (e,f) THDOC binding at interfaces between principal (p) and complementary (c) subunits. Side-views (in the membrane, e) and plan views (extracellular, f) are shown. Dashed lines indicate H-bonding (distances; Q241-steroid, 2.4 - 2.9 Å and T305-steroid, 3.1 - 3.4 Å). Putative hydrophobic interactions (<4 Å) are formed between W245 and rings C and D of THDOC. Labelled residues contribute directly to neurosteroid binding or line the binding pocket. (g) Relative effects of Q241L, W245L and T305W mutations on THDOC (500 nM) potentiation of GLIC-GABAARα1G258Vcryst proton-activated responses. Data shown are means ± sem of biological replicates (Ctrl (G258V) n = 4 oocytes; +Q241L n = 3; +W245L n = 4; +T305W n = 6). Results are representative of injections into oocytes taken from 3 Xenopus laevis performed over 5 separate days.
Figure 6
Figure 6. Inhibitory neurosteroid pregnenolone sulfate binds at a distinct site.
(a) Chemical structure of pregnenolone sulfate (PS) in 2D and 3D. (b) Inhibition of submaximal (pH 5.5) membrane currents for GLIC-GABAARα1G258Vcryst by PS. (c) Intra-subunit binding site for PS (cyan sticks/spheres) located at the lipid face of M3 and M4 α-helices, viewed from the plane of the membrane. Hydrophobic and aromatic residues that line the bilayer-exposed face of M3 and M4 are labelled. These form a smooth groove at the protein surface, with PS bound alongside the α-helices. Residues that bind THDOC (forming the potentiating neurosteroid binding site) are labeled in green, and THDOC orientation is shown by the transparent green oval shape. Cholesteryl hemisuccinate binding is also indicated (orange sticks). (d) Relative effects of Q241L, W245L, and T305W mutations on PS (10 μM) inhibition of GLIC-GABAARα1G258Vcryst proton–activated responses. Values are means ± sem (n = 4, 4, 6 and 3, respectively from independent experiments). (e) Proton-activated (pH4.5) steady-state current inhibition by PS for GLIC-GABAARα1G258Vcryst, and for the chimeras containing either K390A or I391C, A398C, F399C mutations. The curve fits were generated by the inhibition model equation. Note the reduced inhibition for the mutant receptors. Data shown are means of biological replicates (Ctrl (G258V) n = 4 oocytes; +Q241L n = 4; +W245L n = 4; +T305W n = 6; +K390A n = 4; + I391C, A398C, F399C n = 4). Results are representative of injections into oocytes taken from 8 Xenopus laevis performed over 18 separate days.
Figure 7
Figure 7. Inter-subunit anaesthetic binding cavity and aqueous tunnel
(a) Structure of the GLIC-GABAARα1G258Vcryst chimera showing the location of residues involved in anaesthetic binding (magenta, M235 & A290 (equivalent to M286 in β3) and the binding site for potentiating neurosteroids (shown in stick representation, green). Note their accommodation at the same subunit interface. (b) Transverse-view of an aqueous tunnel reveals that it runs close to residues implicated in both anaesthetic (magenta), CHS (teal) and neurosteroid (green) binding sites and would be accessible from the channel pore or the membrane-exposed face of the TMD. (c) Another transverse view of the aqueous tunnel showing the proximity of the CHS binding site (teal) and key residues involved in anesthetic binding (magenta). The tunnel runs from the lipid interface at L231 in M1, through to the back of the ion channel at T264 (10’) in M2, near the 9’ activation gate, and exits into the pore at S269 (15’).

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