Journal: Nature

Article Title: Structure of a human synaptic GABA-A receptor

doi: 10.1038/s41586-018-0255-3

Figure Lengend Snippet: a , FSEC of GABA-A receptor with and without Fab bound and SDS-PAGE analysis of a representative purification (from n>10 purifications). b , Saturation binding assay with [ 3 H]-flumazenil. Single site binding fits for receptor alone and receptor plus Fab both exhibited a Hill slope of ~1 (0.97 and 0.89 respectively). Plotted results are from a representative experiment performed in triplicate. n=3 independent experiments. Data point center is the mean. Error bars are standard deviation, shown for a representative triplicate measurement. c , Competition of 10 nM [ 3 H]-flumazenil with diazepam. Calculated Ki for diazepam assumes a Kd of [ 3 H]-flumazenil of 7.7 nM. n=2 independent experiments in triplicate. Error bars are standard error of the mean (s.d.), shown for a representative triplicate measurement. d , Dose-response experiments in the presence or absence of Fab. HEK cells were transfected with EM constructs and patch-clamped with or without pretreatment of 1 μM Fab for one minute. Hill slopes are 1.7 and 1.4 with and without Fab, respectively. Published values for GABA EC 50 range from 6.6 μM – 107 μM – . n=3 experiments from different cells. Data point center is the mean. Error bars are standard deviation. e , Whole cell patch clamp recording of long application of EM ligands at concentrations used in EM sample to assess conformational state at equilibrium. The two traces shown are from one continuous recording; in between the two responses, Fab was added to 1 μM for one minute to saturate all receptor sites before second application of GABA and flumazenil (including Fab). n=3 independent experiments. f-g , Docking of diazepam at the benzodiazepine binding site based on superposition of benzodiazepine rings. The phenyl ring of diazepam would orient toward the membrane, possibly forming π-π stacking interactions with Y58 on the complementary subunit. Similar to flumazenil, the halogen of diazepam could interact with H102, suggesting this contact is conserved broadly among benzodiazepines and flumazenil. This orientation is largely consistent with predictions from a modeling and docking study and distinct from that suggested by affinity labeling . In this latter prediction, the diazepam phenyl group orients away from the membrane and would require local reorganization of side chains to avoid atomic clashes. h-j , Structural details of Fab-α1 interaction. Labeled residues are on α subunit. i , Top view. j , Side view.

Article Snippet: At the time of transduction, valproic acid (sodium salt, Sigma-Aldrich) was added to 3 mM and flumazenil (Santa Cruz Biotechnology) to 10 μM to boost expression.

Techniques: SDS Page, Purification, Saturation Assay, Binding Assay, Standard Deviation, Transfection, Construct, Patch Clamp, Membrane, Labeling

Journal: Nature

Article Title: Structure of a human synaptic GABA-A receptor

doi: 10.1038/s41586-018-0255-3

Figure Lengend Snippet: a-e , EM density map of the GABA-A receptor conformation B for a representative of each subunit; chain IDs are in parentheses. f-h , EM density segments of Loop C in α1, β2 and γ2 subunits. i-k , EM density segments of M2 helix in α1, β2 and γ2 subunits. l-n , EM density maps of ligand binding sites. l , Flumazenil; m-n , two GABA binding sites.

Article Snippet: At the time of transduction, valproic acid (sodium salt, Sigma-Aldrich) was added to 3 mM and flumazenil (Santa Cruz Biotechnology) to 10 μM to boost expression.

Techniques: Ligand Binding Assay, Binding Assay

Journal: Nature

Article Title: Structure of a human synaptic GABA-A receptor

doi: 10.1038/s41586-018-0255-3

Figure Lengend Snippet: a , b , Top and side views of the 3D reconstruction of GABA-A receptor-Fab complex colored by subunit: α1-green, β2-blue, γ2- magenta; Fab-gray, CHS-yellow. Conformation A is shown. c , d , Top and side view of the atomic model, colored as in a . Flumazenil is shown as cyan spheres, GABA as red spheres and CHS and N-linked glycans are modeled as sticks. e , Structure of single β2 subunit.

Article Snippet: At the time of transduction, valproic acid (sodium salt, Sigma-Aldrich) was added to 3 mM and flumazenil (Santa Cruz Biotechnology) to 10 μM to boost expression.

Techniques:

Journal: Nature

Article Title: Structure of a human synaptic GABA-A receptor

doi: 10.1038/s41586-018-0255-3

Figure Lengend Snippet: a-e , EM density map of the GABA-A receptor conformation A for a representative of each subunit. f-h , EM density segments of Loop C in α1, β2 and γ2 subunits. i-k , EM density segments of M2 helix in α1, β2 and γ2 subunits. l-n , EM density maps of ligand binding sites. l , Flumazenil; m-n , two GABA binding sites.

Article Snippet: At the time of transduction, valproic acid (sodium salt, Sigma-Aldrich) was added to 3 mM and flumazenil (Santa Cruz Biotechnology) to 10 μM to boost expression.

Techniques: Ligand Binding Assay, Binding Assay

Journal: Nature

Article Title: Structure of a human synaptic GABA-A receptor

doi: 10.1038/s41586-018-0255-3

Figure Lengend Snippet: a , Electrophysiology of the cryo-EM construct showing flumazenil (3 μM) blocks GABA-A receptor potentiation by diazepam (1 μM). n=3 independent experiments. b , Flumazenil and diazepam chemical structures. c , View from synapse, as in ; box indicates flumazenil bound at α1-γ2 interface. d , Schematic of flumazenil interactions showing electrostatic (dashes) and hydrophobic interactions (eyelashes). e , f , Detailed architecture of flumazenil binding pocket boxed in c , with orientations and representations as in -f.

Article Snippet: At the time of transduction, valproic acid (sodium salt, Sigma-Aldrich) was added to 3 mM and flumazenil (Santa Cruz Biotechnology) to 10 μM to boost expression.

Techniques: Cryo-EM Sample Prep, Construct, Binding Assay

Journal: Nature

Article Title: Structure of a human synaptic GABA-A receptor

doi: 10.1038/s41586-018-0255-3

Figure Lengend Snippet: a , Synaptic view, with colored boxes indicating the distinct pseudo-agonist sites. b,e , Detailed structural information of pseudo-agonist interfaces α-β and γ-β boxed in a . c,f , Superposition of GABA binding site (in gray) on α-β and γ-β interfaces respectively. d,g , Superposition of flumazenil binding site (in taupe) on the α-β and γ-β interfaces respectively. h , Sequence alignment of the loops involved in ligand binding pockets. Blue residues are involved in flumazenil binding; red residues are involved in GABA binding.

Article Snippet: At the time of transduction, valproic acid (sodium salt, Sigma-Aldrich) was added to 3 mM and flumazenil (Santa Cruz Biotechnology) to 10 μM to boost expression.

Techniques: Binding Assay, Sequencing, Ligand Binding Assay