X-ray structure of the human α4β2 nicotinic receptor

Abstract

Nicotinic acetylcholine receptors are ligand-gated ion channels that mediate fast chemical neurotransmission at the neuromuscular junction and have diverse signalling roles in the central nervous system. The nicotinic receptor has been a model system for cell-surface receptors, and specifically for ligand-gated ion channels, for well over a century. In addition to the receptors' prominent roles in the development of the fields of pharmacology and neurobiology, nicotinic receptors are important therapeutic targets for neuromuscular disease, addiction, epilepsy and for neuromuscular blocking agents used during surgery. The overall architecture of the receptor was described in landmark studies of the nicotinic receptor isolated from the electric organ of Torpedo marmorata. Structures of a soluble ligand-binding domain have provided atomic-scale insights into receptor-ligand interactions, while high-resolution structures of other members of the pentameric receptor superfamily provide touchstones for an emerging allosteric gating mechanism. All available high-resolution structures are of homopentameric receptors. However, the vast majority of pentameric receptors (called Cys-loop receptors in eukaryotes) present physiologically are heteromeric. Here we present the X-ray crystallographic structure of the human α4β2 nicotinic receptor, the most abundant nicotinic subtype in the brain. This structure provides insights into the architectural principles governing ligand recognition, heteromer assembly, ion permeation and desensitization in this prototypical receptor class.

Extended Data Figure 1. Sequence alignment of α4β2 receptor with other Cys-loop receptors and AChBPs

Sequences are numbered starting with the first amino acid in the mature protein. NCBI GI accession numbers are provided for full-length proteins and PDB codes for sequences from crystal structures. Human α4 nAChR (29891586), human β2 nAChR (29891594), human α7 nAChR (29891592), Aplysia californica AChBP (2WN9), Lymnaea stagnalis AChBP (1UW6), human GABA_A β3 (4COF), human glycine α3 (5CFB), Mus musculus 5-HT₃ receptor (4PIR) and Caenorhabditis elegans α (3RHW). Secondary structure, binding pocket loops and other selected structural elements are labeled. Disulfide bonds are highlighted in yellow and residues that lacked electron density and are not present in the model are highlighted in orange. Residues with mutations linked to autosomal dominant nocturnal frontal lobe epilepsy are highlighted in brown.

Extended Data Figure 2. Biochemical analysis

a, FSEC trace of the α4β2 nicotinic receptor. The protein sample used for crystallization was tested by FSEC using an SRT SEC-500 column (0.35 mL/min) monitoring tryptophan fluorescence. The receptor exhibited time-dependent oligomerization/aggregation indicated by an asterisk. Pentamer indicates the elution peak of the heteropentameric assembly. b, SDS-PAGE stained with coomassie of the stages of receptor purification. c, Chemical structures of ligands used in crystallization, electrophysiology and binding assays. d, Saturation binding experiments with [³H]-epibatidine. Binding affinity (K_d) was calculated using the one site binding with variable slope equation in Graphpad Prism. The published range for epibatidine K_i, for reference, is 0.042–0.150 nM (all published values in paper are from a pharmacological review). The experiment was performed in triplicate. Error bars are s.e.m. and n_H is the Hill coefficient.

Extended Data Figure 3. Electron density quality

a and b, 2F_o-F_c electron density maps of Loop C from an α4 and β2 subunit, respectively (contoured at 1 σ), with reference residues indicated. Perspective is from inside binding pocket looking toward receptor periphery. c, View down the channel axis toward the cyotosol. Anomalous difference peaks from co-crystallization with 5-Iodo-A-85380 are shown as red mesh and contoured at 3 σ. No detectable anomalous signal was present in other interfacial pockets. d, Stereo pair of 2F_o–F_c electron density maps (contoured at 1.5 σ) from an interface of α4 and β2 subunits. e, 2F_o–F_c electron density map of an α4 subunit M2 α-helix (contoured at 1.5 σ). Reference residues in the M2 helix are indicated. f, Stereo pair of F_o–F_c omit maps (contoured at 2 σ) of selected residues and nicotine in the neurotransmitter binding pocket. Residues and ligand omitted from map calculation are labeled. g, F_o–F_c omit map (contoured at 2 σ) for nicotine in the α-β interface. h–i, F_o–F_c omit map (contoured at 2 σ) of the ion and waters in the pore. The Na⁺ ion (purple) and water (red) are represented as spheres. The nearest residues on the M2 α-helices are indicated.

Extended Data Figure 4. Structural superimpositions

a, Cα atom r.m.s.d. from pairwise superimpositions of all α4 and β2 chains. b, Backbone comparison of the α4 (green) and β2 (blue) subunits. c, Superimpositions of subunits of representative pentameric ligand gated ion channel structures (magenta) on the chain A α4 subunit (green). PDB codes and Cα r.m.s.d. are listed. Asterisk indicates bulging caused by inserted leucine residue found in the M2–M3 loops of α4 and β2 subunits relative to other receptors shown here (this loop was unmodeled in the 5-HT₃R structure, however that protein has the same loop length as α4 and β2). The most similar subunit structure overall to α4 is GLIC, which has been thought to represent an open state, however studies on its desensitization properties^– and comparison to the α4β2 receptor structure here and in Extended Data Fig. 8 suggest it may rather represent a desensitized conformation. Conversely, the Torpedo nicotinic receptor structure, while clearly adopting the same overall fold, aligns less well structurally with α4 than does GLIC. This difference may relate to the Torpedo receptor being in a closed-resting state; notable differences in the backbone conformation of the Torpedo M2–M3 and Cys-loops (inset) compared to all other structures are less straightforward to interpret.

Extended Data Figure 5. Detailed interface interactions

a–c, Views parallel and perpendicular to the plasma membrane coloring potential van der Waals (gray), H-bonds (orange) and electrostatic (pink) interactions in the subunits interface. Parallel views are from periphery of receptor. d–f, Close-up of the red boxes on the apical receptor surface. g–i, Close-up of the black boxes in the view parallel to the plasma membrane. j–l, Close-up of the yellow boxes in the view parallel to the plasma membrane. Panels j–l highlight the N-capping of the M1 helix by a serine in the M2–M3 loop, an interaction seen in GlyR-closed, but absent in GlyR-open and GABA_AR^17,24. For simplicity, only the residues likely to be involved in forming H-bonds and electrostatic interactions are shown. These potential interactions are shown as dashed lines (2.4–3.9 Å). The subunit interfaces are predominantly stabilized through van der Waals interactions, with interspersed hot spots of hydrogen bonding and electrostatic interactions of known functional importance. The N-terminal helix of the receptor is important in pentameric assembly and mutations in this region of other pentameric receptors results in disease. Loop C is essential for orthosteric ligand binding, the M2–M3 loop is critical for allosteric signal transduction, and residues at the apex of M1 and at the intracellular base of the pore are known to affect desensitization^,.

Extended Data Figure 6. Determinants of nicotine binding

a, Sequence alignment of loops implicated in nicotine binding. The human nicotinic α1 (NCBI GI accession number:87567783), β1 (41327726), γ (61743914), δ (4557461) and ε (4557463) subunits were added to the sequence alignment. Residues making contact with nicotine or stabilizing the binding pocket indirectly are highlighted in yellow and brown, respectively. Determinants indirectly affecting the receptor-nicotine cation-π interaction are highlighted in blue. b, Close-up of the α4β2 nicotinic receptor binding pocket. c, Close-up of the corresponding region in AChBP (PDB: 1UW6). The water in the AChBP pocket is represented as a red sphere and forms a hydrogen bond between the pyridine nitrogen on nicotine and the protein backbone. Potential hydrogen bonding and cation-π interactions are represented as dashed lines (2.7–5 Å).

Extended Data Figure 7. Cys-loop receptor ion channel conformations

a, Sequence alignment of the M2 α-helices. Residues lining the α4β2 receptor pore are highlighted in yellow and the residues lining the pores of GlyR (closed: 3JAD; open: 3JAE), GLIC (4QH5) and GABA_AR (4COF) are highlighted in blue. b–e, View of the M2 α-helices from opposing subunits with side chains shown for pore-lining residues. The blue and yellow spheres represent the internal surface of the transmembrane ion channel. Blue spheres are pore diameters >5.6 Å; yellow are >2.8 Å and <5.6 Å and pink are <2.8 Å.

Extended Data Figure 8. Comparison of Cys-loop receptor conformational states

a, View parallel to the plasma membrane of a superposition of the α4 subunit (green) ECD with the GABA_AR (magenta) and GlyR-open (orange) and GlyR-closed (cyan). b, View parallel to the plasma membrane of a superposition of the TMDs. Asterisk indicates an inserted leucine in the M2–M3 loop of α4β2, which is conserved in 5-HT₃ receptors. In the high-resolution structure of the 5-HT₃R, the majority of the M2–M3 loop including the leucine of interest is not modeled, precluding comparison of the two structures for this analysis. c, Table of Cα r.m.s.d. values between isolated regions of one subunit per structure. d–e, View down the channel axis from the synaptic cleft toward the cyotosol of a superposition of the receptors based on alignments of the TMDs. f–g, Analysis of intrasubunit rotation angles between different conformational states. Rotation axes indicated by yellow bar. In f, the ECD of GlyR-open was superposed on the ECD of α4 and relative displacement of the TMD is shown. In g, the TMD of GABA_AR was superposed on the TMD of α4 and relative displacement of the ECD is shown.

Figure 1. Architecture of the α4β2 nicotinic receptor

a, View parallel to the plasma membrane. α4 subunits are in green and β2 in blue. Nicotine (red) and sodium (pink) are represented as spheres. The Cys-loop and Loop C disulfide bonds are shown as yellow spheres. N-linked glycans (brown) are shown as sticks. Dashed lines indicate approximate membrane position. b, View perpendicular to the plasma membrane looking from the extracellular side. c, Orientation as in a of the individual subunits. Unmodeled residues from the intracellular domain are represented as a dashed line.

Figure 2. Neurotransmitter binding site

a, Competition experiments against [³H]-epibatidine. Calculated inhibition constant (K_i) values assume a K_d for [³H]-epibatidine of 96 pM (Extended Data Fig. 2d). n = 4 independent experiments. Error bars are s.e.m. and n_H is the Hill coefficient. *Published range of the K_i of the ligands against WT α4β2. b, Extracellular view, with colored boxes indicating the three different interface classes. c–e, Architectural details of interfaces boxed in b. The top row is from the same orientation as in b. Nicotine and interacting residues are shown as sticks. Potential hydrogen bonding and cation-π interactions are represented as dashed lines (2.7–5 Å). In the lower row, the loop C backbone is hidden to aid in clarity.

Figure 3. Ion permeation pathway

a, Patch-clamp recordings of the wild type (WT) and crystallized α4β2 receptor. ACh, acetylcholine. b, M2 α-helices from opposing α4 and β2 subunits with side chains shown for pore-lining residues. Blue spheres indicate pore diameters >5.6 Å; yellow are >2.8 Å and <5.6 Å. c, Pore diameter for the α4β2 receptor and representative Cys-loop receptors in distinct functional states: desensitized-closed (GABA_AR + benzamidine; PDB:4COF), activated-open (GlyR + glycine; PDB:3JAE) and resting-closed (GlyR + strychnine; PDB:3JAD). Structures were aligned using the M2 helix 9′ leucine, which occurs at y = ~15 Å. The zero value along the Y-axis in the plot is aligned with the α-carbon of the M2 helix −1′ glutamate residue in α4β2. d, Cutaway of the receptor showing the permeation pathway colored by electrostatic potential.

Figure 4. Rearrangements at the membrane interface underlie desensitization in the α4β2 receptor

a, Reference orientation of the α4β2 receptor. b–d, Superimpositions of whole pentamers based on alignment of transmembrane domains, showing local structural differences at the membrane interface. e–g, Superimpositions of whole pentamers based on alignment of extracellular domains, showing global differences in transmembrane domains. b,e, GlyR-open (orange) vs. GABA_AR (magenta). c,f, GlyR-open vs. α4β2 structure (green, blue). d,g α4β2 vs. GABA_AR.

Figure 5. Conformational changes underlying desensitization

Cartoon illustrates the relative positions of ECD and TMDs in the α4β2 receptor compared to the open conformation of the glycine receptor and the desensitized conformation of the GABA_A receptor.