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 century1,2. 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 surgery2,3,4. The overall architecture of the receptor was described in landmark studies of the nicotinic receptor isolated from the electric organ of Torpedo marmorata5. Structures of a soluble ligand-binding domain have provided atomic-scale insights into receptor–ligand interactions6, while high-resolution structures of other members of the pentameric receptor superfamily provide touchstones for an emerging allosteric gating mechanism7. 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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Langley, J. N. On the reaction of cells and of nerve-endings to certain poisons, chiefly as regards the reaction of striated muscle to nicotine and to curari. J. Physiol. (Lond.) 33, 374–413 (1905)
Changeux, J. P. & Edelstein, S. J. Nicotinic Acetylcholine Receptors: From Molecular Biology to Cognition (Odile Jacob Publishing Corporation, 2005)
Engel, A. G., Shen, X. M., Selcen, D. & Sine, S. M. Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment. Lancet Neurol. 14, 420–434 (2015)
Becchetti, A., Aracri, P., Meneghini, S., Brusco, S. & Amadeo, A. The role of nicotinic acetylcholine receptors in autosomal dominant nocturnal frontal lobe epilepsy. Front. Physiol. 6, 22 (2015)
Unwin, N. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J. Mol. Biol. 346, 967–989 (2005)
Rucktooa, P., Smit, A. B. & Sixma, T. K. Insight in nAChR subtype selectivity from AChBP crystal structures. Biochem. Pharmacol. 78, 777–787 (2009)
Nemecz, Á., Prevost, M. S., Menny, A. & Corringer, P. J. Emerging molecular mechanisms of signal transduction in pentameric ligand-gated ion channels. Neuron 90, 452–470 (2016)
Nelson, M. E., Kuryatov, A., Choi, C. H., Zhou, Y. & Lindstrom, J. Alternate stoichiometries of α4β2 nicotinic acetylcholine receptors. Mol. Pharmacol. 63, 332–341 (2003)
Tapia, L., Kuryatov, A. & Lindstrom, J. Ca2+ permeability of the (α4)3(β2)2 stoichiometry greatly exceeds that of (α4)2(β2)3 human acetylcholine receptors. Mol. Pharmacol. 71, 769–776 (2007)
Lester, H. A. et al. Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry. Implications for drug discovery. AAPS J. 11, 167–177 (2009)
Morales-Perez, C. L., Noviello, C. M. & Hibbs, R. E. Manipulation of subunit stoichiometry in heteromeric membrane proteins. Structure 24, 797–805 (2016)
Zwart, R. et al. 5-I A-85380 and TC-2559 differentially activate heterologously expressed α4β2 nicotinic receptors. Eur. J. Pharmacol. 539, 10–17 (2006)
Zhou, Y. et al. Human α4β2 acetylcholine receptors formed from linked subunits. J. Neurosci. 23, 9004–9015 (2003)
Hassaine, G. et al. X-ray structure of the mouse serotonin 5-HT3 receptor. Nature 512, 276–281 (2014)
Karlin, A. Emerging structure of the nicotinic acetylcholine receptors. Nature Rev. Neurosci. 3, 102–114 (2002)
Sauguet, L. et al. Structural basis for ion permeation mechanism in pentameric ligand-gated ion channels. EMBO J. 32, 728–741 (2013)
Miller, P. S. & Aricescu, A. R. Crystal structure of a human GABAA receptor. Nature 512, 270–275 (2014)
Picciotto, M. R. et al. Acetylcholine receptors containing the β2 subunit are involved in the reinforcing properties of nicotine. Nature 391, 173–177 (1998)
Tapper, A. R. et al. Nicotine activation of α4* receptors: sufficient for reward, tolerance, and sensitization. Science 306, 1029–1032 (2004)
Dougherty, D. A. The cation–π interaction. Acc. Chem. Res. 46, 885–893 (2013)
Mazzaferro, S. et al. Additional acetylcholine (ACh) binding site at α4/α4 interface of (α4β2)2α4 nicotinic receptor influences agonist sensitivity. J. Biol. Chem. 286, 31043–31054 (2011)
Yang, J. Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells. J. Gen. Physiol. 96, 1177–1198 (1990)
Dwyer, T. M., Adams, D. J. & Hille, B. The permeability of the endplate channel to organic cations in frog muscle. J. Gen. Physiol. 75, 469–492 (1980)
Du, J., Lü, W., Wu, S., Cheng, Y. & Gouaux, E. Glycine receptor mechanism elucidated by electron cryo-microscopy. Nature 526, 224–229 (2015)
Gielen, M., Thomas, P. & Smart, T. G. The desensitization gate of inhibitory Cys-loop receptors. Nature Commun. 6, 6829 (2015)
Althoff, T., Hibbs, R. E., Banerjee, S. & Gouaux, E. X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors. Nature 512, 333–337 (2014)
Celentano, J. J. & Wong, R. K. Multiphasic desensitization of the GABAA receptor in outside-out patches. Biophys. J. 66, 1039–1050 (1994)
Léna, C. & Changeux, J. P. Allosteric modulations of the nicotinic acetylcholine receptor. Trends Neurosci. 16, 181–186 (1993)
Paradiso, K. G. & Steinbach, J. H. Nicotine is highly effective at producing desensitization of rat α4β2 neuronal nicotinic receptors. J. Physiol. 553, 857–871 (2003)
Labriola, J. M. et al. Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment. J. Biol. Chem. 288, 11294–11303 (2013)
Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)
Jansen, M., Bali, M. & Akabas, M. H. Modular design of Cys-loop ligand-gated ion channels: functional 5-HT3 and GABA rho1 receptors lacking the large cytoplasmic M3M4 loop. J. Gen. Physiol. 131, 137–146 (2008)
Mukhin, A. G. et al. 5-Iodo-A-85380, an α4β2 subtype-selective ligand for nicotinic acetylcholine receptors. Mol. Pharmacol. 57, 642–649 (2000)
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)
Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 8060–8065 (2006)
Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014)
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
Porebski, P. J., Cymborowski, M., Pasenkiewicz-Gierula, M. & Minor, W. Fitmunk: improving protein structures by accurate, automatic modeling of side-chain conformations. Acta Crystallogr. D 72, 266–280 (2016)
Hibbs, R. E. & Gouaux, E. Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54–60 (2011)
Mnatsakanyan, N. & Jansen, M. Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors. J. Neurochem. 125, 843–854 (2013)
Corringer, P. J. et al. Atomic structure and dynamics of pentameric ligand-gated ion channels: new insight from bacterial homologues. J. Physiol. 588, 565–572 (2010)
Cymes, G. D. & Grosman, C. The unanticipated complexity of the selectivity-filter glutamates of nicotinic receptors. Nature Chem. Biol. 8, 975–981 (2012)
Pei, J. & Grishin, N. V. PROMALS3D: multiple protein sequence alignment enhanced with evolutionary and three-dimensional structural information. Methods Mol. Biol. 1079, 263–271 (2014)
Lee, B. & Richards, F. M. The interpretation of protein structures: estimation of static accessibility. J. Mol. Biol. 55, 379–400 (1971)
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)
Gallivan, J. P. & Dougherty, D. A. Cation-π interactions in structural biology. Proc. Natl Acad. Sci. USA 96, 9459–9464 (1999)
Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, 2256–2268 (2004)
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007)
Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360 (1996)
Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)
Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013)
Hibbs, R. E. et al. Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal α7 nicotinic acetylcholine receptor. EMBO J. 28, 3040–3051 (2009)
Celie, P. H. et al. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41, 907–914 (2004)
Huang, X., Chen, H., Michelsen, K., Schneider, S. & Shaffer, P. L. Crystal structure of human glycine receptor-α3 bound to antagonist strychnine. Nature 526, 277–280 (2015)
Wonnacott, S. & Barik, J. Nicotinic ACh receptors. Tocris Reviews 28, 1–20 (2007)
Gonzalez-Gutierrez, G. & Grosman, C. Bridging the gap between structural models of nicotinic receptor superfamily ion channels and their corresponding functional states. J. Mol. Biol. 403, 693–705 (2010)
Parikh, R. B., Bali, M. & Akabas, M. H. Structure of the M2 transmembrane segment of GLIC, a prokaryotic Cys loop receptor homologue from Gloeobacter violaceus, probed by substituted cysteine accessibility. J. Biol. Chem. 286, 14098–14109 (2011)
Laha, K. T., Ghosh, B. & Czajkowski, C. Macroscopic kinetics of pentameric ligand gated ion channels: comparisons between two prokaryotic channels and one eukaryotic channel. PLoS ONE 8, e80322 (2013)
Bouzat, C., Bartos, M., Corradi, J. & Sine, S. M. The interface between extracellular and transmembrane domains of homomeric Cys-loop receptors governs open-channel lifetime and rate of desensitization. J. Neurosci. 28, 7808–7819 (2008)
Fourati, Z., Sauguet, L. & Delarue, M. Genuine open form of the pentameric ligand-gated ion channel GLIC. Acta Crystallogr. D 71, 454–460 (2015)
Acknowledgements
We thank J. Lindstrom for providing the α4 and β2 receptor genes, D. Borek and Z. Otwinowski for guidance in structural analyses, J. Cabrera for assistance with illustrations, and members of the Hibbs laboratory for comments on the manuscript. X-ray diffraction experiments at the Argonne National Laboratory’s Advanced Photon Source 24-ID-C beamline were supported by the National Institutes of Health (NIH; GM103403 and RR029205) and the Department of Energy (DE-AC02-06CH11357). This research project was supported by an NIH training grant (T32 NS069562) and a Howard Hughes Medical Institute Gilliam Fellowship to C.L.M.-P.; R.E.H. is supported by a McKnight Scholar Award, a Klingenstein-Simons Fellowship Award in the Neurosciences, The Welch Foundation (I-1812), The Friends of the Alzheimer’s Disease Center and the NIH (DA037492, DA042072 and NS077983).
Author information
Authors and Affiliations
Contributions
C.L.M.-P., C.M.N. and R.E.H. contributed to all aspects of the project.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Additional information
Reviewer Information
Nature thanks M. Akabas, R. Dutzler and A. Karlin for their contribution to the peer review of this work.
Extended data figures and tables
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 accessions are provided for sequences from crystal structures. Human α4 nAChR (29891586), human β2 nAChR (29891594), human α7 nAChR (29891592), Aplysia californica AChBP (2WN9)54, Lymnaea stagnalis AChBP (1UW6)55, human GABAA β3 (4COF)17, human glycine α3 (5CFB)56, Mus musculus 5-HT3 receptor (4PIR)14 and Caenorhabditis elegans α (3RHW)41. Secondary structure, binding-pocket loops and other selected structural elements are labelled. 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, Fluorescence-detection size-exclusion chromatography (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−1) 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–polyacrylamide gel electrophoresis (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 (Kd) was calculated using the one site binding with variable slope equation in Graphpad Prism. The published range for epibatidine Ki, for reference, is 0.042–0.150 nM (all published values are from a pharmacological review57). The experiment was performed in triplicate. nH, Hill coefficient. Error bars are s.e.m.
Extended Data Figure 3 Electron density quality.
a, b, 2Fo − Fc electron density maps of loop C from an α4 and β2 subunit, respectively (contoured at 1σ), with reference residues indicated. Perspective is from inside binding pocket looking towards receptor periphery. c, View down the channel axis towards the cyotosol. Anomalous difference peaks from co-crystallization with 5-Iodo-A-85380 are shown as red mesh and contoured at 3σ. No detectable anomalous signal was present in other interfacial pockets. d, Stereo pair of 2Fo − Fc electron density maps (contoured at 1.5σ) from an interface of α4 and β2 subunits. e, 2Fo − Fc electron density map of an α4 subunit M2 α-helix (contoured at 1.5σ). Reference residues in the M2 helix are indicated. f, Stereo pair of Fo − Fc omit maps (contoured at 2σ) of selected residues and nicotine in the neurotransmitter-binding pocket. Residues and ligand omitted from map calculation are labelled. g, Fo − Fc omit map (contoured at 2σ) for nicotine in the α–β interface. h–i, Fo − Fc omit map (contoured at 2σ) 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 accessions and Cα r.m.s.d. values 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 unmodelled 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 properties58,59,60 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, colouring potential van der Waals (grey), hydrogen 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 GABAAR17,24. For simplicity, only the residues likely to be involved in forming hydrogen 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 disease17. Loop C is essential for orthosteric ligand binding, the M2–M3 loop is critical for allosteric signal transduction7, and residues at the apex of M1 and at the intracellular base of the pore are known to affect desensitization25,61.
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 accession 1UW6)55. 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: PDB accession 3JAD; open: 3JAE)24, GLIC (4QH5)62 and GABAAR (4COF)17 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 GABAAR (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 modelled, 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 towards the cyotosol of a superposition of the receptors based on alignments of the TMDs. f, g, Analysis of intra-subunit 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 GABAAR was superposed on the TMD of α4 and relative displacement of the ECD is shown.
Rights and permissions
About this article
Cite this article
Morales-Perez, C., Noviello, C. & Hibbs, R. X-ray structure of the human α4β2 nicotinic receptor. Nature 538, 411–415 (2016). https://doi.org/10.1038/nature19785
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature19785
This article is cited by
-
An original potentiating mechanism revealed by the cryo-EM structures of the human α7 nicotinic receptor in complex with nanobodies
Nature Communications (2023)
-
The modes of action of ion-channel-targeting neurotoxic insecticides: lessons from structural biology
Nature Structural & Molecular Biology (2023)
-
Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology
The Journal of Membrane Biology (2023)
-
Structural mechanism of muscle nicotinic receptor desensitization and block by curare
Nature Structural & Molecular Biology (2022)
-
Structural basis of human α7 nicotinic acetylcholine receptor activation
Cell Research (2021)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.