Journal of Molecular Biology
Refined Structure of the Nicotinic Acetylcholine Receptor at 4 Å Resolution
Introduction
The nicotinic ACh receptor is a member of the pentameric “Cys-loop” superfamily of transmitter-gated ion channels, which includes neuronal ACh receptors, GABAA receptors, 5-HT3 receptors and glycine receptors.1, 2, 3, 4, 5 The channel is found in high concentrations at the nerve–muscle synapse, where it mediates fast chemical transmission of electrical signals in response to ACh released from the nerve terminal into the synaptic cleft. It is a large (290 kDa) glyco-protein, assembled from a ring of homologous subunits (α, γ, α, β, δ) and divided into three domains: a large N-terminal extracellular ligand-binding domain, a membrane-spanning pore, and a smaller intracellular domain, giving it a total length of about 160 Å normal to the membrane plane. The ligand-binding domain shapes a long, ∼20 Å diameter central vestibule and has two binding sites for ACh, which are about 40 Å from the membrane surface on opposite sides of the pore. The pore makes a narrow water-filled path across the membrane and contains the gate, which opens when ACh occupies both binding sites. The intracellular domain shapes another, smaller vestibule, having narrow lateral openings for the ions.
The receptor subunits in the ligand-binding domain are each organised around two sets of β-sheets packed into a curled β-sandwich and joined through the disulphide bridge forming the Cys loop, as was shown by the structure of the closely related soluble protein, AChBP.6 The ACh-binding sites lie at the α–γ and α–δ subunit interfaces, and are contributed mainly by residues from loops A, B and C, connecting β-strands in the α subunits.7, 8, 9 The subunits in the membrane-spanning domain are each made from four α-helical segments (M1–M4).10 The helical segments are arranged symmetrically, forming an inner ring of helices (M2), which shape the pore, and an outer shell of helices (M1, M3 and M4), which coil around each other and shield the inner ring from the lipids. In the closed channel, the inner ring of helices come together near the middle of the membrane to make a constricting hydrophobic girdle, which constitutes an energetic barrier to ion permeation11, 12 and may function as the gate of the channel.10, 13 The subunits in the intracellular domain each contribute one α-helix (part of the M3–M4 loop), which together make the wall of the vestibule.14
Insight into the structural mechanism of gating has been obtained by electron microscopical experiments on helical tubes grown from Torpedo postsynaptic membranes,15, 16 using a rapid spray-freezing technique to mimic the synaptic release of ACh and trap the open-channel form.17 These experiments showed that binding of ACh initiates two interconnected events in the ligand-binding domain. One is a local disturbance in the region of the ACh-binding sites, and the other a larger-scale conformational change, involving rotational movements predominantly in the two α subunits. The inner M2 helices also change their configuration in response to ACh, widening the lumen of the pore at the middle of the membrane. Higher resolution studies of the extended conformational change18 and of the structure in the membrane10 suggested a simplified mechanical model for the channel opening mechanism, whereby ACh triggers rotations of the inner β-sheets of the α subunits and the twisting movement, communicated through the inner helices, breaks the gate apart.
In addition to the structural details, summarised above, the roles played by individual amino acid residues in determining the ligand-binding, gating and cation-conduction properties of the ACh receptor have been extensively characterised by chemical labelling and by site-directed mutagenesis experiments combined with electrophysiological study of function.19, 20, 21, 22, 23, 24, 25, 26, 27, 28 Other experiments of this kind, performed on GABAA, glycine, 5-HT3 and neuronal α7 receptors constitute a wealth of complementary information.
We report here a preliminary three-dimensional framework for relating these biochemical and physiological data, based on refinement of a 4 Å structure obtained from electron images of the tubular Torpedo membranes frozen in a near-physiological ionic environment.10 The refined model enables a detailed description of the whole receptor in the closed-channel form, including the ligand-binding region and vestibular entrances, which have not previously been interpreted at a chemical level. We confirm that the two ligand-binding α subunits have a different extended conformation from the three other subunits in the closed-channel form of the receptor,18 and identify several interactions at the subunit interfaces, and within the α subunits, which may be responsible for their “distorted” structures. The ACh-binding site itself, which was not correctly identified in the 9 Å map,13 shows many features that are apparent in the structure of AChBP. However, the organisation of the B and C loops at the binding site of the closed channel differs from that in AChBP, where ligand is present, indicating that the binding reaction is accompanied by a local structural rearrangement. A comparison of the two structures suggests how the local rearrangement associated with ACh binding stabilises the alternative open-channel form of the receptor. Given our improved understanding of this initial step, it is now possible to sketch a complete picture of the series of coordinated events leading to opening of the channel. Finally, we discuss the role of the vestibules, the ionic surfaces of which create a strongly electronegative environment at either entrance of the narrow membrane pore.
Section snippets
Structure refinement
The original 4 Å data set was from 359 images of tubes,10 grown from Torpedo marmorata postsynaptic membranes.15 The tubes have four distinct helical symmetries, with individual molecules arranged on a p2 surface lattice15 such that the inside of the tube corresponds to the inside of the cell.29 The receptors come closest to each other near radial 2-fold axes (Figure 1(a)). A disulphide bridge between the δ subunits of neighbouring receptors30, 31 lies near the membrane at one such axis; the
Symmetry
The approximate 5-fold symmetry of the receptor was examined further by determining the angles required to achieve optimal least-squares superposition of the subunits around the pentamer. Deviations from 5-fold were found to be smallest in the membrane-spanning domain, where each subunit assumed an orientation lying within 2° (s.d.=1.61°) of the value required for exact register with a 5-fold-averaged structure. These deviations appeared to be a consequence of structural variations (which are
Discussion
The refined 4 Å structure reported here provides a chemical interpretation of all the main functional regions of the ACh receptor, as they would appear under near-physiological ionic conditions in Torpedo postsynaptic membranes. Although the final crystallographic R-factor was only 36.7%, limited by the quality of the amplitudes from images, we demonstrated that the polypeptide chains could now be traced with reasonable confidence over the entire length of the molecule (Figure 2, Figure 12).
General conclusions
This analysis extends earlier electron microscopic analyses of the ACh receptor in Torpedo postsynaptic membranes, imaged either in the absence of ACh, or following brief exposure to ACh to trap the open-channel form. The results together suggest that the channel has the following properties that are fundamental to the way it works:
The main ligand-binding α subunits, in the closed channel, are in a “distorted” state, which is stabilised by inter and intra-subunit interactions.
In the
Model building
The amino acid sequences of the four T. marmorata polypeptide chains67, 68 were used to create the starting receptor structure for the refinement. This structure was modelled initially by fitting fragments of the chains to the experimental densities using the program O.32 The membrane-spanning region was modelled from the original coordinates (PDB entry 1OED). The extracellular region was modelled from the coordinates of the separately aligned inner and outer β-sheet fragments of AChBP,18
Acknowledgements
The structure refinement was based on data from electron images, all of which had been recorded in Japan using microscopes incorporating a liquid helium-cooled stage. I am particularly grateful to Yoshi Fujiyoshi, who designed the stage, and to Atsuo Miyazawa, who recorded most of the images, for their continued support and advice. The work has also benefited from many helpful discussions with colleagues at the Laboratory of Molecular Biology, Cambridge, UK, and at the Scripps Research
References (76)
- et al.
Cys-loop receptors: new twists and turns
Trends Neurosci.
(2004) - et al.
Function and structure in glycine receptors and some of their relatives
Trends Neurosci.
(2004) - et al.
Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures
Neuron
(2004) Theoretical conformation of the closed and open states of the acetylcholine receptor channel
Biochim. Biophys. Acta
(2004)Nicotinic acetylcholine receptor at 9 Å resolution
J. Mol. Biol.
(1993)- et al.
Nicotinic acetylcholine receptor at 4.6 Å resolution: transverse tunnels in the channel wall
J. Mol. Biol.
(1999) - et al.
Activation of the nicotinic acetylcholine receptor involves a switch in conformation of the α subunits
J. Mol. Biol.
(2002) - et al.
The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices MII of the receptor subunits
FEBS Letters
(1986) - et al.
Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist
J. Biol. Chem.
(1992) - et al.
Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the α subunit
Neuron
(1994)
Disulfide bond cross-linked dimer in acetylcholine receptor from Torpedo californica
Biochem. Biophys. Res. Commun.
Distortion correction of tubular crystals: improvements in the acetylcholine receptor structure
Ultramicroscopy
Lysine scanning mutagenesis delineates structural model of the nicotinic acetylcholine receptor ligand binding domain
J. Biol. Chem.
Three-dimensional location of the main immunogenic region of the acetylcholine receptor
Neuron
On the nature of allosteric transitions: a plausible model
J. Mol. Biol.
Mutation of the acetylcholine receptor alpha subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity
Neuron
Mutational analysis of ligand-induced activation of the Torpedo acetylcholine receptor
J. Biol. Chem.
Activation kinetics of recombinant mouse nicotinic acetylcholine receptors: mutations of α-subunit tyrosine 190 affect both binding and gating
Biophys. J.
A mutational analysis of the acetylcholine receptor channel transmitter binding site
Biophys. J.
Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor
J. Biol. Chem.
The GABAA receptor α1 subunit Pro174-Asp191 segment is involved in GABA binding and channel gating
J. Biol. Chem.
Design, synthesis and functional characterisation of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor
FEBS Letters
GABAA receptor M2–M3 loop secondary structure and changes in accessibility during channel gating
J. Biol. Chem.
Allosteric receptors after 30 years
Neuron
M2 pore mutations convert the glycine receptor channel from being anion- to cation-selective
Biophys. J.
Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily
J. Biol. Chem.
SETOR: hardware lighted three-dimensional solid model representations of macromolecules
J. Mol. Graph.
Nicotinic receptors in wonderland
Trends Biochem. Sci.
Mechanisms of channel gating of the ligand-gated ion channel superfamily inferred from protein structure
Expt. Physiol.
Nicotinic acetylcholine receptors
Burger's Med. Chem. Drug Discov.
Emerging structure of the nicotinic acetylcholine receptors
Nature Rev. Neurosci.
Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors
Nature
Nicotinic receptors at the amino acid level
Annu. Rev. Pharmacol. Toxicol.
The nicotinic receptor ligand binding domain
J. Neurobiol.
Structure and gating mechanism of the acetylcholine receptor pore
Nature
The influence of geometry, surface character, and flexibility on the permeation of ions and water through biological pores
Phys. Biol.
Tubular crystals of acetylcholine receptor
J. Cell Biol.
Three-dimensional structure of the acetylcholine receptor by cryoelectron microscopy and helical image reconstruction
J. Cell Biol.
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