Nicotinic acetylcholine receptors
The nicotinic acetylcholine receptor (nAChR), a key player in neuronal communication, converts neurotransmitter binding into membrane electrical depolarization. This protein combines binding sites for the neurotransmitter acetylcholine (ACh) and a cationic transmembrane ion channel. The nAChR also binds the addictive drug nicotine. It mediates synaptic transmission at the junction between nerve and muscle cells and various types of nAChR are expressed in the brain. It is involved in several neurological pathologies.
The nicotinic acetylcholine receptor was the first membrane receptor of a neurotransmitter and ion channel that was characterized as a protein. Its biochemical isolation in 1970 constitutes a landmark in the history of pharmacology. The concept of a pharmacological receptor harkens back to Claude Bernard’s (1857) early attempt to "localize" the physiological action of "toxic substances", such as curare, at the junction between motor nerve and muscle, then to Paul Ehrlich (1885) who suggested that cells carry specific "side-chains" on their surface that would react specifically against chemical groups in toxin molecules, and finally most notably to John Newport Langley (1905), for his proposal that there is, in the muscle, a "receptive substance" presently designated as receptor "which combines with nicotine and curare", and which "receives the stimulus from the nerve and transmits it to the effector cell". Initially postulated to be a protein by David Nachmansohn (1955), the molecule was first biochemically characterized by Changeux, Kasai & Lee (1970) using jointly a tissue particularly rich in cholinergic synapses, the electric organ of fish, and a highly specific α-neurotoxin from the venom of the snake Bungarus multicinctus. The nAChR was demonstrated to be a protein distinct from the enzyme acetylcholinesterase that breaks down ACh. In 1983, the nAChR was the first ligand–gated ion channel for which the DNA and protein were further defined by using molecular genetics. Its functional organisation and biophysical properties make the nAChR a typical allosteric membrane protein.
The nicotinic acetylcholine receptor protein
The nicotinic acetylcholine receptor protein contains a binding site(s) for the neurotransmitter ACh and an intrinsic cationic channel specific for Na+, K+ and Ca2+ ions (in some brain subtypes) together with the physical mechanism that links the binding of ACh to the opening of the ion channel. In some invertebrates the nAChR contains an anion channel. The nAChR mediates in less than 1 ms, the transduction of neurotransmitter binding into an electrical signal and on a longer ms to min time-scale it is desensitized toward a high affinity closed state. The nAChR is a transmembrane pentameric glycoprotein of about 300,000 Da that appears by electron microscopy as a cylinder of ca 16nm in length and ca 8nm in diameter, the long axis being perpendicular to the membrane plane. Along the symmetry axis, a low electron density path delineates the ion channel, which mainly consists of a water pore running through the entire length of the protein. Viewed from the synaptic cleft the nAChR suggests a pseudo-symmetrical pentameric rosette, each petal of the rosette corresponding to a single nAChR subunit. The muscle/electric organ nicotinic acetylcholine receptor is composed of four different types of subunits, named α1, β1, γ1 and δ1 according to their apparent molecular weight, that associate into a pentamer with an α2βγδ stoichiometry and a (αγαδβ) organisation. Neuronal nicotinic receptors are made up of different subunits associated in a variety of combinations.
The superfamily of "cys loop" receptors
Cloning of nAChR subunit cDNAs in vertebrates revealed up to a total of 10 different α subunits and four β subunits whose combinatorial assembly contributes to a broad diversity of nAChR oligomers with distinct pharmacological, physiological and kinetic properties and diverse distributions in the nervous system. In contrast to α2βγδ muscle-type nAChR, the simplest receptors are homo-pentameric nAChRs made up of α7-10 subunits with a perfect five-fold symmetry. A wide diversity of neuronal nAChRs hetero-oligomers is also generated by any combination of one or several "tribe I" subunits (α 2,3,4,6), one or several "tribe II" subunit (β 2,4), and one "tribe III" structural subunits (α5, β3) with variable stoichiometries. This rich repertoire of nAChRs, each with distinct features, contributes to the specific physiology of nAChRs in cells or subregions of the brain. More distant homologs of nAChR subunits have also been identified that compose a large superfamily of ligand-gated ion channels currently called cys-loop receptors, by reference to a canonical 13 amino-acid motif flanked in most cases by a disulfide bridge. All subunits are 300 to 600 residues in length, contain a signal peptide, an extracellular hydrophilic domain and four hydrophobic transmembrane segments. In eukaryotes, cys-loop receptor subunits were found only in multicellular animals, with no member known in plants and yeasts. In vertebrates, more than 40 subunits have been identified and regrouped into distinct families, named according to neurotransmitter pharmacology: acetylcholine (nAChR), serotonin (5HT3R) and Zinc (ZAC) receptors that are linked to a cationic channel, and γ-aminobutyric acid (GABAAR and GABACR) and glycine receptors possessing an anionic Cl-channel. In invertebrates, γ-aminobutyric, serotonin, glutamate, histamine and proton-gated channels were also identified, as well as nAChRs with anion conductance.
An extensive search in the databases of all the genomes sequenced led to the discovery of prokaryotic homologs of nAChRs including one from the cyanobacterium Gloeobacter violaceus which has been shown to form a homooligomer functioning as a proton-gated ion channel. Comparative genomic of the whole family reveals a high degree of mobility and losses in the course of evolution suggesting that animals acquired these genes through early lateral transfer from bacteria.
Prokaryotic cys-loop receptors are possibly involved in cell adaptation towards the outside medium and chemotaxis. In animals, cys-loop receptors are found at the plasma membrane of a large diversity of cell types, including neurons, muscle fiber, epithelial cells, and immune cells where they are involved in inter-cellular communication, but they are particularly abundant in central and peripheral neurons, where they mediate and/or modulate synaptic transmission, neurotransmitter release or neuronal excitability depending on their sub-cellular localization.
Atomic structure of the nicotinic acetylcholine receptor
Protein chemistry and sequence analysis revealed a general scheme for each subunit polypeptide consisting of 1. a globular extracellular N-terminal domain (ECD); 2. a trans-membrane domain (TMD) made of a bundle of four transmembrane α-helices (M1 to M4) delineating the ion channel and 3. a cytoplasmic domain, inserted between M3 and M4 thought to contribute to the anchoring of the nAChRs to the plasma membrane and to modulate channel activity. Yet, in the prokaryotic homologue of nAChR this cytoplasmic domain is missing, indicating that it is not essential for signal transduction.
The complete high resolution structure of the nAChR molecule is not available to-date, but only of some of its components: 1. the 1.76 Å resolution X-ray structure of a soluble protein, a truncated homolog of the ECD of nAChRs: the acetylcholine binding protein from snail (AChBP); 2. the 1.94 Å resolution crystal structure of the ECD of a mouse α1-subunit; 3. the up to 4 Å resolution electron microscopy data collected with two-dimensional arrays of whole nAChR molecules in Torpedo marmorata receptor-rich membranes. In particular, the high resolution structures of (1) and (2) show a strong conservation of the monomer fold and of the architecture of the binding site.
In the ECD (around 200 residues in total), the peptide chain folds, from the N- to the C- terminus, into an α-helix followed by ten β-strands arranged in two sets joined through the canonical cys-loop and forming a β-sandwich. The peptide chain then crosses the membrane three times through α-helical segments of 20-30 residues, after which it forms, in eukaryotes, a hydrophilic cytoplasmic domain that is hypervariable both in composition and length (from 10 to 300 residues). A last α-helical transmembrane segment crosses the membrane with the C-terminus facing the extracellular side.
ACh binding sites
The ECD carries from 2 to 5 ACh binding sites, which were initially mapped by photoaffinity labeling on Torpedo nAChR. Each binding site is located at the interface between two subunits: a principal subunit that carries a signature disulfide bridge between two vicinal cysteines (α1−10) and a complementary subunit (δ, γ, ε, β2, β4). Each subunit contributes three segments or “loops” to the binding site, called A (Tyr89), B (Trp143) and C (Tyr185, the double Cys187-188 and Tyr192) for the principal subunit, and D (Trp 53 and Gln 55), E (Arg 104, Val 116, Leu 112 and Met 114) and F (Tyr 164) for the complementary subunit (numbering according to the nAChR from Torpedo). The X-ray structure of nicotinic agonists and competitive antagonist-AChBP co-crystals revealed excellent agreement with these data. The binding cavity consists of well conserved residues, mainly aromatic from the principal side and loop D from the complementary side, which confer nicotinic specificity through characteristic hydrogen bonds, hydrophobic contacts, stabilization of the quaternary/tertiary ammonium moiety of nicotinic ligands via cation-pi interaction and a possible complementary positive charge interaction from a tryptophan carbonyl oxygen. The complementary side is much less conserved, thus creating specificity or differences in affinities among pharmacological ligands of the diverse nAChR subtypes. In all known agonist-bound structures, the loop C from the principal side caps the entrance of the binding site and traps the agonist within the protein. By contrast, in the apo forms and its complexes with antagonists (including polypeptidic toxins from conus and snakes), loop C significantly moves away from the protein.
Within the 20 α-helice bundles of the transmembrane domain, the M2 segment of each subunit borders the ion channel along the axis of symmetry. The channel contributes to nicotinic acetylcholine receptor function in three critical aspects: 1) it contains a gating mechanism that is shut in the resting and desensitized states of the channel, but open in its active state, 2) it contains a water pore that stabilizes ions within the hydrophobic environment of the membrane, 3) it carries a selectivity filter that selects ions mainly according to their charge. Site-directed mutagenesis located the selectivity filter at the cytoplasmic border of the M2 segment, with key contributions of the cytoplasmic end of the M2 segment, as well as of the loop linking the M1 and M2 segments, where discrimination among permeant ions is achieved through partial dehydration and ionic interactions. The location of the gate remains controversial and has been proposed to lie near the selectivity filter, in the middle of the membrane segment or, en bloc, throughout the whole segment.
The ACh binding site and the ion channel are topologically distant by about 35-50 Å within the receptor. This kind of elementary molecular mechanism which links such distinct sites, referred to as an allosteric interaction, has been investigated in detail with globular regulatory proteins and shown to be mediated by a conformational change of the protein. The fast (ms) and slow (10s of ms to min) processes of activation/desensitization in response to ACh involve several discrete conformations of the protein: a basal state with high affinity for antagonists and a closed channel, an active state with low affinity for agonists and little if any affinity for antagonists and an open channel, and one (or several) desensitized states with a closed channel and high affinity for agonists and antagonists. Two principal models have been proposed for the transition between these states. The concerted Monod-Wyman-Changeux model postulates that the protein is in spontaneous equilibrium between the different “discrete” states, and that agonists and antagonists differentially stabilize the state for which their affinity is the highest. Second, the sequential model postulates that at rest, the protein is entirely in the basal state and that the binding of agonist induces a progressive change of conformation which ultimately opens the ion channel.
The observation that spontaneous openings of the ion channel as well as desensitized states (up to 20%) occur in the absence of agonist as well as the identification of mutations which constitutively open the channel without the agonist being present are consistent with the concerted scheme. Normal mode analysis suggests, as well that, in the course of activation, the entire pentamer undergoes a twist motion that involves mainly a quaternary reorganization. On the other hand, electron microscopy data, under rather ill-defined physiological conditions, suggest that ACh binding would trigger only a local distortion of the β-sandwich within the ECD that is transmitted to the M2 segment. Higher resolution studies are required to clarify the precise mechanism of action.
Biosynthesis/receptor trafficking and aggregation
Within the cell, nAChRs are synthesized in the endoplasmic reticulum as core-glycosylated high-mannose subunits. Within this compartment, a fraction of the subunits assemble into homo and hetero-pentameric complexes, the misfolded proteins being degraded by the proteasome machinery. For the Torpedo receptor, assembly likely proceeds through a sequential pathway, from αβγ trimers, to αβγδ tetramers and αβγδα pentamers. Only pentameric complexes exit from the endoplasmic reticulum compartment to reach the cell surface. This latter stage involves targeting of the receptors to relevant subcellular compartments, for instance post-synaptic, pre-synaptic or somatic for neurons. At the neuromuscular junction, nicotinic acetylcholine receptors are located mainly in the postsynaptic membrane under the motor nerve terminal. This distribution results from a differential transcriptional regulation of nAChR genes at the level of the sub-junctional nuclei as well as from their targeting through a local secretory pathway. Local immobilisation of AChR molecules involve a large assembly of cytoskeletal proteins, including the 43K-rapsyn protein that is thought to directly cross-link nAChR molecules.
Physiopathology of muscle and brain nAChRs
At the neuro-muscular junction, nAChR is the target of curare-mimetic muscle relaxants currently used in anesthesiology. Moreover, its functional alterations, through anti-receptor autoantibodies or congenital mutations, generate myasthenic pathologies.
Multiple nAChR subunit compositions are expressed in the central and peripheral nervous system, but the most represented receptors are α4β2 and α7 in the brain and α3β4 in the peripheral nervous system. nAChRs are involved in a wide range of physiological and pathological processes, including learning and memory, reward, motor control and analgesia. Mutations of α4β2 nAChR cause in humans autosomal dominant nocturnal frontal lobe epilepsy. Deletion of the β2-subunit gene in the mouse is accompanied by deficits in exploratory behavior and in nicotine self administration. Its re-expression by stereotaxic lentiviral injection in the ventral tegmental area restores these functions. The modulatory role of nAChRs on dopamine release in the mesolimbic pathway is viewed as a major contributing factor to nicotine addiction.
An important role of nAChRs in the brain is, indeed, to modulate the release of neurotransmitters such as dopamine but also serotonin, glutamate and γ-aminobutyric acid. As a consequence, they are the privileged targets for the treatment of pain, neurodegenerative and psychiatric disorders including Alzheimer and Parkinson diseases, schizophrenia and nicotine addiction.
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