Nicotinic ACh Receptors Susan Wonnacott and Jacques Barik Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK Susan Wonnacott is Professor of Neuroscience in the Department of Biology and Biochemistry at the University of Bath. Her research focuses on understanding the roles of nicotinic acetylcholine receptors in the mammalian brain and the molecular and cellular events initiated by acute and chronic nicotinic receptor stimulation. Jacques Barik was a PhD student in the Bath group and is continuing in addiction research at the Collège de France in Paris. Introduction and there followed detailed studies of the properties The nicotinic acetylcholine receptor (nAChR) is the of nAChRs mediating synaptic transmission at prototype of the cys-loop family of ligand-gated ion these sites. nAChRs at the muscle endplate and in sympathetic ganglia could be distinguished channels (LGIC) that also includes GABAA, GABAC, by their respective preferences for C10 and C6 glycine, 5-HT3 receptors, and invertebrate glutamate-, histamine-, and 5-HT-gated chloride channels.1,2 polymethylene bistrimethylammonium compounds, 7 nAChRs in skeletal muscle have been characterised notably decamethonium and hexamethonium. This DRIVING RESEARCH FURTHER in detail whereas mammalian neuronal nAChRs provided the first evidence that muscle and neuronal DRIVING RESEARCH FURTHER in the central nervous system have more recently nAChRs are structurally different. become the focus of intense research efforts. This In the 1970s, elucidation of the structure and function was fuelled by the realisation that nAChRs in the brain of the muscle nAChR, using biochemical approaches, and spinal cord are potential therapeutic targets for was facilitated by the abundance of nicotinic synapses a range of neurological and psychiatric conditions. akin to the muscle endplate in electric organs of the The generation of transgenic mice with deleted or electric ray, Torpedo, and eel, Electrophorus. High mutated nAChR subunits3 and the development affinity snake α-toxins, including α-bungarotoxin of subtype-selective ligands to complement the (α-Bgt), enabled the nAChR protein to be purified generous armamentarium of natural products that and subsequently resolved into 4 different subunits, target nAChRs,4 support this research. Progress is designated α,β,γ and δ.8 An additional subunit, ε, was being made in understanding the physiological roles subsequently identified in adult skeletal muscle. In of nAChRs in the brain and the underlying molecular the early 1980s, these subunits were cloned and the and cellular mechanisms, and the contribution of era of the molecular analysis of nAChRs commenced. nAChRs to pathological conditions. The muscle endplate nAChR has the subunit combination and stoichiometry (α1) β1εδ, whereas Muscle nAChR 2 the extrajunctional nAChR (α1)2β1γδ predominates nAChRs in vertebrate skeletal muscle have been in foetal or denervated muscle, and (muscle-derived) studied for over a century; this preparation was electric organs. The high density of nAChRs in Torpedo pivotal in Langley’s formulation of the concept electric organ has facilitated high resolution structural 5 of a ‘receptive substance’. In these studies he studies using electron microscopy.9 Together with showed that ‘nicotine causes tonic contraction of biochemical and biophysical approaches to studying certain muscles of fowl, frog and toad, and that this structure-function relationships, this has resulted in a contraction is prevented .... by curare’. This was detailed molecular description of the nAChR.1 the first notion that the action of a neurotransmitter or pharmacological agonist is transduced into an Molecular Architecture of the nAChR intracellular response by interaction with a molecular (Figure 1) entity (‘receptor’) in the membrane of the responsive Each of the five subunits comprising the nAChR span cell. Dale distinguished the actions of muscarine the lipid bilayer to create a water-filled pore. Each and nicotine, leading to the recognition of two subunit consists of 4 transmembrane segments, pharmacologically distinct (and structurally and the second transmembrane segment (M2) lines the functionally unrelated) families of receptors for the ion channel. The extracellular N-terminal domain neurotransmitter acetylcholine (ACh), that take their of every subunit contains a ‘cys-loop’ that is the names from these natural products.6 Neuromuscular signature sequence of this LGIC family: two cysteine and ganglionic preparations lend themselves to residues, separated by 13 amino acids (Cys 128, physiological and pharmacological investigations, 142, Torpedo α subunit numbering), form a disulphide Tocris Bioscience Scientific Review Series Tocris Bioscience Scientific Review Series Figure 1 | General structure of nAChRs1 Non-competitive Antagonist K+ Positive Allosteric Agonist / Competitive Complementary Modulator Antagonist binding site: γ/(δ) Channel Blocker Primary E D binding site: α Y B Y111 117 W149 W55/(57) Y151 F Nic D180/(182) Y190 C C192 C193Y198 Y W86 a) N b) 93 A ACh binding protein Cys-loop C C 2+ + Ca , Na C M1 M2 M3 M4 M2 lines c) the channel a) Schematic of a nAChR with one subunit removed to reveal the ion channel lumen. Notional sites of action of interacting drugs in the extracellular domain or within the channel lumen are indicated. b) Agonist binding site loop model. The agonist binding site is enlarged to show the contributing polypeptide loops forming the primary and complementary components, with key amino acids indicated on the loops. c) The topography of a single subunit. bond to create a loop that has been implicated in californica and Bulinus truncatus.12,13 Each subunit the transduction of agonist binding into channel of this pentameric secreted protein is homologous opening.10 The principal agonist binding site resides to the N-terminal domain of a nAChR subunit, with in the N-terminal domain of α subunits, close to a pair conservation of all the residues implicated in ACh of adjacent (‘vicinal’) cysteine residues (Cys 192, binding to muscle nAChRs. These proteins provide a 193, Torpedo numbering) that define an α subunit. high resolution view of the extracellular portion of the Mutagenesis and photoaffinity labelling experiments receptor, notably of the binding sites at the interface have highlighted the importance of 4 aromatic between adjacent subunits, and the interaction of residues (Tyr 93, Trp 149, Tyr 190, Tyr 198, Torpedo agonists with these sites.10 numbering), consistent with 3 polypeptide loops of Upon agonist binding, nAChRs undergo an allosteric the α subunit (loops A-C) contributing to the primary 11 transition from the closed, resting conformation to agonist binding site (see Figure 1). The adjacent an open state that allows an influx of Na+, and to a subunit (γ/ε or δ) also contributes to the binding site lesser extent Ca2+, and an efflux of K+ under normal (complementary site: ‘loops’ D-F, now recognised to physiological conditions. In the closed state the ion be mostly β strands). One consequence of this is that channel is occluded by a ‘hydrophobic girdle’ that the αγ/ε and αδ binding sites are not identical with 1 constitutes a barrier to ion permeation. Agonist respect to ligand affinity. However, occupancy of binding in the extracellular domain promotes a both binding sites is required to open the channel. conformational change that results in a rotational Knowledge of ligand binding to nAChRs has been movement of the M2 helices lining the pore. Twisting greatly augmented by the crystal structure of an ACh of the girdle widens the pore by ~3 Å, sufficient for binding protein first identified in the snail Lymaea ion permeation.9 At the muscle endplate, the ensuing stagnalis and subsequently also cloned from Aplysia depolarisation elicits muscle contraction. Despite the | Nicotinic Receptors presence of agonist, the nAChR channel closes within seconds to minutes, to enter a desensitised state. In Figure 2 | Relationship between the major this condition, the nAChR is refractory to activation. conformational states of a nAChR Multiple desensitised states have been proposed to agonist exist.14 In the active (open) conformation, the nAChR RESTING ACTIVE Channel closed Channel open Agonist binds binds agonists with low affinity (Figure 2; e.g. Kd for with low affinity ACh ~50 μM). The desensitised states display higher affinity for agonist binding (Kd for ACh ~1-5 μM), thus the desensitised nAChRs can retain bound agonist despite its non-conducting state. DESENSITISED Fast onset Sites on the Muscle nAChR for Ligand Agonist binds Interactions (Figure 1) with high affinity In addition to agonists binding to the agonist binding sites in the extracellular domain, competitive DESENSITISED antagonists also bind at or close to these sites, Slow onset preventing access to agonists. Their antagonism can be overcome by increasing the agonist concentration (unless the antagonist binds irreversibly, as is the agonist binding sites, and include channel blocking case for α-Bgt), hence competitive antagonism is drugs that occlude the channel. Their inhibition is not referred to as ‘surmountable’. The concentration surmountable with increasing agonist concentration. of competitive antagonist necessary for nAChR In addition to compounds that interact specifically blockade will depend on the experimental conditions. with residues in the mouth or lumen of the pore, any Non-competitive antagonists bind to sites distinct from small positively