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Nicotinic ACh Receptors - · PDF file Nicotinic ACh Receptors Introduction The nicotinic acetylcholine receptor (nAChR) is the prototype of the cys-loop family of ligand-gated ion

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    Tocris Bioscience Scientific Review Series

    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.

    Nicotinic ACh Receptors

    Introduction The nicotinic acetylcholine receptor (nAChR) is the prototype of the cys-loop family of ligand-gated ion channels (LGIC) that also includes GABAA, GABAC, glycine, 5-HT3 receptors, and invertebrate glutamate-, histamine-, and 5-HT-gated chloride channels.1,2 nAChRs in skeletal muscle have been characterised in detail whereas mammalian neuronal nAChRs in the central nervous system have more recently become the focus of intense research efforts. This was fuelled by the realisation that nAChRs in the brain and spinal cord are potential therapeutic targets for a range of neurological and psychiatric conditions. The generation of transgenic mice with deleted or mutated nAChR subunits3 and the development of subtype-selective ligands to complement the generous armamentarium of natural products that target nAChRs,4 support this research. Progress is being made in understanding the physiological roles of nAChRs in the brain and the underlying molecular and cellular mechanisms, and the contribution of nAChRs to pathological conditions.

    Muscle nAChR nAChRs in vertebrate skeletal muscle have been studied for over a century; this preparation was pivotal in Langley’s formulation of the concept of a ‘receptive substance’.5 In these studies he showed that ‘nicotine causes tonic contraction of certain muscles of fowl, frog and toad, and that this contraction is prevented .... by curare’. This was the first notion that the action of a neurotransmitter or pharmacological agonist is transduced into an intracellular response by interaction with a molecular entity (‘receptor’) in the membrane of the responsive cell. Dale distinguished the actions of muscarine and nicotine, leading to the recognition of two pharmacologically distinct (and structurally and functionally unrelated) families of receptors for the neurotransmitter acetylcholine (ACh), that take their names from these natural products.6 Neuromuscular and ganglionic preparations lend themselves to physiological and pharmacological investigations,

    and there followed detailed studies of the properties of nAChRs mediating synaptic transmission at these sites. nAChRs at the muscle endplate and in sympathetic ganglia could be distinguished by their respective preferences for C10 and C6 polymethylene bistrimethylammonium compounds, notably decamethonium and hexamethonium.7 This provided the first evidence that muscle and neuronal nAChRs are structurally different.

    In the 1970s, elucidation of the structure and function of the muscle nAChR, using biochemical approaches, was facilitated by the abundance of nicotinic synapses akin to the muscle endplate in electric organs of the electric ray, Torpedo, and eel, Electrophorus. High affinity snake α-toxins, including α-bungarotoxin (α-Bgt), enabled the nAChR protein to be purified and subsequently resolved into 4 different subunits, designated α,β,γ and δ.8 An additional subunit, ε, was subsequently identified in adult skeletal muscle. In the early 1980s, these subunits were cloned and the era of the molecular analysis of nAChRs commenced. The muscle endplate nAChR has the subunit combination and stoichiometry (α1)2β1εδ, whereas the extrajunctional nAChR (α1)2β1γδ predominates in foetal or denervated muscle, and (muscle-derived) electric organs. The high density of nAChRs in Torpedo electric organ has facilitated high resolution structural studies using electron microscopy.9 Together with biochemical and biophysical approaches to studying structure-function relationships, this has resulted in a detailed molecular description of the nAChR.1

    Molecular Architecture of the nAChR (Figure 1) Each of the five subunits comprising the nAChR span the lipid bilayer to create a water-filled pore. Each subunit consists of 4 transmembrane segments, the second transmembrane segment (M2) lines the ion channel. The extracellular N-terminal domain of every subunit contains a ‘cys-loop’ that is the signature sequence of this LGIC family: two cysteine residues, separated by 13 amino acids (Cys 128, 142, Torpedo α subunit numbering), form a disulphide

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    Tocris Bioscience Scientific Review Series

    bond to create a loop that has been implicated in the transduction of agonist binding into channel opening.10 The principal agonist binding site resides in the N-terminal domain of α subunits, close to a pair of adjacent (‘vicinal’) cysteine residues (Cys 192, 193, Torpedo numbering) that define an α subunit. Mutagenesis and photoaffinity labelling experiments have highlighted the importance of 4 aromatic residues (Tyr 93, Trp 149, Tyr 190, Tyr 198, Torpedo numbering), consistent with 3 polypeptide loops of the α subunit (loops A-C) contributing to the primary agonist binding site (see Figure 1).11 The adjacent subunit (γ/ε or δ) also contributes to the binding site (complementary site: ‘loops’ D-F, now recognised to be mostly β strands). One consequence of this is that the αγ/ε and αδ binding sites are not identical with respect to ligand affinity.1 However, occupancy of both binding sites is required to open the channel.

    Knowledge of ligand binding to nAChRs has been greatly augmented by the crystal structure of an ACh binding protein first identified in the snail Lymaea stagnalis and subsequently also cloned from Aplysia

    californica and Bulinus truncatus.12,13 Each subunit of this pentameric secreted protein is homologous to the N-terminal domain of a nAChR subunit, with conservation of all the residues implicated in ACh binding to muscle nAChRs. These proteins provide a high resolution view of the extracellular portion of the receptor, notably of the binding sites at the interface between adjacent subunits, and the interaction of agonists with these sites.10

    Upon agonist binding, nAChRs undergo an allosteric transition from the closed, resting conformation to an open state that allows an influx of Na+, and to a lesser extent Ca2+, and an efflux of K+ under normal physiological conditions. In the closed state the ion channel is occluded by a ‘hydrophobic girdle’ that constitutes a barrier to ion permeation. Agonist binding in the extracellular domain promotes a conformational change that results in a rotational movement of the M2 helices lining the pore. Twisting of the girdle widens the pore by ~3 Å, sufficient for ion permeation.9 At the muscle endplate, the ensuing depolarisation elicits muscle contraction. Despite the

    Figure 1 | General structure of nAChRs1

    M2 lines the channel

    ACh binding protein

    M2 M3 M4

    C

    C C

    N

    Cys-loop

    Primary binding site: α

    Complementary binding site: γ/(δ)

    Agonist / Competitive Antagonist

    Non-competitive Antagonist

    Positive Allosteric Modulator

    Channel Blocker B

    C Nic

    A

    E

    F

    D W55/(57)

    Y117Y111

    Y151

    Y190

    Y198 Y93

    C192 C193

    W149

    W86

    D180/(182)

    M1

    Ca2+, Na+

    K+

    a) b)

    c)

    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.

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    Nicotinic Receptors

    presence of agonist, the nAChR channel closes within seconds to minutes, to enter a desensitised state. In this condition, the nAChR is refractory to activation. Multiple desensitised states have been proposed to exist.14 In the active (open) conformation, the nAChR binds agonists with low affinity (Figure 2; e.g. Kd for 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.

    Sites on the Muscle nAChR for Ligand Interactions (Figure 1) In addition to agonists binding to the agonist binding sites in the extracellular domain, competitive antagonists also bind at or close to these sites, preventing access to agonists. Their antagonism can be overcome by increasing the agonist concentration (unless the antagonist binds irreversibly, as is the case for α-Bgt), hence competitive antagonism is referred to as ‘surmountable’. The concentration of competitive antagonist necessary for nAChR blockade will depend on the experimental conditions. Non-competitive antagonists bind to sites distinct fro