NEURONAL NICOTINIC RECEPTORS Dr Christopher G V Sharples and preparations lend themselves to physiological and pharmacological investigations, and there followed a Professor Susan Wonnacott period of intense study of the properties of nAChR- mediating transmission at these sites. nAChRs at the Department of Biology and Biochemistry, muscle endplate and in sympathetic ganglia could be University of Bath, Bath BA2 7AY, UK distinguished by their respective preferences for C10 and C6 polymethylene bistrimethylammonium Susan Wonnacott is Professor of compounds, notably decamethonium and Neuroscience and Christopher Sharples is a hexamethonium,5 providing the first hint of diversity post-doctoral research officer within the among nAChRs. Department of Biology and Biochemistry at Biochemical approaches to elucidate the structure the University of Bath. Their research and function of the nAChR protein in the 1970’s were focuses on understanding the molecular and facilitated by the abundance of nicotinic synapses cellular events underlying the effects of akin to the muscle endplate, in electric organs of the acute and chronic nicotinic receptor electric ray,Torpedo , and eel, Electrophorus . High stimulation. This is with the goal of affinity snakea -toxins, principallyaa -bungarotoxin ( - Bgt), enabled the nAChR protein to be purified, and elucidating the structure, function and subsequently resolved into 4 different subunits regulation of neuronal nicotinic receptors. designateda ,bg , and d .6 An additional subunit, e , was subsequently identified in adult muscle. In the early 1980’s, these subunits were cloned and sequenced, The nicotinic acetylcholine receptor (nAChR) arguably and the era of the molecular analysis of the nAChR has the longest history of experimental study of any commenced. The muscle endplate nAChR has the receptor, and is the prototype ligand-gated ion subunit combination and stoichiometry (a 1)bed 1 , channel (LGIC). nAChRs in muscle and, to a lesser 2 whereas the extrajunctional nAChR (a 1)2bgd 1 extent, in autonomic neurones have been predominates in foetal or denervated muscle. characterised in detail. In contrast, nAChRs in the CNS have only lately become the focus of intense Neuronal nAChRs research efforts. The family of neuronal nAChRs in the brain and spinal cord is increasingly recognised to The biochemical and molecular approaches to have therapeutic potential in a wide range of studying the muscle nAChR eclipsed interest in conditions, yet the physiological functions of these neuronal nAChRs, as no model systems existed for nAChRs remain enigmatic and their subunit these receptors. The credentials for ganglionic composition, assembly, trafficking and regulation, and nAChRs were firmly established from the significance of their heterogeneity, are still to be electrophysiological and pharmacological approaches fully elucidated. Progress in these endeavours has but the notion of nAChRs in the brain was viewed with been hampered by a dearth of subtype-selective scepticism. Whilea -Bgt, the defining ligand of muscle tools. Historically, Nature has provided the majority of nAChRs, specifically bound to brain tissue, it did not nAChR-selective compounds, but the nicotinic antagonise central cholinergic functions.7 However, pharmacopoeia is now expanding with a number of the tobacco smoking habit was increasingly accepted novel synthetic ligands that have been developed in to be an addiction to nicotine, implying that nicotine the last few years. exerts a powerful central effect. In 1980, [3 H]-nicotine was reported to specifically label sites in the rat brain Historical perspective1 that have a unique nicotinic pharmacology.83 [ H]- The use of nicotine as an experimental tool was Nicotine binding was not blocked bya -Bgt, and the pivotal in Langley’s formulation of the concept of a anatomical distributions of binding sites for these two nicotinic ligands were quite distinct, raising the (then) ‘receptive substance’.2 In these studies he showed that ‘nicotine causes tonic contraction of certain novel and controversial prospect of nAChR muscles of fowl, frog and toad, and that this heterogeneity in the brain. The first publication of a contraction is prevented… by curare’. Subsequently, cloned neuronal nAChR subunit (a 3) appeared in Loewi and Dale developed the theory of 1986.9,10 The nAChR credentials of thea -Bgt binding neurohumoural transmission, and recognised site in neurones were conclusively provided by acetylcholine (ACh) as an endogenous transmitter.3 cloning of thea 7 nAChR subunit gene in 1990.11 The Dale distinguished the actions of muscarine and current tally of neuronal nAChR subunits in mammals nicotine, leading to the recognition of two is eleven (aaaa 2- 7, 9, 10,bb 2- 4), with an additional pharmacologically distinct (and now known to be subunit,a 8, identified in avian species.12,13 structurally and functionally unrelated) families of receptors for ACh, that take their names from these A distinct but related gene family ofa andb subunits natural products.4 Neuromuscular and ganglionic has been revealed in invertebrates.14 The C. elegans Tocris Cookson Ltd., UK Tocris Cookson Inc., USA Tel: + 44 (0)117 982 6551 Tel: (800) 421-3701 Fax: + 44 (0)117 982 6552 www.tocris.com Fax: (800) 483-1993 e-mail: [email protected] [email protected] e-mail: [email protected] genome sequence incorporates 17 candidate nAChR nAChRs are present (albeit in relatively low subunits; nAChRs in nematodes are the target for the abundance) throughout the central and peripheral anthelmintic levamisole. In insects, nAChRs are the nervous systems. In contrast to their pivotal role in principal excitatory receptors in the CNS, and autonomic neurotransmission and in the initiation of constitute a major pesticide target (e.g. for muscle contraction, nAChRs in the CNS are imidacloprid). Here we will focus on vertebrate considered to exert a more modulatory influence.16,17 nAChRs; the reader is referred to a recent review14 Neuronal nAChRs are gaining credence as significant for a comprehensive account of insect nAChRs. players in the nervous system from their relationship with a number of disease states in which they are Heterogeneity of neuronal nAChR subunits and perceived as novel drug targets.18 These include subtypes Alzheimer’s and Parkinson’s diseases, schizophrenia, nAChRs are pentameric LGICs and members of the Tourette’s Syndrome and attention deficit disorder. In superfamily of neurotransmitter receptors that addition, neuronal nAChRs are also targets for includes the GABA , glycine and 5-HT receptors.15 analgesia, anxiolysis, neuroprotection and smoking A3 cessation. Figure 1. nAChR structure and heterogeneity A. Structure of a neuronal nAChR subunit. N-Terminal Glycosylation Cys Cys C-Terminal M1 M2 M3 M4 P Phosphorylation B. Structural overview of a nAChR with one subunit removed revealing channel lumen. Shown are binding sites for agonists (including agonist binding site loop model), competitive antagonists, non-competitive antagonists and positive allosteric modulators.38 A F Agonist/Competitive Non-competitive antagonist antagonist interacting with B E K+ extracellular domain CD Positive allosteric modulator a b subunit subunit AGONIST BINDING SITE LOOP MODEL Channel blocking non-competitive antagonist Ca2+/Na+ C. Major nAChR subtypes present in the CNS and peripheral nervous system (PNS). Blue circles represent putative agonist binding sites at subunit interfaces. b2 a7 b4 a4 a4 a7 a7 a3 a3 b2 b2 a7 a7 a5 bx CNS CNS/PNS PNS 2 Neuronal nAChRs are composed of pentameric (M1-M4) that transverse the plasma membrane combinations ofab and subunits, giving the potential (Figure 1A); evidence indicates that M2 isa -helical for a great variety of native neuronal nAChRs.12 and lines the cation pore.1,37 M3 and M4 are Heterologous expression of nAChRs in Xenopus separated by a large, variable, intracellular loop which oocytes and mammalian cell lines has established contains putative phosphorylation sites for Ser/Thr certain rules of assembly that limit the subtypes of kinases. native nAChRs that may exist. Pairwise combinations Five subunits assemble to form a pentameric nAChR. ofab 2,aa 3 or 4 subunits with 2 and b 4 subunits can Cryo-electron microscopy at 4.6Å resolution of form functional nAChRs, whereas thea 5 andb 3 Torpedo nAChRs in a resting, non-activated state has subunits are generally unable to participate revealed a membrane spanning protein in which the successfully in pairwise combinations, but they have bulk of the structure is extracellular.39 It been expressed in heteromers with at least two other accommodates a cation conducting pore which types of subunits.12,19,20,21,22 Thea 6 subunit has been narrows to a putative gate within the membrane shown to form a functional nAChR in combination bilayer. Two agonist binding sites are located at the withb 4, but higher levels of expression were interfaces of theTorpedo aa /gd and / subunits observed whena 6 was co-expressed with at least (analogous toa /b subunit interfaces in neuronal two other types of subunit, includingb 3.23,24 In nAChRs, Figure 1C), approximately 20-40 Å from the contrast, theaa 7, 8 and a 9 subunits are ion channel.37,40 Most features deduced from this distinguished by their ability to form robust homomeric moderately resolvedTorpedo nAChR structure have receptors in expression systems, althoughaa 7 8 been confirmed with the determination of a high heteromers also occur in avian tissues.11,25,26,27