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Nicotinic acetylcholine receptors: Introduction
Nicotinic acetylcholine receptors (nAChRs) are a family of multi-subunit transmembrane neurotransmitter receptors that are members of the ‘Cys-loop’ superfamily [1,33]. They serve diverse and often critical functions throughout the central and peripheral nervous systems, and have emerging roles in non-neuronal systems [10,38]. Nicotinic receptors contain five subunits and assemble into both heteromeric and homomeric pentamers . Sixteen subunits (see tabulated entries) have been identified in mammals  and are designated by Greek letters, followed by Arabic numerals (not subscript) to distinguish variants (α1-α7, α9-10, β1-β4, γ, δ, ε) [1,29]. Note: an α8 subunit (similar in subunit composition to the α7 subunit) is expressed in avian but not in mammalian species. Muscle cells and neurones express distinct complements of subunits, resulting in the classification of muscle-type and neuronal-type nAChRs. Receptor subtypes are named according to their known subunit composition (e.g. α1β1γδ-nAChRs (muscle-type), α4β2- and α7-nAChRs (neuronal-type)). An asterisk is employed as a 'wild-card' to indicate that one or more additional subunit type may also be present (e.g. α3β4*-nAChRs) .
nAChRs mediate some of the effects of the endogenous neurotransmitter acetylcholine (other effects of acetylcholine being mediated by muscarinic acetylcholine receptors), and these actions of acetylcholine at nAChRs are mimicked (to varying degrees) by the tobacco alkaloid nicotine (except in the case of α9/10 nAChRs where nicotine has only antagonist actions [12,26]). nAChRs mediate classical excitatory neurotransmission at the vertebrate neuromuscular junction, in autonomic ganglia, and at selected synapses in the brain and spinal cord[1,10]. Neuronal nAChRs more commonly have a modulatory role through the modulation of neurotransmitter release or second messenger systems; actions typically mediated by calcium signalling . Transgenic mice with subunit deletion, mutation or overexpression have been useful in defining the contribution of nAChR subtypes to specific functions [7,13].
Expression of certain nAChR subunits and receptor subtypes in brain and muscle is developmentally regulated, suggesting that nAChRs can influence (and/or may be influenced by) cell maturation and innervation [6,20,23]. An emerging literature indicates that certain nAChRs (principally neuronal-type) are expressed in a variety of other tissues and cell types, including glia, lymphocytes, fibroblasts, pulmonary neuroendocrine cells, spermatozoa, keratinocytes, granulocytes, chondrocytes, and placenta, as well as in several sensory organs . nAChRs are proposed to serve a modulatory function in these cells [4,17].
RECEPTOR SUBUNIT TOPOGRAPHY AND ASSEMBLY
nAChR subunits are structurally homologous and share the same topographical features as subunits of other ‘Cys-loop’ receptors such as ionotropic glycine, γ-aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) receptors, and invertebrate glutamate-gated Cl- channels. These features include an extensive extracellular N-terminal domain, four transmembrane segments (TM1-TM4), a cytoplasmic loop of variable length and amino acid sequence between the third and fourth transmembrane segments, and a short extracellular C-terminus . The different subunits are believed to have arisen by gene duplication events . The N-terminal domain contains a 15 amino acid loop delimited by two cysteine residues that form a disulphide bridge: this is the ‘Cys-loop’ that defines all subunits in this receptor family . The Cys-loop is believed to be important in the transduction of agonist binding into channel opening . nAChR subunits designated ‘α’ are unique in having a pair of adjacent cysteine residues in the extracellular N-terminal domain . These cysteines reside at, or close to, the binding site for ACh and other agonists. Biochemical and mutagenesis studies established that this binding site occurs at the interface between an α subunit and its neighbour, with each contributing three non-contiguous loops of key aromatic amino acid residues to the binding pocket . The binding site model has been corroborated by structural studies on a novel soluble acetylcholine binding protein that shares homology with the extracellular domain of a nAChR [32,34]. Other, modulatory sites on nAChRs are also emerging: for example binding sites for positive allosteric modulators [14,39].
Structural studies have confirmed that nAChRs are assembled from five subunits, arranged like staves of a barrel to create a central pore lined by the TM2 segment of each of the constituent subunits [5,36]. Of the mammalian nAChR subunits, only α7 and α9 can assemble as homopentamers (and only α7 is believed to form native homomeric receptors, while α9 pairs with the α10 subunit in native receptors) [29,37]. This property is believed to reflect the ancestral position of these subunits in phylogenetic analyses of the nAChR subunit family . All other functional nAChRs require combinations of α and non-α subunits. From heterologous expression systems, it is evident that not all subunits will cooperate to form a functional nAChR, and some combinations express more efficiently than others . The subunit composition of native nAChR has been more difficult to determine, but a combination of knockout mice and imunoprecipitation with subunit-specific antibodies has made a big contribution, for example with respect to unveiling the surprising diversity of β2* nAChR subtypes that modulate dopamine release in the rodent mesostriatal system [15-16,24]. Disparities between results for in vitro expression systems and native nAChR may indicate that subunit combinations introduced in a heterologous system never encounter each other in vivo, or are regulated in a cell-specific manner. On the other hand, the failure to express in heterologous systems nAChRs that are known to occur in nature suggests that additional factors crucial for assembly, maturation, trafficking, membrane insertion, stabilisation or function are absent from the experimental system [3,29]. The requirement for the chaperone RIC-3 for the effective expression of α7 nAChRs is a case in point .
By analogy with muscle nAChRs which have the stoichiometry α12β1γδ or α12β1δε, two α subunits are believed to be a minimum requirement, generating two agonist binding sites that must both be occupied by agonist for efficient channel opening to occur . The number of binding sites that must be occupied in a homomeric Cys-loop receptor (i.e. α7) is still debated [2,31]. As both α and non-α subunits contribute to the binding site, both determine the pharmacological profile of a heteromeric nAChR, and this is illustrated by the distinct differences exhibited by the two agonist binding sites of the immature muscle nAChR, conferred by αγ and αδ interfaces . Interestingly, the α5 subunit lacks certain key residues from the binding site loops and is incapable of contributing to an agonist binding site; like the β1 and β3 subunits, it can only occupy the 5th position in the pentamer. α5 and and β3 are referred to as ‘accessory subunits’ . All neuronal α and β subunits can also occupy the 5th position and thereby influence receptor properties . Indeed, the stoichiometry of neuronal nAChRs is emerging as a critical determinant of receptor properties. For example, (α4β2)2α4 nAChRs gain an additional binding site and altered function compared with (α4β2)2β2 nAChRs [18,27]. However, in contrast to muscle receptors, the precise stoichiomentry of native neuronal nAChRs remains largely unknown.
SUMMARY OF NATIVE SUBTYPES
Autonomic neurons (including SH-SY5Y, PC12 and IMR32 cell lines) typically express α3β4* (may include α5, β2) and α7 nAChRs.
Brain and spinal cord neurons express various complements of subunits, with α4β2* and α7 nAChRs being most abundant. Some subunits have very restricted expression patterns (e.g. α6 and β3 are restricted to catecholaminergic neurones and retina where they form α6β2β3* nAChRs). Expression patterns can vary between species (e.g. α2 is much more widespread in primate compared with rodent brain).
α9α10 nAChRs are limited to cochlear hair cells and some sensory neurons.
Presently the subunit composition and stoichiometry of nAChRs in other, non-neuronal cell types is not well-defined, although many subunits have been reported and α7 and α4β2* nAChRs have been implicated in some responses.
nAChRs continue to attract considerable interest: as the prototype ligand-gated ion channel providing superior structural and biophysical information relevant to other Cys-loop receptors [2,11,20], and from a clinical perspective of their involvement in disease and addiction to their potential for therapeutic interventions [19,35]. The selected reviews and research papers cited here are merely a snapshot to provide access to further details about nAChRs and bibliographies that highlight the very extended literature. The individual subunit entries that form part of the IUPHAR database contain many additional details and references.
To cite this family introduction, please use the following:
Neil S. Millar, Cecilia Gotti, Michael. J. Marks, Susan Wonnacott.
Nicotinic acetylcholine receptors, introductory chapter. Last modified on 07/12/2011. Accessed on 21/05/2013. IUPHAR database (IUPHAR-DB), http://www.iuphar-db.org/DATABASE/FamilyIntroductionForward?familyId=76.