General

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 [29]. Sixteen subunits (see tabulated entries) have been identified in mammals [29] 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) [25].

Functional roles

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 [9]. 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 [38]. 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 [20]. The different subunits are believed to have arisen by gene duplication events [22]. 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 [30]. The Cys-loop is believed to be important in the transduction of agonist binding into channel opening [33]. nAChR subunits designated ‘α’ are unique in having a pair of adjacent cysteine residues in the extracellular N-terminal domain [20]. 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 [8]. 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 [22]. 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 [29]. 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 [28].

Subunit stoichiometry

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 [20]. 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 [20]. 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’ [21]. All neuronal α and β subunits can also occupy the 5th position and thereby influence receptor properties [29]. 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.

Concluding remarks

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 contain many additional details and references.

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4. Arias HR, Richards VE, Ng D, Ghafoori ME, Le V, Mousa SA. (2009) Role of non-neuronal nicotinic acetylcholine receptors in angiogenesis. Int J Biochem Cell Biol, 41 (7): 1441-51. [PMID:19401144]

5. Baenziger JE, Corringer PJ. (2011) 3D structure and allosteric modulation of the transmembrane domain of pentameric ligand-gated ion channels. Neuropharmacology, 60 (1): 116-25. [PMID:20713066]

6. Campbell NR, Fernandes CC, Halff AW, Berg DK. (2010) Endogenous signaling through alpha7-containing nicotinic receptors promotes maturation and integration of adult-born neurons in the hippocampus. J Neurosci, 30 (26): 8734-44. [PMID:20592195]

7. Champtiaux N, Changeux JP. (2004) Knockout and knockin mice to investigate the role of nicotinic receptors in the central nervous system. Prog Brain Res, 145: 235-51. [PMID:14650919]

8. Corringer PJ, Le Novère N, Changeux JP. (2000) Nicotinic receptors at the amino acid level. Annu Rev Pharmacol Toxicol, 40: 431-58. [PMID:10836143]

9. Dajas-Bailador F, Wonnacott S. (2004) Nicotinic acetylcholine receptors and the regulation of neuronal signalling. Trends Pharmacol Sci, 25 (6): 317-24. [PMID:15165747]

10. Dani JA, Bertrand D. (2007) Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Annu Rev Pharmacol Toxicol, 47: 699-729. [PMID:17009926]

11. Dellisanti CD, Yao Y, Stroud JC, Wang ZZ, Chen L. (2007) Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution. Nat Neurosci, 10 (8): 953-62. [PMID:17643119]

12. Elgoyhen AB, Vetter DE, Katz E, Rothlin CV, Heinemann SF, Boulter J. (2001) alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells. Proc Natl Acad Sci USA, 98 (6): 3501-6. [PMID:11248107]

13. Fowler CD, Arends MA, Kenny PJ. (2008) Subtypes of nicotinic acetylcholine receptors in nicotine reward, dependence, and withdrawal: evidence from genetically modified mice. Behav Pharmacol, 19 (5-6): 461-84. [PMID:18690103]

14. Gill JK, Savolainen M, Young GT, Zwart R, Sher E, Millar NS. (2011) Agonist activation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site. Proc Natl Acad Sci USA, 108 (14): 5867-72. [PMID:21436053]

15. Gotti C, Clementi F, Fornari A, Gaimarri A, Guiducci S, Manfredi I, Moretti M, Pedrazzi P, Pucci L, Zoli M. (2009) Structural and functional diversity of native brain neuronal nicotinic receptors. Biochem Pharmacol, 78 (7): 703-11. [PMID:19481063]

16. Grady SR, Salminen O, Laverty DC, Whiteaker P, McIntosh JM, Collins AC, Marks MJ. (2007) The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of mouse striatum. Biochem Pharmacol, 74 (8): 1235-46. [PMID:17825262]

17. Grando SA. (2008) Basic and clinical aspects of non-neuronal acetylcholine: biological and clinical significance of non-canonical ligands of epithelial nicotinic acetylcholine receptors. J Pharmacol Sci, 106 (2): 174-9. [PMID:18285656]

18. Harpsøe K, Ahring PK, Christensen JK, Jensen ML, Peters D, Balle T. (2011) Unraveling the high- and low-sensitivity agonist responses of nicotinic acetylcholine receptors. J Neurosci, 31 (30): 10759-66. [PMID:21795528]

19. Jensen AA, Frølund B, Liljefors T, Krogsgaard-Larsen P. (2005) Neuronal nicotinic acetylcholine receptors: structural revelations, target identifications, and therapeutic inspirations. J Med Chem, 48 (15): 4705-45. [PMID:16033252]

20. Karlin A. (2002) Emerging structure of the nicotinic acetylcholine receptors. Nat Rev Neurosci, 3 (2): 102-14. [PMID:11836518]

21. Kuryatov A, Onksen J, Lindstrom J. (2008) Roles of accessory subunits in alpha4beta2(*) nicotinic receptors. Mol Pharmacol, 74 (1): 132-43. [PMID:18381563]

22. Le Novère N, Changeux JP. (1995) Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells. J Mol Evol, 40 (2): 155-72. [PMID:7699721]

23. Liu Z, Zhang J, Berg DK. (2007) Role of endogenous nicotinic signaling in guiding neuronal development. Biochem Pharmacol, 74 (8): 1112-9. [PMID:17603025]

24. Livingstone PD, Wonnacott S. (2009) Nicotinic acetylcholine receptors and the ascending dopamine pathways. Biochem Pharmacol, 78 (7): 744-55. [PMID:19523928]

25. Lukas RJ, Changeux JP, Le Novère N, Albuquerque EX, Balfour DJ, Berg DK, Bertrand D, Chiappinelli VA, Clarke PB, Collins AC et al.. (1999) International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharmacol Rev, 51 (2): 397-401. [PMID:10353988]

26. Maehle AH, Prüll CR, Halliwell RF. (2002) The emergence of the drug receptor theory. Nat Rev Drug Discov, 1 (8): 637-41. [PMID:12402503]

27. Mazzaferro S, Benallegue N, Carbone A, Gasparri F, Vijayan R, Biggin PC, Moroni M, Bermudez I. (2011) Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity. J Biol Chem, 286 (35): 31043-54. [PMID:21757735]

28. Millar NS. (2008) RIC-3: a nicotinic acetylcholine receptor chaperone. Br J Pharmacol, 153 Suppl 1: S177-83. [PMID:18246096]

29. Millar NS, Gotti C. (2009) Diversity of vertebrate nicotinic acetylcholine receptors. Neuropharmacology, 56 (1): 237-46. [PMID:18723036]

30. Miller PS, Smart TG. (2010) Binding, activation and modulation of Cys-loop receptors. Trends Pharmacol Sci, 31 (4): 161-74. [PMID:20096941]

31. Palma E, Bertrand S, Binzoni T, Bertrand D. (1996) Neuronal nicotinic alpha 7 receptor expressed in Xenopus oocytes presents five putative binding sites for methyllycaconitine. J Physiol (Lond.), 491 ( Pt 1): 151-61. [PMID:9011607]

32. Rucktooa P, Smit AB, Sixma TK. (2009) Insight in nAChR subtype selectivity from AChBP crystal structures. Biochem Pharmacol, 78 (7): 777-87. [PMID:19576182]

33. Sine SM, Engel AG. (2006) Recent advances in Cys-loop receptor structure and function. Nature, 440 (7083): 448-55. [PMID:16554804]

34. Smit AB, Brejc K, Syed N, Sixma TK. (2003) Structure and function of AChBP, homologue of the ligand-binding domain of the nicotinic acetylcholine receptor. Ann N Y Acad Sci, 998: 81-92. [PMID:14592865]

35. Steinlein OK, Bertrand D. (2008) Neuronal nicotinic acetylcholine receptors: from the genetic analysis to neurological diseases. Biochem Pharmacol, 76 (10): 1175-83. [PMID:18691557]

36. Unwin N. (2005) Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J Mol Biol, 346 (4): 967-89. [PMID:15701510]

37. Vetter DE, Katz E, Maison SF, Taranda J, Turcan S, Ballestero J, Liberman MC, Elgoyhen AB, Boulter J. (2007) The alpha10 nicotinic acetylcholine receptor subunit is required for normal synaptic function and integrity of the olivocochlear system. Proc Natl Acad Sci USA, 104 (51): 20594-9. [PMID:18077337]

38. Wessler I, Kirkpatrick CJ. (2008) Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans. Br J Pharmacol, 154 (8): 1558-71. [PMID:18500366]

39. Young GT, Zwart R, Walker AS, Sher E, Millar NS. (2008) Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site. Proc Natl Acad Sci USA, 105 (38): 14686-91. [PMID:18791069]