79Primer on the Autonomic Nervous System. DOI: © 2012 Elsevier Inc. All rights reserved.201210.1016/B978-0-12-386525-0.00016-0

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Nicotinic acetylcholine receptors are members of a large pentameric super family of ligand-gated ion channel receptors that include the 5-hydoxytryptamine3 (5-HT3), glycine, γ-aminobutyric acid (GABA), and Zn-activated families of receptors along with several invertebrate and prokaryotic receptors [1–4]. Less closely related are the ligand-gated ion channels that respond to the excitatory amino acids and adenosine. Owing to the abundance of nicotinic receptors in the electric fish and findings show-ing that several peptide toxins that block motor activity bind to subtypes of nicotinic receptor with high affinity and selectivity, the nicotinic receptor was the first pharma-cologic receptor to be purified and the cDNAs encoding its subunits cloned. Appropriately, the nicotinic receptor became the prototype for the ligand-gated ion channel family.

STRUCTURAL CONSIDERATIONS

Nicotinic receptors assemble as pentamers of individ-ual subunits. Assembly occurs in a precise manner and order such that the assembled subunits encircle an inter-nal membrane pore and the extracellular vestibule lead-ing to the pore. The replicated pattern of front to back assembly of subunits insures that identical subunit inter-faces are formed from the pentameric assembly of identi-cal subunits. Moreover, the amino acid residues found at homologous positions in the receptors with heteromeric subunit assemblies should occupy the same position in three dimensional space (Fig. 16.1A).

Each subunit encodes a protein with four transmem-brane spans. The amino-terminal ~210 amino acids lie in the extracellular domain followed by three tightly threaded transmembrane spans. A relatively large cyto-plasmic loop is found between transmembrane spans 3 and 4 with a short carboxyl terminus winding up on the extracellular side. The overall structure has been detailed in a series of electron microscopy imaging studies (Fig. 16.2) and reveals a large extracellular domain, a wide diameter vestibule on the extracellular side and the gorge constriction region controlling the gating function to exist within the transmembrane spanning region [3]. Rapid and slow binding of ligands appear to induce shape changes within the receptor structure reflective of the individual

conformational states that lead to activation and desensi-tization [1–5].

More recently, a soluble protein, termed the acetylcho-line binding protein, exported from glial cells of snails, has been shown to bind acetylcholine and many of the clas-sical nicotinic agonists and antagonists [6]. Homologous proteins have been found in other invertebrate species as well. This protein, with identical subunits of slightly over 200 amino acids, is composed of residues with homology to the amino-terminal, extracellular domain of family of nicotinic receptors. Its structural characterization by X-ray crystallography (Fig. 16.3) shows it to be pentameric and to contain an arrangement of amino acid residues consis-tent with findings of protein modification and site-specific mutagenesis studies conducted with acetylcholine recep-tors isolated from neuronal and muscle systems [5,6]. The binding protein protein is homomeric, and its five binding sites reside at the five subunit interfaces with binding site determinants coming from both of the proximal subunit surfaces. The ligands bind from an outer radial direction [6,7] and enlodge behind a loop that contains selective binding determinants and proximal cysteines at or near its tip (Fig. 16.3). Hence, consistent with other proteins that exhibit homotropic cooperativity, the binding protein and the nicotinic receptors have their binding sites located at the subunit interfaces.

SUBTYPE DIVERSITY OF NICOTINIC RECEPTORS

The receptor from skeletal muscle and its homolo-gous forms in the fish electric organ consist of four dis-tinct subunits, where the pentamer has two copies of the α subunit and one copy of β, γ and δ subunits. When mus-cle becomes innervated, the γ subunit is replaced by an subunit. Within this pentameric structure, the two ace-tylcholine binding sites exist at the αδ and αγ() subunit interfaces. Opposing faces of the homologous α and γ, δ or subunits make up the two binding sites.

The subunit compositions of the receptors expressed in the nervous system are far more complex where we find nine different α subtypes and four different β sub-types [8] (Table 16.1). The α subtypes have been defined as those containing vicinal cysteines on a loop on which

Nicotinic ReceptorsPalmer Taylor

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reside several binding site determinants. All α subunits, except α5, use the face with the vicinal cysteines to form the binding site. The β subunits which lack this sequence and presumably the α5 subunit use the opposite or com-plementary face to form the binding site (Fig. 16.1).

The α subunits, α2, α3, α4 and α6, combine with certain β subunits, mainly β2 and β4, to form pentameric recep-tors. α5 has the capacity to substitute for a β subunit in the pentameric assembly. Typically, it is thought that two of the neuronal α subunits assemble with three β subunits. Hence, typical stoichiometries (subscripted) would be α2β3 or α2α51β3 where the generic α subtypes are usually α3 or α4 and the β subtypes β2 or β4. Each of these hetero-meric receptors would have two binding sites. Receptors composed of α7 subunits appear as functional homomeric entities where five copies of a single subunit confer func-tion, when assembled. α9 and α10 subunits assemble uniquely in a pentamer and may control nociceptive responses.

While the assembly patterns of the neuronal nicotinic receptor subunits are diverse, only certain combinations yield functional receptors, and particular combinations seem to be prevalent within regional areas of the nervous system. For example, pentamers of α3β4 are prevalent in ganglia of the autonomic nervous system, α4β2 is most prevalent in the central nervous system, and α7 appears widely distributed [8]. Central and peripheral nicotinic receptors play a discrete role in nicotine addiction and cardiovascular actions [9]. Mutations in nicotinic recep-tors give rise to congenital myasthenic syndromes [10] and other manifestations that affect autonomic and central ner-vous system function.

ELECTROPHYSIOLOGIC EVENTS ASSOCIATED WITH RECEPTOR

ACTIVATION

All of the nicotinic receptors that are well character-ized, to date, are cationic channels resulting in depolariza-tion after activation. They differ substantially with respect to the permeabilities of the open channel state to Na and Ca. Na permeability is most effective in initiating a rapid depolarization, whereas Ca permeability could

α1

γ β1

α1

αβγδAChR

α4 α

α

αα

αβ2

αβNeuronal AChR

β2

β2α4

αAChR

H2N

COOH

1 2 3 4

δ

(A)

(B)

FIGURE 16.1 Structure of the nicotinic acetylcholine receptor. (A) Arrangement of receptor subunits as pentamers in muscle and neuro-nal receptors. The muscle receptor exists as a pentamer with two copies of α1 and one of β, γ and δ, as seen for the receptor found in embryonic muscle. The binding sites as designated are found at the αγ and αδ inter-faces. In the case of the innervated receptor in skeletal muscle, an sub-unit of different composition replaces γ at the same position. Two types of neuronal receptors are shown: the heteromeric type is thought to have usually two copies of α, where α is α2,α3,α4, or α6, and three copies of β, where β can be β2, β3 or β4. α5 is thought to substitute for a β subunit at one of the non-binding positions. The homomeric neuronal receptors are made up of α7 pentamers. α7 subunits may form heteromeric sub-types with certain β subunits remains open, but not proven. α9 and α10 subunits associate with each other in presumed variable ratios to form pentamers. (B) Threading pattern of the α-carbon chain of the subunits of the nicotinic receptor. The first ~210 amino acids form virtually all of the extracellular domain, contain the residues on the primary and com-plementary subunit interfaces forming the binding site determinants and govern the assembly process. Transmembrane span 2 from the five subunits forms the inner perimeter of the channel and is involved in the ligand gating. The other transmembrane segments contribute to the structural integrity of the receptor. The extended segment between trans-membrane spans 3 and 4 forms the bulk of the cytoplasmic domain.

TABLE 16.1 subtypes of Nicotinic Acetylcholine Receptors found in Muscle and Neuronal systems

Composition Assembly

Muscle α1, β1, γ, δ, α2βγδ or α2βδNeuronal α2 α6 β2 Various

pentameric assemblies of α and β or α alone

α3 α7 β3α4 β4α5 α9

α10

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serve as an intrinsic activating function in cell signaling or in triggering an excitation step. However, depolarization per se through an increase in Na permeability can also mobilize Ca through voltage sensitive Ca channels. Typically postsynaptic nicotinic receptors, such as those found in ganglia and the neuromuscular junction, function through the simultaneous occupation of the receptor by more than a single agonist molecule. Agonist association and the ensuing channel opening reveals positive coopera-tivity in ligand binding. These rapid binding events cause channel openings to occur in a millisecond time frame. Several openings and closings may occur during the short interval of agonist occupation, and the intrinsic efficacy of an agonist may relate to whether the ligand-receptor com-plex favors an open or closed state.

Upon continuous exposure to agonists, most recep-tors desensitize. Desensitization confers a receptor state wherein the agonist has a high affinity, but the receptor is locked in a closed channel state. Desensitization provides an additional means by which temporal responses to an agonist can be regulated.

Antagonists may be competitive or non-competitive. Competitive antagonists show binding that is mutually exclusive with agonists and exhibit a surmountable block of the receptor in which the dose–response curve to the agonist is shift rightward in a parallel fashion. Occupation by a single antagonist molecule is sufficient to block func-tion. Non-competitive antagonists typically block the channel through which ions pass in the open state of the receptor. In the case of ganglionic nicotinic receptors,

trimethaphan is a competitive agonist, while hexametho-nium and mecamylamine are non-competitive, therein blocking channel function.

Great interest has developed in allosteric sites where binding of a ligand at a non-agonist site, enhances or dimin-ishes agonist function and are termed allosteric activators or inhibitors. Hence, they are distinguished from orthosteric ligands that occupy the primary agonist or antagonist sites.

DISTRIBUTION OF NICOTINIC RECEPTORS

Nicotinic receptors are widely distributed in the cen-tral and peripheral nervous systems. In innervated skel-etal muscle they are found in very high density localized to the motor end plate. Also, certain motor neurons may contain receptors at the presynaptic nerve ending to con-trol release. In ganglia, the primary nicotinic receptor is found on the postsynaptic dendrite and nerve cell body. Others may exist presynaptically to control release from the presynaptic nerve ending. In the CNS, one finds that the majority of receptors are presynaptic or prejunctional. As such, they control the release of other transmitters or in the case of released acetylcholine, they play an autorecep-tor role. Presynaptic nicotinic receptors found in the spinal cord and in higher centers of the brain have a functional role in modulating central control of autonomic function and sensory to autonomic control of reflexes and other functions.

(A) (B) (C)M2

I

synapse

IV

EL

LL

V

VL

L

S

M

GE

K

S

N cytoplasmic face

synaptic face

� �

cytoplasm

� �

TP

D

TL

SV

I

L ST

F

PS

C

FIGURE 16.2 Overall dimensional characteristics of the nicotinic receptor. Image reconstruction of electron micrographs shows the receptor to be some ~140 Angstroms in length and perpendicular to the membrane. Its diameter is 80–90 Angstroms and contains a large central channel on the membrane surface. Rapid and prolonged exposure to acetylcholine followed by rapid freezing has yielded distinctive structures for the agonist bound, unligated and desensitized states of the receptor [3,6].

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References [1] Changeux J-P, Edelstein SJ. Nicotinic acetylcholine receptors.

New York: Odie Jacob; 2005. [2] Karlin A. Emerging structures of nicotinic acetylcholine receptors.

Nat Rev Neurosci 2002;3:102–14. [3] Changeux J-P. Allosteric receptors: From electric organ to cognition.

Annu Rev Pharmacol Toxicol 2010;50:1–38. [4] Thompson AJ, Lester HA, Lummis SCR. The structural basis of

function in Cys-loop receptors. Q Rev Biophys 2010;43:449–99. [5] Unwin N. Refined structure of the nicotinic acetylcholine receptor

at 4A resolution. J Mol Biol 2005;346:967–89. [6] Brejc K, van Dijk WJ, Klaasen RV, Shuurmans M, van der Oost J,

Smit AB, et al. Crystal structure of an acetylcholine binding protein

FIGURE 16.3 X-ray crystallographic structure of the acetylcholine binding protein. The structure was produced from crystallographic coordinates of Sixma and colleagues [5]. Surfaces are represented as Connolly surfaces. Left, colors are used to delineate the subunit interfaces in the homomeric pentamer. The vestibule on the extracellular surface that represents the entry to the internal channel in the receptor is shown by the arrow. Right, a single subunit interface Note the residues that have been found to be determinants in the binding of agonists and alkaloid and peptidic antagonists to the receptor. Hence, the numbers delineate the probable binding surface(s) for large and small antagonists. These residues come from seven distinct segments of amino acid sequence in the subunit, as determined from extensive mutagenesis, labeling and crystallographic studies [3,5,6]. The right hand panel shows a superimposition of the acetylcholine binding protein with the presumed structure of the muscle receptor.

(A) (B)

reveals the ligand binding domain of nicotinic receptors. Nature (London) 2001;411:269–76.

[7] Hibbs RE, Sulzenbacher G, Shi J, Talley T, Conrod S, Kem WR, et al. Structural determinants for interaction of partial agonist with the acetylcholine binding protein and the neuronal alpha7 acetylcholine receptor. EMBO J 2009;28:3040–51.

[8] Various authors. Neuronal nicotinic receptors. In: Clementi F, Fornasi D, Gotti C, editors. Handbook of experimental pharmacol-ogy, vol. 144. Berlin: Springer-Verlag, 2000. 821 pp.

[9] Benowitz NL. Nicotine Addiction. N Eng J Med 2010;362:2295–303.[10] Engel AG, Shen X-M, Selcen D, Sine SM. What we have learned

from congenital myasthenic syndromes. J Mol Neurosci 2010;40:143–53.