The nicotinic acetylcholine receptor is the best characterized neurotransmitter receptor

The nicotinic receptor was purified about a decade before purification of other neurotransmitter receptors. The electric organ of Torpedo, consisting of stacks of electrocytes that have differentiated from tissue of embryonic origin common to that of skeletal muscle, is a rich source of nicotinic receptors. Upon differentiation, the electrogenic bud in the electrocyte proliferates, but the contractile elements atrophy. The excitable membrane encompasses the entire ventral surface of the electrocyte rather than being localized to small, focal junctional areas, as found in skeletal muscle. The electrical discharge in Torpedo relies solely on a PSEP resulting from depolarization of the postsynaptic membrane, rather than propagation from an action potential. The density of receptors in the Torpedo electric organ approaches 100 pmol/mg protein, which may be compared with 0.1 pmol/mg protein in skeletal muscle.

In the early 1960s, it was established that snake α-toxins, such as α-bungarotoxin, irreversibly inactivate receptor function in intact skeletal muscle, and this finding led directly to the identification and subsequent isolation of the nicotinic ACh receptor from Torpedo [3]. By virtue of their high affinity and very slow rates of dissociation, labeled α-toxins serve as markers of the receptor during solubilization and purification.

Purification of the nicotinic acetylcholine receptor facilitated examination of its overall structure

Antibodies were raised to the purified protein, and sufficient amino acid sequence of the receptor itself became available to permit the cloning and sequencing of the genes encoding the individual subunits of the receptor [4]. As a consequence of the high density of nicotinic ACh receptors in the postsynaptic membranes of Torpedo, sufficient order of the receptor molecules is achieved in isolated membrane fragments such that image reconstructions from electron microscopy have allowed a more detailed analysis of structure [24]. Finally, labeling of functional sites, determination of subunit composition and structure modification through mutagenesis contributed to our understanding of the structure of nicotinic receptors [25].

The nicotinic acetylcholine receptor consists of five subunits arranged around a pseudoaxis of symmetry

The subunits display homologous amino acid sequences with 30 to 40% identity of amino acid residues [4]. In muscle, one subunit, designated α, is expressed in two copies; the other three, β, γ and δ, are present as single copies (Fig. 11-7). Thus, the receptor is a pentamer of molecular mass of approximately 280 kDa. Structural studies show the subunits to be arranged around a central cavity, with the largest portion of the protein exposed toward the extracellular surface. The central cavity is believed to lead to the ion channel, which in the resting state is impermeable to ions; upon activation, however, it opens to a diameter of 6.5 Å. The open channel is selective for cations. The two α subunits and the opposing face of the γ and δ subunits form the two sites for binding of agonists and competitive antagonists and provide the primary surface with which the larger snake α-toxins associate. The sites for ligand binding are localized toward the external perimeter of each of the α subunits; occupation of both sites is necessary for receptor activation. Electrophysiological and ligand-binding measurements together with analysis of the functional states of the receptor indicate positive cooperativity in the association of agonists; Hill coefficients greater than unity have been described for agonist-elicited channel opening, agonist binding and agonist-induced desensitization of the receptor [3,25]. Noncompetitive inhibitor sites within various depths of the internal channel also have been defined and are the sites of local anesthetic inhibition of receptor function.

Figure 11-7

A: Longitudinal view of the muscle nicotinic acetylcholine receptor with the γ subunit removed. The remaining subunits, two copies of α, one of β and one of δ, surround an internal channel with outer vestibule and its constriction (more...)

Sequence identity among the subunits appears to be greatest in the hydrophobic regions. Various models for the disposition of the peptide chains have been proposed on the basis of hydropathy and reactivity of certain residues to modifying agents and antibodies (Fig. 11-8). Four candidate membrane-spanning regions are predicted, although only one clear α-helical segment is evident in the electron microscopic reconstruction of the channel [24]. All of these potential membrane-spanning domains appear after residue 210, with the amino-terminal portion of the molecule on the extracellular surface. The homology among the four subunits strongly suggests that the same folding pattern is found in all subunits.

Figure 11-8

Features of the sequence of the acetylcholine receptor. A: Schematic drawing of the sequence showing candidate regions for spanning the membrane. The region M₂ is believed to be an α helical segment and lines the internal pore of the receptor. (more...)

Site-directed labeling, chemical cross-linking, homology modeling, antibody association, fluorescence energy transfer and site-specific mutagenesis represent techniques that have made incremental contributions to the understanding of nicotinic receptor structure [25]. Analysis with techniques achieving atomic level resolution has not been possible for an integral membrane protein of this size.

A disulfide loop between Cys 128 and 142 in the α subunit is conserved in the entire receptor channel family (Fig. 11-8A). A second disulfide is found in the α subunits between vicinal Cys 192 and 193, and this structural feature has been used to identify α subunits. Early studies showed that reduction of the Cys 192–193 bond allowed for labeling by the site-directed sulfhydryl-reactive agonist and antagonist, respectively, bromoacetylcholine and m-maleimidobenzyl trimethylammonium (Fig. 11-8B) [25]. Subsequent studies involving photolytic labeling, labeling by the natural coral toxin lophotoxin and site-specific mutagenesis identified the region between residues 185 and 200 in the α subunit as being important for forming part of the agonist- and antagonist-binding surface. Two other segments of sequence in the α subunit and four discrete segments on the opposing face of the γ and δ subunits also have been identified as forming loops that contribute to the binding surfaces at the αγ and αδ interfaces [26].

Four candidate membrane-spanning regions are found after residue 210 with a large cytoplasmic loop between membrane spans 3 and 4 (Fig. 11-8A). Based on labeling experiments and site-specific mutagenesis, membrane span 2 was found to be proximal to the ion channel. This span, when constructed as an α helix, is amphipathic, with an abundance of serine and threonine residues pointed toward the channel lumen. Positions corresponding to α-Thr 244, α-Leu 251, α-Val 255 and α-Glu 262 in this transmembrane span have been labeled with the noncompetitive, channel-blocking inhibitors chlorpromazine and tetraphenyl phosphonium [3,25]. Mutation of several of the hydroxyl groups on residues at these positions affects channel kinetics. The channel gate, or constriction, is thought to lie deep within the channel either at the boxed leucine in Figure 11-8C or even farther to the cytoplasmic side. The ion selectivity of the channels appears to be controlled in part by rings of charges formed by all five subunits at the extracellular surface of the channel corresponding to α-Glu 262 and at the cytoplasmic exit corresponding to α-Glu241. Exposed amide backbone hydrogens and carbonyl groups and a ring of hydroxylated amino acids corresponding to α-Thr 244 also contribute to ion selectivity and permeation [25].

Analysis of the opening and closing events of individual channels has provided information about ligand binding and activation of the receptor

Electrophysiological studies utilize high-resistance patch electrodes of 1 to 2 μM diameter, which form tight seals on the membrane surface [27]. They have the capacity to record conductance changes of individual channels within the lumen of the electrode (see Chap. 10). The patch of membrane affixed to the electrode may be excised, inverted or studied on the intact cell. The individual opening events for ACh achieve a conductance of 25 pS across the membrane and have an opening duration that is distributed exponentially around a value of about 1 msec. The duration of channel opening is dependent on the particular agonist, whereas the conductance of the open-channel state is usually agonist-independent. Analyses of the frequencies of opening events have permitted an estimation of the kinetic constants for channel opening and ligand binding, and these numbers are in reasonable agreement with estimates of ligand binding and activation from rapid kinetic, or stopped-flow, studies. Overall, activation events can be described by Scheme 1 [3,27].

Two ligands (L) associate with the receptor (R) prior to the isomerization step to form the open-channel state L₂R*. For ACh, the forward rate constant for binding, k₊₁, is 1 to 2 × 10⁸ M ⁻¹ sec⁻¹; k₊₂ and k₋₂, forward and reverse rate constants for isomerization, yield rates of isomerization consistent with opening events in the millisecond time frame. Since k₊₂ and k₋₂ are greater than k₋₁, the rate constant for ligand dissociation, several opening and closing events with the fully liganded receptor occur prior to dissociation of the first ligand. Binding of the first and second ligands appears not to be identical, even allowing for the statistical differences arising from the two sites. Such a conclusion is consistent with receptor structure since different subunits, such as the γ and δ subunits in muscle, are adjacent to the same face of the α subunits in the pentamer.

Continued exposure of nicotinic receptors to agonist leads to desensitization of the receptors

This diminution of the response occurs even though the concentration of agonist available to the receptor has not changed. Katz and Thesleff examined the kinetics of desensitization with microelectrodes and found that a cyclic scheme in which the receptor existed in two states, R and R′, prior to exposure to the ligand best described the process.

To achieve receptor desensitization and activation by a single ligand, multiple conformational states of the receptor are required. The binding steps represented in horizontal equilibria are rapid; vertical steps reflect the slow, unimolecular isomerizations involved in desensitization (Scheme 2). Rapid isomerization to the open channel state (Scheme 1) should be added. To accommodate the additional complexities of the observed fast and slow steps of desensitization, additional states have to be included.

A simplified scheme, in which only one desensitized and one open-channel state of the receptor exist, is represented in Scheme 2, where R is the resting (activatable) state, R* the active (open channel) state and R′ the desensitized state of the receptor; M is an allosteric constant defined by R′/R, and K and K′ are equilibrium dissociation constants for the ligand.

In this scheme, M < 1 and K′ < K. Addition of ligand eventually will result in an increased fraction of R′ species due to the values dictated by the equilibrium constants. Direct binding experiments have confirmed the generality of this scheme for nicotinic receptors. Thus, distinct conformational states govern the different temporal responses that ensue on addition of a ligand to the nicotinic receptor. No direct energy input or covalent modification of the receptor channel is required.

Nicotinic receptor subunits are part of a large superfamily of ligand-gated channels

Nicotinic receptors on neurons, such as those originating in the CNS or neural crest, show ligand specificities distinct from the nicotinic receptor in the neuromuscular junction. One of the most remarkable differences is the resistance of most nicotinic neuronal receptors containing α2 through α6 subunits to α-bungarotoxin and related snake α-toxins. This fact and the lack of an abundant source of neuronal CNS receptors limited initial progress in their isolation and characterization. However, low-stringency hybridization with cDNAs encoding the subunits of electric organ and muscle receptors provided a means to clone neuronal nicotinic receptor genes. Isolation of the candidate cDNA clones, their expression in cell systems to yield functional receptors and the discrete regional localizations of the endogenous mRNAs encoding these receptor subunits revealed that the nicotinic receptor subunits are part of a large and widely distributed gene family. They are related in structure and sequence to receptors for inhibitory amino acids (GABA and glycine), to 5-hydroxytryptamine type 3 (5HT₃) receptors and, somewhat more distantly, to glutamate receptors.

At least 11 distinct genes encoding neuronal nicotinic receptor subunits α2 through α9 and β2 through β4 have been identified in the central and peripheral nervous systems (Fig. 11-2). The α subunits are similar in sequence to the muscle α1 subunit and contribute to the ligand-binding interface. The β subunits fulfill the role of β1, γ and δ subunits in the muscle receptor. When certain pairs or triplets of cDNAs encoding neuronal α and β subunits are cotransfected into cells or their corresponding mRNAs are injected into oocytes, characteristic ACh-gated channel function can be achieved. The α5 subunit appears unique in that it will not contribute to function in the absence of other α subunits; its global sequence features are more similar to those of the β subunits. The α7 and α8 subunits display function as homologous pentamers. Receptors containing α7 subunits have a high Ca²⁺ permeability, and Ca²⁺ entry may be integral to their function in vivo. While not all combinations of α and β mRNAs lead to the expression of functional receptors on the cell surface, the number of permutations is large [10,11,25]. A future challenge is the assignment of pharmacological and biophysical signatures to all of the subunit combinations found in vivo.

The α3 subunit is prevalent in peripheral ganglia, usually with β2, β4, and α5 subunits, while the α4β2 subunit combination predominates in the CNS. The α6 subunit appears to localize with biogenic amine-containing neurons, while α9 is found in vestibular sensory and cochlear hair cells. Receptors containing the α9 subunit may have some muscarinic receptor characteristics.

Substantial evidence points to nicotinic receptors in the CNS functioning at presynaptic locations to regulate release of several CNS transmitters [10]. Electrophysiological and microdialysis studies provide evidence that glutamatergic, dopaminergic, serotonergic, peptidergic and cholinergic pathways are under the control of presynaptic nicotinic receptors. Hence, nicotinic receptors appear to play an important amplification and modulatory role in the CNS.

Both nicotinic receptors and acetylcholinesterase are regulated tightly during differentiation and synapse formation

At present, we understand more about tissue-specific gene expression in muscle than in nerve [28,29]. Both of the above proteins show enhanced expression during myogenesis upon differentiating from a mononucleated myoblast to a multinucleated myotube. Curiously, enhanced receptor expression occurs largely by transcriptional activation, while the increase in cholinesterase expression arises from stabilization of the mRNA [30]. The receptor appears to cluster spontaneously, which involves a protein on the cytoplasmic side of the membrane, termed 43K or rapsyn [28,29]. This protein links the receptor to cytoskeletal elements and restricts its diffusional mobility. Following innervation and synaptic activity, expression of the receptor and AChE persists in endplate, or junctional, regions and disappears in extrajunctional regions. The collagen-tail-containing species of AChE is localized to the basal lamina in the neuromuscular synapse.

With innervation and the development of electrically excitable synapses, the γ subunit of the receptor is replaced by an ϵ subunit; small changes in the biophysical properties of the receptor occur concomitantly. Upon denervation, many of the developmental changes associated with innervation are reversed and there is again an increase in expression of extrajunctional receptors containing the γ subunit. In multinucleated muscle cells, particular subsynaptic nuclei drive the expression of these synapse-specific proteins. The factors controlling these regulatory events are incompletely understood, but calcitonin gene-related peptide (CGRP) and the protein ACh receptor-inducing activity (ARIA) may be extracellular mediators of expression. In addition, intracellular Ca²⁺, membrane depolarization and protein kinase C play distinct roles in maintaining junctional expression of synapse-localized proteins.

A neurally derived signaling protein, agrin, acts through a receptor tyrosine kinase, MuSK, in the formation of the specialized postsynaptic endplate by interaction with rapsyn. Thus, MuSK—rapsyn interactions are critical in forming the local scaffold for postsynaptic components in the motor endplate [29,31].