Nicotinic Receptors in Wonderland by Thomas Grutter and Jean-Pierre Changeux
This article will appear in a forthcoming issue of Trends in Biochemical Sciences.
Posted July 6, 2001 · Issue 106

Abstract

The structure of a soluble homopentameric homologue of the N-terminal extracellular domain of the nicotinic acetylcholine (ACh) receptor has recently been determined at the atomic level. These data reveal the three-dimensional structure of the binding site for ACh and nicotinic ligands. The ACh-binding sites are located at subunit boundaries in an equatorial position and are framed by residues previously identified in nicotinic receptors, by photoaffinity labeling and mutagenesis experiments, as being crucial for ligand binding. On this basis, a hypothetical mechanism for the allosteric transitions of the nicotinic receptors is suggested.

Despite the proposal by Langley (1905) of the concept of a 'receptive substance' mediating the effect of nicotine and curare on muscle, receptors remained enigmatic entities. Their identification was made possible largely by a few wonders of the animal world. First, the electric organ of fish offers both an extremely rich and homogeneous source of synaptic material and single cells (or electroplaques) for pharmacological studies, notably the response to acetylcholine (ACh) in vivo [1]. However, none of the many pharmacological agents, including thiol group reagents, tested in vivo by following the electrophysiological response of single electroplaques was selective enough to characterize the detergent-solubilized protein isolated from crude extracts of electric organs that bound ACh in vitro. Second, because of its exquisite selectivity and high affinity, an α-toxin from the venom of the snake Bungarus [2] enabled the isolation and purification of the nicotinic ACh receptor (nAChR) from the electric organ [3,4]. Finally, a soluble protein that binds nicotinic ligands and a-bungarotoxin has recently been discovered in a snail (Lymnaea stagnalis) from European fresh waters [5]. This ACh-binding protein (AChBP) is produced and stored in glial cells and is released in an ACh-dependent manner in the synaptic cleft where it regulates synaptic transmission [5]. X-ray crystallographic analysis of AChBP at 2.7 Å resolution reveals a striking resemblance between the AChBP structure and the organization for the ligand-binding domain of nAChR proposed from biochemical and structure prediction experiments [6].

Molecular Structure of Nicotinic Receptors

The nAChR from fish electric organ is a heteropentamer of ∼300 kDa with an (α1)2.b1.γ/ε.δ stoichiometry. The receptor has two distinct ACh-binding sites (located at interfaces between the α and γ, and the α and δ, subunits), a unique ion channel along the transmembrane axis of (pseudo)symmetry, and all the structural elements that mediate allosteric coupling of the two binding sites during activation and desensitization [4]. In response to ACh, single nAChR molecules undergo a conformational transition to an open-channel low-affinity state, associated with slower modulatory transitions to (or from) desensitized closed high-affinity states. These structural and dynamic properties are consistent with an allosteric mechanism [7,8] based on concerted transitions between pre-existing conformational states that might involve 'rigid body motion' of the subunits [7]. First, the distance separating the ion channel and the ACh-binding site is in the range of 20-40 Å [9], indicating that the coupling of binding and channel opening is mediated by an indirect, conformational mechanism. Second, spontaneous all-or-nothing openings of single nAChR channels have been recorded by voltage clamp in the absence of ligand for both wild-type and mutant receptors, demonstrating that the transition between resting and active states occurs independently of ligand binding. Third, pleiotropic phenotypes have been discovered that are caused by single point mutations in the channel domain; these abolish desensitization, increase the apparent affinity for agonists, convert competitive antagonists into partial agonists and are readily accounted for by an allosteric mechanism [10]. In addition, direct structural evidence for a concerted two-state transition has been demonstrated recently in the model system of Escherichia coli aspartate transcarbamoylase [11].

Sequence analysis reveals homologies between the nAChR subunits from electric organ, skeletal muscle and brain of higher vertebrates, including humans [12]. The 17 nAChR subunits whose sequence is known (α1-α10, β1-β4, γ, δ, ε) form multiple functional homopentamers (α7, α8 and α9) or heteropentamers (with one or several different a subunits) [4,12,13]. The different nAChRs mediate the pharmacological action of drugs, such as the effects of curare on muscle and of nicotine in brain (including addiction as well as analgesia, enhanced cognition, memory and attention). Neuronal 5-HT₃, GABA_A, GABA_C and glycine receptors share many features with nAChR and thus belong to a common superfamily of ligand-gated ion channels.

All nAChR subunits share a similar hydropathy profile, with a large N-terminal hydrophilic domain (comprising ∼210 amino acids) that faces the synaptic cleft and carries the ACh-binding site, four hydrophobic transmembrane stretches (one of which, M2, lines the ion channel) and a small hydrophilic domain that is exposed to the cytoplasm where it can be phosphorylated and, as a consequence, enhances the desensitization of the physiological response.

Attempts to solve the entire structure of nAChR failed, possibly because of the amphiphilic nature and allosteric behavior of the receptor. Structural studies were thus limited to electron microscopy [14] down to 4.6 Å resolution [15] and to the recombinant N-terminal synaptic domain of nAChR. Circular dichroism revealed a high content of β-sheet structure in the isolated extracellular domain of the α1-subunit of the mouse muscle receptor [16] (and in the a1-subunit of the GABAA receptor [17]), a finding consistent with recent electron microscopy (EM) images that revealed tunnels in the α1-subunit N-terminal domain of Torpedo receptor that were framed by seven twisted β strands running approximately perpendicular to the membrane plane [15]. In addition, secondary structure predictions suggested an immunoglobulin-like folding, almost exclusively of β sheets, for the N-terminal domain of the nicotinic receptor [18]. High resolution data at the atomic level are now available for AChBP.

Atomic Structure of AChBP is a Naturally Occurring Analogue of the nAChR Ligand-binding Domain

The crystal structure of AChBP at 2.7 Å resolution reported by Brejc et al. [6] reveals an oligomer of five identical asymmetric subunits arranged in an anticlockwise manner along the fivefold axis (figure 1a) with dimensions (80 Å diameter, 62 Å height and a rosette-like motif with a central hole of ∼18 Å diameter) consistent with EM data for the Torpedo nAChR N-terminal domain [14,15]. Each subunit comprises an N-terminal, a helix, two short 310 helices, and a core of ten b strands folded into α twisted β sandwich. The order of β strands conforms, as predicted [4,18], to an immunoglobulin topology, but in a nonclassical orientation [6] (figure 1c,d). The two twisted β sheets of the β sandwich are connected by a characteristic disulfide bond referred to as the Cys-loop (figure 1d). This structure, which appears crucial for the stabilization of the tertiary folding [19], consists of highly conserved hydrophobic residues in the nAChR family and is hypothesized to interact with some channel-forming elements [6]. By contrast, the sequence in the Cys-loop is not conserved in AChBP family members and possesses hydrophilic amino acids that, together with the presence of glycosylation sites, might be sufficient to yield a soluble homopentameric complex [6].

The subunit interfaces are of considerable interest as they might play a crucial role in allosteric transitions [8,20]. The plus side of the interface (figure 1d) is framed essentially by loops, whereas the minus side mostly presents secondary structure elements. The residues located at the interface [6] are not well conserved among nAChRs and probably contribute to the diversity of subunit combinations. By contrast, specific interactions (e.g. hydrophobic, polar, hydrogen bonds) have to be at least partially conserved to mediate common features of the allosteric transitions (see below). Hydrophobic residues in AChBP are essentially located inside the subunit interface whereas polar residues are exposed to the outside where they promote oligomerization.

Cavities are located at each interface between AChBP subunits (figure 1b). These are lined by residues formerly shown to belong to the ACh-binding site of nAChR on the basis of photoaffinity labeling [4,21,22] and mutagenesis [4,23] experiments, thus a posteriori validating these experimental approaches (figure 2a,b). The location of these cavities at overlapping domains between subunits fits with early a-bungarotoxin binding data [24] and their equatorial position was also predicted by energy transfer experiments [9]. Thus, AChBP data do not validate the suggestion that the 'tunnels' seen in Torpedo nAChR EM pictures are the access routes to the ACh-binding site [15]. Two components of the ACh-binding domain have been distinguished in nAChR: a 'principal component' on the α subunit formed by the three loops - A [Tyr93 (25)], B [Trp149 (26)] and C [Tyr190, Cys192, Cys193 and Tyr198 (26-28)] - and a 'complementary component' made up of loops D [Trp55, Glu57 (29)], E [Leu109, Tyr111, Tyr117, Leu119 (22,29,30,31)] and F [Asp174, Glu176 (32)] on the neighboring subunit (figure 2b). AChBP structure reveals that loops A and B are not involved in a periodic structure whereas loop C is folded in a β hairpin connected at the turn by the two vicinal Cys residues (Cys187, Cys188) homologous to the initially identified Cys pair [27]. The unexpected folding of loop C might act as a gate restricting access to the ligand-binding site [6]. The loop B amino acid Trp143 (homologous to Trp149 in Torpedo) probably exerts cation-π interactions with the quaternary ammonium group of ACh (or with HEPES in the crystal structure). This interaction has been previously observed for acetylcholinesterase [33] and suggested for nicotinic receptors [34]. AChBP was crystallized in the absence of nicotinic ligands (although a HEPES buffer molecule was resolved in the crystal at the level of the putative ACh-binding site) and, therefore, precise positioning of the nicotinic ligands within this site remains to be elucidated. However, it is possible to dock agonists within the site based on studies proposing the relative orientation of ACh (21,34,35). One residue (Trp86), identified by photoaffinity labeling in Torpedo receptor [25], is found in the crystal structure of AChBP within a hydrophobic pocket located at a distance (∼15 Å) from the ACh site but this residue might contribute to a second cationic subsite postulated to fit bisquaternary compounds [36].

Unlike the principal component, the complementary one is folded in a series of β strands, thus making a relatively large binding surface area. Indeed, loop E was predicted to be in an antiparallel β-sheet structure [18,30]. By contrast, loop D does not fold into an a helix [4]. Although positions identified by affinity labeling and mutagenesis experiments are conserved in AChBP (figure 2a), the chemical nature of the residues are not conserved in loops B and E, further suggesting that these loops contribute to pharmacological diversity in the entire family of nAChRs (4,30,37). Moreover, the residue Asp160 in loop F, homologous to δAsp180 and γAsp174 identified by affinity labeling and mutagenesis in Torpedo [32], does not participate directly in the ACh-binding site in AChBP (figure 2b). Nevertheless, as loop F is not folded in a periodic structure in AChBP, local conformational changes in Torpedo nAChR might bring this residue closer to the ACh-binding site. One residue located in loop F in AChBP (Tyr164) was not previously identified by chemical labeling in nAChR, but might contribute to the ligand-binding area because of a Ca²⁺ site that orientates its lateral chain towards loop C (figure 2b). The aromatic character of the amino acid (Tyr or Phe) conserved at this position in the superfamily of ligand-gated ion channels, suggests a functional role for the ligand-binding site geometry. Finally, the Ca²⁺ site identified near loop F in AChBP does not correspond to the site of Ca²⁺ potentiation identified in α7 receptors [38].

A Hypothetical Mechanism for the Allosteric Transitions of Nicotinic Receptors

With the crystal structure data in hand, the next major issue is to decipher the allosteric mechanism that mediates the activation and desensitization of the nAChR ion channel from a 20-30 Å distant agonist-binding site. The high affinity of AChBP for nicotinic ligands (agonists and antagonists) and Hill coefficients that are either equal or below unity [5], suggest (but do not prove) that AChBP was crystallized in a frozen 'desensitized' (D) state. Therefore, one might further hypothesize that the basal (B) activatable conformation of nAChR derives from AChBP structure by a reorganization of the quaternary structure, possibly involving a concerted anticlockwise rotation of all the N-terminal domains (figure 2c) as supported by low resolution EM (15). In the basal state, the a-toxin would have easier access to the external surface of the nAChR. The hypothesis of a more compact conformation of the D state (as proposed for allosteric proteins [8] and haemoglobin [20]) is consistent with two observations. First, during desensitization of Torpedo receptor, dynamic photoaffinity labeling reveals an increased accessibility of the complementary component from the adjacent subunits [i.e. the δ (39) and γ (Grutter et al., unpublished) subunits]. Accordingly, the two components of the ACh-binding pockets would move closer during the B → D transition (figure 2c). Second, some amino acids that selectively alter desensitization are common to the ligand-binding site and intersubunit association area. One of them, namely Trp143 in loop B of AChBP (37), might make a hydrogen bond with the peptide bond of the complementary component on the neighbouring subunit and thus be at a strategic position to play a role in the allosteric transitions. However, to date, the actual mechanisms for the allosteric transitions of activation and desensitization of the nAChR remain to be elucidated.

Concluding Remarks

The three-dimensional structure at the atomic level of a soluble, homopentameric homologue of the N-terminal extracellular domain of the nAChRs brings an outstanding insight to the knowledge of neurotransmitter receptor mechanisms. The high resolution data pave the way to the elucidation of the still pending enigma of nAChR channel activation and desensitization mechanisms and are anticipated to facilitate drug design against, for example, nicotine addiction, anxiety, pain, neurodegeneration and Alzheimer's disease.

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