nAChRs (nicotinic acetylcholine receptors) are neuron receptor proteins that signal for muscular contraction upon a chemical stimulus. They are cholinergic receptors that form ligand-gated ion channels in the plasma membranes of certain neurons and on the presynaptic and postsynaptic sides of the neuromuscular junction ^[1]. 17 vertebrate nAChR subunits have been identified, which are divided into 5 main subunits (α1-9, β1-4, δ, γ, ε) ^[2]. Thus, there is immense potential of variation of these subunits, some of which are more commonly found than others (α4β2, α2β2, α9β10, (α1)2β1δγε, α2β4, α4β4, α3β4, α3β2, α6β2, and α5α3β4). Moreover, there are some other subtypes include (α1)2β1δε (adult muscle-type), (α3)2(β4)3 (ganglion-type), (α4)2(β2)3 (CNS-type) and (α7)5 (another CNS-type).

Nicotinic receptors, with a molecular mass of 290 kDa ^[3], are made up of five subunits, arranged symmetrically around a central pore. Each subunit comprises four transmembrane domains with both the N- and C-terminus located extracellularly. They possess similarities with GABA_A receptors, glycine receptors, and the type 3 serotonin receptors (which are all ionotropic receptors), or the signature Cys-loop proteins ^[4]. 17 vertebrate nAChR subunits have been identified, which are divided into muscle-type and neuronal-type subunits. Although an α8 subunit/gene is present in avian species such as the chicken, it is not present in human or mammalian species ^[5]. The nAChR subunits have been divided into four subfamilies (I–IV) based on similarities in protein sequence. In addition, subfamily III has been further divided into three types ^[6]. Neuronal nAChRs are transmembrane proteins that form pentameric structures assembled from a family of subunits composed of α2–α10 and β2–β4 ^[7]. These subunits were discovered from the mid-1980s through the early 1990s, when cDNAs for multiple nAChR subunits were cloned from rat and chicken brains, leading to the identification of eleven different genes (twelve in chickens) that code for neuronal nAChR subunits; The subunit genes identified were named α2–α10 (α8 only found in chickens) and β2–β4 ^[8]. It has also been discovered that various subunit combinations could form functional nAChRs that could be activated by acetylcholine and nicotine, and the different combinations of subunits generate subtypes of nAChRs with diverse functional and pharmacological properties. When expressed alone, α7, α8, α9, and α10 are able to form functional receptors, but other α subunits require the presence of β subunits to form functional receptors ^[7]. In mammals, nAchR subunits have been found to be encoded by 17 genes, and of these, nine genes encoding α-subunits and three encoding β-subunits are expressed in the brain. β2 subunit-containing nAChRs (β2nAChRs) and α7nAChRs are widely expressed in the brain, whereas other nAChR subunits have more restricted expression ^[9]. The pentameric assembly of nAChRs is subjected to the subunits that are produced in various cell types such as in the human lung where epithelial and muscular pentamers largely differ ^[10].

Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(-), the α9(+)/α10(-), and the α10(+)/α9(-). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown by the crystal structures of the monomeric α9-ECD with these molecules. RgIA binding at α9(+)/α9(-) or α10(+)/α9(-) rather than the α9(+)/α10(-) interface ^[11].

At the contact zone, Ala(7), Ser(9), and Ile(11) in finger I and Arg(36), Lys(38), Val(39), and Val(40) in finger II of alpha-bungarotoxin interface with Phe(186), Tyr(187), Glu(188), and Tyr(194) in the alpha7 19-mer underscoring the importance of receptor aromatic residues as critical neurotoxin-binding determinants. Superimposing the structure of the complex onto that of the acetylcholine-binding protein (1I9B), a soluble homologue of the extracellular domain of the alpha7 receptor, places alpha-bungarotoxin at the peripheral surface of the inter-subunit interface occluding the agonist-binding site ^[14].

Yellow Sphere (Residue of Ile165, Ser124, Ile94, Met39, Asn45, Gln37) form the binding pocket of Epibatidine in α7 ^[12]. Blue Sphere (Residue of W149, Y93, W59,L123, Y197, C192, C193) form the binding pocket of nicotine in α3β4 ^[13].

X-ray crystallography was uesd to identify allosteric binding sites of α7 nicotinic acetylcholine receptor. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site ^[15].

Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors ^[16].

Its azabicyclo moiety points toward the principal (abbreviated +) subunit and interacts with the conserved aromatic cage. The chloropyridine ring faces the complementary (abbreviated −) subunit. An ordered water molecule bridges the hydroxyl group of T105, the backbone carbonyl of N106, and the chloropyridine nitrogen. In the α7-epi/NS1738 complex, the modulator is shifted extracellularly compared with the macrolide core of IVM and is nestled just beneath the M2-M3 loop. This binding site is consistent with earlier work mplicating the M2-M3 loop in NS1738 binding.46 NS1738 binding triggers striking flexibility within the coupling region that links the ECD and TMD. It disrupts interactions between R132-Q272 and R204-D477 and causes a detachment of the latch helix that is well-ordered in all other modulator bound structures. Structural analysis of the α7-epi/PAM complexes highlights three α7 residues that likely contribute to regulating modulator activity: N213, M253, and A275.34 N213 in (−)M1 is positioned to interact with all four modulators ^[17].