Nicotinic acetylcholine receptors (nAChRs) owe their name to their activation by the endogenous ligand acetylcholine (ACh) as well as the alkaloid nicotine and are among the most studied neuroreceptors. nAChRs belong to the superfamily of “Cys-Loop” receptors that are so termed due to the presence of a disulfide bond between two conserved cysteine residues separated by 13 amino acids. In addition to nAChRs, other members of the Cys-Loop superfamily include 5-hydroxytryptamine type 3 (5-HT3) receptors, γ-aminobutyric acid type A and type C (GABAA and GABAC) receptors, glycine receptors, and invertebrate glutamate and histidine receptors. Receptors are further classified into excitatory or inhibitory, the former correspond to cation permeable channels (Na+, K+ and Ca2+), which promote the firing of an action potential, and the latter conduct anions (Cland HCO3-), discouraging the firing of an action potential. nAChRs and 5-HT3 receptors are excitatory, glycine receptors are inhibitory and GABA are mostly inhibitory. Furthermore, nAChRs mediate fast synaptic transmission and are involved in a wide range of physiological and pathophysiological processes.
Figure 1 nAChR Agonists. These four nicotinic agonists share structural features consisting of a cationic nitrogen (blue) and a hydrogen bond acceptor moiety (red).
nAChRs are distinguished into muscle or neuronal nAChRs according to their localization in the neuromuscular junction or peripheral and central nervous systems, respectively. The muscle-type nAChR is found postsynaptically at the neuromuscular junction where it mediates the chemical to electrical signal transduction resulting in skeletal muscle tone. Neuronal nAChRs have been identified in numerous subtypes that reside at presynaptic and postsynaptic densities in autonomic ganglia and cholinergic neurons throughout the CNS.
These receptors have been implicated in various processes related to cognitive functions, learning and memory, arousal, reward, motor control and analgesia. Due to their role regulating the neurotransmission of dopamine (DA), norepinephrine (NE), serotonin (5-hydroxytryptamine, 5-HT), glutamate (Glu) and γ-aminobutyric acid (GABA), neuronal nAChRs represent potential therapeutic targets for the treatment of pain, epilepsy, Alzheimer’s disease, Parkinson’s disease, Tourette’s syndrome, schizophrenia, anxiety, depression and nicotine addiction. As such, there is considerable interest in gaining nAChR subtype specific structural and functional information. The studies portrayed in this thesis aim to further our knowledge in this area.
Membrane proteins such as the nAChR are notoriously hard to crystallize, and therefore structural information on these proteins is scarce when compared to soluble proteins. Despite the fact that membrane proteins represent ~30% of genetically encoded proteins and ~60% of pharmaceutical targets, they correspond to less than 1% of the structures in the Protein Data Bank (PDB). Presently, structural information on the nAChR stems mainly from two sources, the crystal structure of the acetylcholine binding protein (AChBP) at 2.7Å resolution, and the 4Å resolved cryo-electron microscopy images of the nAChR from the electric organ of Torpedo californica (Pacific electric ray). Isolated from the snail Lymnaea stagnalis, AChBP is a soluble protein that forms stable homopentamers and shares 20-24% sequence homology with the extracellular domain (ECD) of nAChRs; thus making it a useful model of the nAChR ECD. It is worth noting that since AChBP lacks a transmembrane domain (TMD) it is not an ion channel and thus no information on nAChR receptor activation can be obtained from this model. However, Unwin et al. solved high resolution cryo-EM images of the muscle-type nAChR, revealing structural insights into the TMD of nAChRs. In conjunction, these studies depict the architecture of nAChR subunits consisting of a large N-terminal extracellular domain comprised mainly of β-sheets, a transmembrane domain consisting of 4 membrane spanning α-helices (M1-M4) and a small extracellular C-terminal domain. nAChRs are pentameric, containing five homologous subunits arranged pseudosymmetrically around a central ion conducting pore lined by the M2 α-helices of each subunit.
Figure 2 nAChR structure. Left: Cartoon depiction of a prototypical pentameric nAChR. Middle: Subunit topology showing the pore lining M2 transmembrane domain in green. Right: Muscle-type nAChR image based on Unwin’s model of the Torpedo receptor (pdb file 2BG9). Red star denotes agonist binding site and yellow rhombus indicates channel gate separated by 60Å.
The nAChR family shows considerable diversity. To date, seventeen different vertebrate nAChR subunits have been identified and cloned: α1-α10, β1- β4, γ, δ and ε. The subunits are divided into muscle-type (α1, β1, γ, δ and ε) and neuronal (α2-α10, β2-β4) subunits according to the receptor subtypes that they are known to form. The muscle-type subunits make up the two forms of muscle-type receptor; (α1)2β1γδ (embryonic form) and (α1)2β1γε (adult form). Subunits are organized clockwise as α1β1γα1δ(or ε) and ACh binds to two orthosteric sites located at the α1/γ and α1/δ interfaces. A total of two molecules of ACh must bind, each occupying one of the two binding sites before the channel gate opens and allows the passage of cations, in favor of their electrochemical gradient, thereby translating a chemical signal into an electrical one.
In contrast to the precise stoichiometry of the muscle-type nAChR, the 12 neuronal subunits can form a wide variety of different nAChR subtypes, each of which shows different characteristics in terms of ligand pharmacology, activation and desensitization kinetics as well as cation permeability. Over 20 different neuronal nAChR subtypes have been identified throughout the nervous systems of various vertebrates and at least 22 neuronal subtypes have been successfully expressed in heterologous systems such as Xenopus laevis (African clawed frog) oocytes. Of the neuronal subunits, subunits α2-α6 and β2-β4 assemble in heteropentameric complexes of variable stoichiometry, the prevalent stoichiometry being (α)2(β)3 arranged as αβαββ but the (α)3(β)2 stoichiometry has also been reported. ACh and other agonists bind at α/β interfaces thereby giving rise to two orthosteric binding sites per receptor. Subunits α3 and α5 are considered structural subunits because they lack the amino acid residues critical for agonist binding and as such cannot participate in the formation of binding sites. It is generally accepted that the more complex neuronal nAChRs are formed by two pairs of α(2,3,4,6)/β(2,4) and only one structural subunit, though there may be exceptions where two structural subunits are present.