Neuronal nicotinic acetylcholine receptors (nAChRs) are prototypical cation-selective, ligand-gated ion channels that mediate fast neurotransmission in the central and peripheral nervous systems. nAChRs are involved in a range of physiological and pathological functions and hence are important therapeutic targets. Their subunit homology and diverse pentameric assembly contribute to their challenging pharmacology and limit their drug development potential. Toxins produced by an extensive range of algae, plants and animals target nAChRs, with many proving pivotal in elucidating receptor pharmacology and biochemistry, as well as providing templates for structure-based drug design. The crystal structures of these toxins with diverse chemical profiles in complex with acetylcholine binding protein (AChBP), a soluble homolog of the extracellular ligand-binding domain of the nAChRs and more recently the extracellular domain of human α9 nAChRs, have been reported. These studies have shed light on the diverse molecular mechanisms of ligand-binding at neuronal nAChR subtypes and uncovered critical insights useful for rational drug design. This review provides a comprehensive overview and perspectives obtained from structure and function studies of diverse plant and animal toxins and their associated inhibitory mechanisms at neuronal nAChRs.
nAChRs are formed by the assembly of five transmembrane subunits. Seventeen different nAChR subunits have been identified so far in mammals, including ten α (α1–10), four β (β1–4), γ, δ, and ε subunits. Neuronal nAChRs are assembled either as homo-pentamers of α7, α8, and α9 or hetero-pentamers of α2–α6 in combination with β2–β4 or α9 with α10 subunits. In contrast, the hetero-pentameric muscle nAChRs comprise two α1 plus a β1, δ, and γ (fetal) or ε (adult) subunits. The ligand binding pocket (LBP) for agonists or antagonist in nAChRs is at the interface between two neighboring subunits with one subunit being the principal face and the other being the complementary face. In heteromeric nAChRs, the principal face comes from one α subunit, while the complementary face arises from non-α subunit. The binding of ligand stimulates different functional states of nAChRs via the conformational changes induced by the relative movement of the five subunits to each other. The structural characters of the LBP and the specific amino acid interactions between ligands and this site determine the conformational transitions that lie behind the pharmacological properties of a specific neuronal nAChR subtype. Thus, different pharmacological and biophysical properties are displayed by a diverse range of neuronal nAChR subtypes underpinned by the different subunit combinations. A complex expression profile in the nervous system is also exhibited by different subtypes of neuronal nAChRs. Together, this contributes to the complexity in the structure and function of neuronal nAChRs and their roles in the CNS.
nAChRs regulate the flow of mainly sodium, potassium and calcium ions across the cell membrane. The binding of ligands triggers a tertiary conformational transition of nAChRs among functionally distinct resting, open and desensitized states, with subunit composition and class of agonists influencing the kinetics of these conformational state transitions. Agonists bound at the orthosteric site of nAChRs initially stabilize the open state and later a desensitized closed state, while effectors bound at the allosteric site can modify the energy barriers between transitions that shifts the equilibrium between states. Desensitization state may encompass short-and long-lived states of desensitization where the latter state is favored by long exposure to low concentration of agonists. Electrophysiology has been pivotal in determining the biophysical and pharmacological properties of different nAChRs subtypes. For example, the α7 nAChR is characterized by a low affinity for agonists, rapid activation, large conductance, high permeability to Ca 2+ and fast desensitization, while α4β2 nAChRs and α3β4 nAChRs have slow inactivating nicotinic responses. Interestingly, mutation of a single amino acid (L247T) in the ionic pore of chick α7 nAChRs caused pleiotropic effects on the nature of this receptor subtype, specifically the suppression of receptor desensitization, the increase in ligand affinity and the change in pharmacological profile of certain ligands from competitive antagonist into full agonists. These properties of this mutant are suggested to render a desensitized conductive state based on the basis of the allosteric model. This phenomenon has, in turn, shed light on the antagonism mechanism of certain antagonists from natural toxins, which are discussed later in this review.
nAChRs are broadly distributed across the peripheral nervous system (PNS) and central nervous system (CNS) of both simple and complex organisms. This highlights the importance of nAChRs in the nervous system where they play a wide range of functions from the mediation of different cognitive processes to synaptic transmission from nerves to muscle. Homomeric α7 nAChRs and heteromeric α4β2∗ nAChRs are predominantly expressed in the human brain where they contribute to the pathogenesis of a range of neurological disorders including Alzheimer’s disease, schizophrenia, Parkinson’s disease and depression. α7 and α4β2 nAChRs also contribute to other non-neurological diseases, including a correlation of both subtypes with nicotine addiction and nicotine-induced behaviors and the overexpression of α7 nAChRs associated with small-cell lung carcinomas. Given their potential roles in disease development and progression, α7 and α4β2 nAChRs are currently one of the most studied nAChR subtypes. Recent studies are now starting to delineate roles for other nAChRs subtypes in a number of diseases. For example, despite the limited neuronal distribution of α6β2∗ subtypes, expression of the α6 subunit in nociceptors suggests it could contribute to sensory processing and pain, with an inverse correlation between CHRNA6 expression and neuropathic pain found in mice and humans. More recently, the α9∗ has also been implicated in modulating the pathophysiology of neuropathic pain. In contrast, dysfunction of muscle nAChRs results in the impaired neuromuscular transmission and muscle weakness typically associated with inherited mutations and acquired diseases such as myasthenia gravis or congenital myasthenic syndromes.
The therapeutically significant role of the nAChR subtypes in several pathophysiological conditions, together with the diversity in the subtype combinations, biophysical properties and expression patterns present a formidable challenge in rational drug discovery and design for this receptor family. This urges for thorough insights into molecular and structural mechanisms governing nAChR subtype selectivity to facilitate successful therapeutic strategies for nAChR associated neuronal diseases.
A breakthrough in characterization of nAChRs-ligand interactions came with the determination of the X-ray structure of acetylcholine binding protein (AChBP), a naturally occurring soluble protein homolog of nAChR. Despite a low sequence similarity, AChBPs and nAChRs show remarkable structural homology, including the orthosteric ligand recognition site formed by aromatic side chain residues found in nAChRs. However, the ligand-bound AChBPs still require the translation of information into individual nAChR subtypes via homology modeling in order to build a more accurate model for the interactions of ligands at targeted nAChRs.
A step forward in modeling the binding mechanism of ligands at nAChRs is to make AChBP resemble a given nAChR subtype. The crystal structure of the chimeric ligand binding domain of the human α7 AChR with AChBP was introduced via the substitution of selected native human α7 residues into Lymnaea Stagnalis (Ls) or Aplysia californica (Ac) AChBP. An alternative approach is the crystallization of an isolated component of the full length nAChR in complex with ligands at atomic level, which has been performed with neuronal nAChR α9 subunit extracellular domain (ECD). This approach could, in turn, improve the modeling of other neuronal nAChR ECDs. Taken together, the co-crystal structure of nAChR structural surrogates (AChBP, chimera AChBP or nAChR ECD) in complex with different nAChR ligands is currently one of the most popular approaches for structure-function studies of nAChRs. Importantly, inhibitors from natural toxins take up a high percentage of the co-crystal structures of ligands with nAChR structural surrogates.
