Diversity of vertebrate nicotinic acetylcholine receptors

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Potentiation and allosteric agonist activation of α7 nicotinic acetylcholine receptors: binding sites and hypotheses

Considerable progress has been made in recent years towards the identification and characterisation of novel subtype-selective modulators of nicotinic acetylcholine receptors (nAChRs). In particular, this has focussed on modulators of α7 nAChRs, a nAChR subtype that has been identified as a target for drug discovery in connection with a range of potential therapeutic applications. This review focusses upon α7-selective modulators that bind to receptor sites other than the extracellular 'orthosteric' agonist binding site for the endogenous agonist acetylcholine (ACh). Such compounds include those that are able to potentiate responses evoked by orthosteric agonists such as ACh (positive allosteric modulators; PAMs) and those that are able to activate α7 nAChRs by direct allosteric activation in the absence of an orthosteric agonist (allosteric agonists or 'ago-PAMs'). There has been considerable debate about the mechanism of action of α7-selective PAMs and allosteric agonists, much of which has centred around identifying the location of their binding sites on α7 nAChRs. Based on a variety of experimental evidence, including recent structural data, there is now clear evidence indicating that at least some α7-selective PAMs bind to an inter-subunit site located in the transmembrane domain. In contrast, there are differing hypotheses about the site or sites at which allosteric agonists bind to α7 nAChRs. It will be argued that the available evidence supports the conclusion that direct allosteric activation by allosteric agonists/ago-PAMs occurs via the same inter-subunit transmembrane site that has been identified for several α7-selective PAMs

Nicotinic acetylcholine receptors (nACh) in GtoPdb v.2021.3

Nicotinic acetylcholine (ACh) receptors are members of the Cys-loop family of transmitter-gated ion channels that includes the GABAA, strychnine-sensitive glycine and 5-HT3 receptors [215, 3, 159, 225, 259]. All nicotinic receptors are pentamers in which each of the five subunits contains 4 TM domains. Genes encoding a total of 17 subunits (α1-10, β1-4, γ, δ and ε) have been identified [120]. All subunits with the exception of α8 (present in avian species) have been identified in mammals. All α subunits possess two tandem cysteine residues near to the site involved in acetylcholine binding, and subunits not named α lack these residues [159]. The orthosteric ligand binding site is formed by residues within at least three peptide domains on the α subunit (principal component), and three on the adjacent subunit (complementary component). Nicotinic ACh receptors contain several allosteric modulatory sites. One such site, for positive allosteric modulators (PAMs) and allosteric agonists, has been proposed to reside within an intrasubunit cavity between the 4 TM domains [264, 87]; see also [106]). The high resolution crystal structure of the molluscan ACh binding protein, a structural homologue of the extracellular binding domain of a nicotinic receptor pentamer, in complex with several nicotinic receptor ligands (e.g.[35]) and the crystal structure of the extracellular domain of the α1 subunit bound to α-bungarotoxin at 1.94Â resolution [55], has revealed the orthosteric binding site in detail (reviewed in [215, 120, 39, 198]). Nicotinic receptors at the somatic neuromuscular junction of adult animals have the stoichiometry (α1)2β1δε, whereas an extrajunctional (α1)2β1γδ receptor predominates in embryonic and denervated skeletal muscle and other pathological states. Other nicotinic receptors are assembled as combinations of α(2-6) and β(2-4) subunits. For α2, α3, α4 and β2 and β4 subunits, pairwise combinations of α and β (e.g. α3β4 and α4β2) are sufficient to form a functional receptor in vitro, but far more complex isoforms may exist in vivo (reviewed in [96, 93, 159]). There is strong evidence that the pairwise assembly of some α and β subunits can occur with variable stoichiometry [e.g. (α4)2(β2)2 or (α4)3(β2)2] which influences the biophysical and pharmacological properties of the receptor [159]. α5 and β3 subunits lack function when expressed alone, or pairwise, but participate in the formation of functional hetero-oligomeric receptors when expressed as a third subunit with another α and β pair [e.g. α4α5αβ2, α4αβ2β3, α5α6β2, see [159] for further examples]. The α6 subunit can form a functional receptor when co-expressed with β4 in vitro, but more efficient expression ensues from incorporation of a third partner, such as β3 [263]. The α7, α8, and α9 subunits form functional homo-oligomers, but can also combine with a second subunit to constitute a hetero-oligomeric assembly (e.g. α7β2 and α9α10). For functional expression of the α10 subunit, co-assembly with α9 is necessary. The latter, along with the α10 subunit, appears to be largely confined to cochlear and vestibular hair cells. Comprehensive listings of nicotinic receptor subunit combinations identified from recombinant expression systems, or in vivo, are given in [159]. In addition, numerous proteins interact with nicotinic ACh receptors modifying their assembly, trafficking to and from the cell surface, and activation by ACh (reviewed by [158, 9, 118]).The nicotinic receptor Subcommittee of NC-IUPHAR has recommended a nomenclature and classification scheme for nicotinic acetylcholine (nACh) receptors based on the subunit composition of known, naturally- and/or heterologously-expressed nACh receptor subtypes [143]. Headings for this table reflect abbreviations designating nACh receptor subtypes based on the predominant α subunit contained in that receptor subtype. An asterisk following the indicated α subunit denotes that other subunits are known to, or may, assemble with the indicated α subunit to form the designated nACh receptor subtype(s). Where subunit stoichiometries within a specific nACh receptor subtype are known, numbers of a particular subunit larger than 1 are indicated by a subscript following the subunit (enclosed in parentheses- see also [46])

Stoichiometry-Selective Antagonism of α4β2 Nicotinic Acetylcholine Receptors by Fluoroquinolone Antibiotics

Quinolone antibiotics disrupt bacterial DNA synthesis by interacting with DNA gyrase and topoisomerase IV. However, in addition, they have been shown to act as inhibitors of pentameric ligand-gated ion channels such as GABAA receptors and the α7 nicotinic acetylcholine receptor (nAChR). In the present study, we have examined the effects of quinolone antibiotics on the human α4β2 nAChR, an important subtype that is widely expressed in the central nervous system. A key feature of α4β2 nAChRs is their ability to coassemble into two distinct stoichiometries, (α4)_{2}(β2)_{3} and (α4)_{3}(β2)_{2}, which results in differing affinities for acetylcholine. The effects of nine quinolone antibiotics were examined on both stoichiometries of the α4β2 receptor by two-electrode voltage-clamp recording. All compounds exhibited significant inhibition of α4β2 nAChRs. However, all of the fluoroquinolone antibiotics examined (ciprofloxacin, enoxacin, enrofloxacin, difloxacin, norfloxacin, pefloxacin, and sparfloxacin) were significantly more potent inhibitors of (α4)_{2}(β2)_{3} nAChRs than of (α4)_{3}(β2)_{2} nAChRs. This stoichiometry-selective effect was most pronounced with pefloxacin, which inhibited (α4)_{2}(β2)_{3} nAChRs with an IC_{50} of 26.4 ± 3.4 μM but displayed no significant inhibition of (α4)_{3}(β2)_{2} nAChRs. In contrast, two nonfluorinated quinolone antibiotics (cinoxacin and oxolinic acid) exhibited no selectivity in their inhibition of the two stoichiometries of α4β2. Computational docking studies suggest that pefloxacin interacts selectively with an allosteric transmembrane site at the β2(+)/β2(−) subunit interface, which is consistent with its selective inhibition of (α4)_{2}(β2)_{3}. These findings concerning the antagonist effects of fluoroquinolones provide further evidence that differences in the subunit stoichiometry of heteromeric nAChRs can result in substantial differences in pharmacological properties

nicotinic acetylcholine receptors version 2019 4 in the iuphar bps guide to pharmacology database

Nicotinic acetylcholine receptors are members of the Cys-loop family of transmitter-gated ion channels that includes the GABAA, strychnine-sensitive glycine and 5-HT3 receptors [210, 3, 155, 220, 252]. All nicotinic receptors are pentamers in which each of the five subunits contains four α-helical transmembrane domains. Genes encoding a total of 17 subunits (α1-10, β1-4, γ, δ and ε) have been identified [117]. All subunits with the exception of α8 (present in avian species) have been identified in mammals. All α subunits possess two tandem cysteine residues near to the site involved in acetylcholine binding, and subunits not named α lack these residues [155]. The orthosteric ligand binding site is formed by residues within at least three peptide domains on the α subunit (principal component), and three on the adjacent subunit (complementary component). nAChRs contain several allosteric modulatory sites. One such site, for positive allosteric modulators (PAMs) and allosteric agonists, has been proposed to reside within an intrasubunit cavity between the four transmembrane domains [257, 85]; see also [103]). The high resolution crystal structure of the molluscan acetylcholine binding protein, a structural homologue of the extracellular binding domain of a nicotinic receptor pentamer, in complex with several nicotinic receptor ligands (e.g.[33]) and the crystal structure of the extracellular domain of the α1 subunit bound to α-bungarotoxin at 1.94 Å resolution [53], has revealed the orthosteric binding site in detail (reviewed in [210, 117, 37, 193]). Nicotinic receptors at the somatic neuromuscular junction of adult animals have the stoichiometry (α1)2β1δε, whereas an extrajunctional (α1)2β1γδ receptor predominates in embryonic and denervated skeletal muscle and other pathological states. Other nicotinic receptors are assembled as combinations of α(2-6) and &beta(2-4) subunits. For α2, α3, α4 and β2 and β4 subunits, pairwise combinations of α and β (e.g. α3β4 and α4β2) are sufficient to form a functional receptor in vitro, but far more complex isoforms may exist in vivo (reviewed in [94, 91, 155]). There is strong evidence that the pairwise assembly of some α and β subunits can occur with variable stoichiometry [e.g. (α4)2(β2)2 or (α4)3(β2)2] which influences the biophysical and pharmacological properties of the receptor [155]. α5 and β3 subunits lack function when expressed alone, or pairwise, but participate in the formation of functional hetero-oligomeric receptors when expressed as a third subunit with another α and β pair [e.g. α4α5αβ2, α4αβ2β3, α5α6β2, see [155] for further examples]. The α6 subunit can form a functional receptor when co-expressed with β4 in vitro, but more efficient expression ensues from incorporation of a third partner, such as β3 [256]. The α7, α8, and α9 subunits form functional homo-oligomers, but can also combine with a second subunit to constitute a hetero-oligomeric assembly (e.g. α7β2 and α9α10). For functional expression of the α10 subunit, co-assembly with α9 is necessary. The latter, along with the α10 subunit, appears to be largely confined to cochlear and vestibular hair cells. Comprehensive listings of nicotinic receptor subunit combinations identified from recombinant expression systems, or in vivo, are given in [155]. In addition, numerous proteins interact with nicotinic ACh receptors modifying their assembly, trafficking to and from the cell surface, and activation by ACh (reviewed by [154, 9, 115]).The nicotinic receptor Subcommittee of NC-IUPHAR has recommended a nomenclature and classification scheme for nicotinic acetylcholine (nACh) receptors based on the subunit composition of known, naturally- and/or heterologously-expressed nACh receptor subtypes [139]. Headings for this table reflect abbreviations designating nACh receptor subtypes based on the predominant α subunit contained in that receptor subtype. An asterisk following the indicated α subunit denotes that other subunits are known to, or may, assemble with the indicated α subunit to form the designated nACh receptor subtype(s). Where subunit stoichiometries within a specific nACh receptor subtype are known, numbers of a particular subunit larger than 1 are indicated by a subscript following the subunit (enclosed in parentheses – see also [44])

Differential Contribution of Subunit Interfaces to α 9 α 10 Nicotinic Acetylcholine Receptor Function

Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.Fil: Boffi, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Marcovich, Irina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gill Thind, JasKiran K.. University College London; Reino UnidoFil: Corradi, Jeremias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Collins, Toby. University College London; Reino UnidoFil: Lipovsek, Maria Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Moglie, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Plazas, Paola Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Craig, Patricio Oliver. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Millar, Neil S.. University College London; Reino UnidoFil: Bouzat, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Inhibition of Human α7 Nicotinic Acetylcholine Receptors by Cyclic Monoterpene Carveol

Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol\u3ethymoquinone\u3ecarvacrol\u3ementhone\u3ethymol\u3elimonene\u3eeugenole\u3epulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3 µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies

Understanding key constraints and practice design in Rugby Union place kicking : experiential knowledge of professional kickers and experienced coaches

Place kicks present valuable opportunities to score points in Rugby Union, which are typically performed under varying constraints in competitive performance environments. Previous quantitative studies suggest these interacting constraints can influence fluctuations in place kick success. To further the understanding of how fluctuations in place kicking success emerge, our aim was twofold: i) to explore and identify the key constraints that professional place kickers and experienced place kicking coaches perceive to influence the difficulty of a place kick and ii) to understand the level to which current place kicking practice environments represent these key constraints experienced in performance environments. Six professional place kickers and six experienced place kicking coaches were interviewed. Using a deductive thematic analysis, 11 key constraints were identified: individual constraints of expectation for success and fatigue, task constraints of angle and distance to goalposts, environmental constraints of wind, weather, pitch, and crowd, and situational constraints of previous kicking performance, time remaining and current score margin. Place kicking is typically practised individually or with a small number of place kickers in isolation from team sessions. Where possible, coaches should be encouraged to include place kicking in simulated game scenarios during practice to represent key constraints from performance environments. Our study demonstrates how experiential knowledge can enrich the understanding of sport performance and inform the design of practice environments which simulate relevant constraints of competitive performance to enhance skill adaptation of athletes

Diversity of nicotinic acetylcholine receptor positive allosteric modulators revealed by mutagenesis and a revised structural model

By combining electrophysiological and computational approaches we have examined a series of positive allosteric modulators (PAMs) acting on the human α7 nicotinic acetylcholine receptor (nAChR). Electrophysiological studies have focussed on three α7-selective PAMs (A-867744, TBS-516 and TQS) that display similar effects on wild-type α7 nAChRs. In addition to potentiating agonist-evoked responses, all three compounds reduce receptor desensitisation and, consequently, are classed as type II PAMs. Despite having similar effects on wild-type receptors, A-867744 was found to have profoundly differing effects to TBS-516 and TQS on mutated receptors, a finding that is consistent with previous studies indicating that A-867744 may have a different mechanism of action to other α7-selective type II PAMs. Due to evidence that these PAMs bind within the α7 nAChR transmembrane region, we generated and validated new structural models of α7. Importantly, we have corrected a previously identified error in the transmembrane region of the original cryo-EM Torpedo model; the only pentameric ligand-gated ion channel imaged in a native lipid membrane. Real-space refinement was used to generate closed and open conformations on which the α7 models were based. Consensus docking with an extended series of PAMs with chemical similarity to A-867744, TBS-516 and TQS suggests that all bind to a broadly similar inter-subunit transmembrane site. However, differences in the predicted binding of A-867744, compared with TBS-516 and TQS, may help to explain the distinct functional effects of A-867744. Thus, our revised structural models may provide a useful tool for interpreting functional effects of PAMs

Evaluation of pragmatic oxygenation measurement as a proxy for Covid-19 severity

Choosing optimal outcome measures maximizes statistical power, accelerates discovery and improves reliability in early-phase trials. We devised and evaluated a modification to a pragmatic measure of oxygenation function, the [Formula: see text] ratio. Because of the ceiling effect in oxyhaemoglobin saturation, [Formula: see text] ratio ceases to reflect pulmonary oxygenation function at high [Formula: see text] values. We found that the correlation of [Formula: see text] with the reference standard ([Formula: see text]/[Formula: see text] ratio) improves substantially when excluding [Formula: see text] and refer to this measure as [Formula: see text]. Using observational data from 39,765 hospitalised COVID-19 patients, we demonstrate that [Formula: see text] is predictive of mortality, and compare the sample sizes required for trials using four different outcome measures. We show that a significant difference in outcome could be detected with the smallest sample size using [Formula: see text]. We demonstrate that [Formula: see text] is an effective intermediate outcome measure in COVID-19. It is a non-invasive measurement, representative of disease severity and provides greater statistical power