Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Neuropharmacology
2015 Oct 01;97:75-85. doi: 10.1016/j.neuropharm.2015.05.006.
Show Gene links
Show Anatomy links
The influence of allosteric modulators and transmembrane mutations on desensitisation and activation of α7 nicotinic acetylcholine receptors.
Chatzidaki A
,
D'Oyley JM
,
Gill-Thind JK
,
Sheppard TD
,
Millar NS
.
???displayArticle.abstract???
Acetylcholine activates nicotinic acetylcholine receptors (nAChRs) by binding at an extracellular orthosteric site. Previous studies have described several positive allosteric modulators (PAMs) that are selective for homomeric α7 nAChRs. These include type I PAMs, which exert little or no effect on the rate of receptor desensitisation, and type II PAMs, which cause a dramatic loss of agonist-induced desensitisation. Here we report evidence that transmembrane mutations in α7 nAChRs have diverse effects on receptor activation and desensitisation by allosteric ligands. It has been reported previously that the L247T mutation, located toward the middle of the second transmembrane domain (at the 9' position), confers reduced levels of desensitisation. In contrast, the M260L mutation, located higher up in the TM2 domain (at the 22' position), does not show any difference in desensitisation compared to wild-type receptors. We have found that in receptors containing the L247T mutation, both type I PAMs and type II PAMs are converted into non-desensitising agonists. In contrast, in receptors containing the M260L mutation, this effect is seen only with type II PAMs. These findings, indicating that the M260L mutation has a selective effect on type II PAMs, have been confirmed both with previously described PAMs and also with a series of novel α7-selective PAMs. The novel PAMs examined in this study have close chemical similarity but diverse pharmacological properties. For example, they include compounds displaying effects on receptor desensitisation that are typical of classical type I and type II PAMs but, in addition, they include compounds with intermediate properties.
Fig. 1. Chemical structures of the allosteric modulators examined in this study.
Fig. 2. Potentiation of responses to acetylcholine on wild-type α7 nAChRs by TBS compounds. Representative traces are shown illustrating responses to acetylcholine (100 μM) together with responses from the same oocyte to acetylcholine (100 μM) after pre- and co-application of 10 μM TBS-346 (A), TBS-546 (B), TBS-345 (C), TBS-556 (D) or TBS-516 (E).
Fig. 3. Influence of TBS compounds on the recovery of α7 nAChRs from desensitisation. Representative traces showing prolonged exposure of α7 nAChRs to acetylcholine (100 μM), causing activation, followed by rapid desensitisation. In the continued presence of acetylcholine, application of (A) TBS-346 (10 μM) and (B) TBS-546 (10 μM) does not result in reactivation of the receptor. However, application of (C) TBS-345 (10 μM), (D) TBS-556 (10 μM) and (E) TBS-516 (10 μM) results in reactivation of desensitised receptors. Traces have been scaled to their response to acetylcholine.
Fig. 4. Competition radioligand binding. Equilibrium radioligand binding was performed with [3H]-α-bungarotoxin (1 nM) with mammalian tsA201 cells transiently transfected with human α7 nAChR subunit and with human RIC-3 cDNAs (1:1 ratio). TBS-345, TBS-346, TBS-516, TBS-546 and TBS-556 caused no significant displacement of [3H]-α-bungarotoxin binding, whereas MLA caused complete displacement of specific radioligand binding. Data points are means of triplicate samples (± SD) from a single experiment, and data are typical of three independent experiments.
Fig. 5. The influence of α7 nAChR mutations (L247T and M260L) on activation by acetylcholine. A) The location of L247T (9′) and M260L (22′) mutations in the α7 nAChR subunit transmembrane (TM2) domain. The transmembrane region of an α7 nAChR subunit homology model (Young et al., 2008) is shown viewed from the top (left hand image) and from the side (right hand image). The α-helical transmembrane regions are illustrated as ribbon structures with the side chains of the two mutated amino acids shown as space-filling models (L247 in red and M260 in blue). B) Representative traces are shown illustrating responses to maximal concentrations of acetylcholine on human wild-type (WT) α7 nAChRs and α7 nAChRs containing the M260L mutation (M260L) and the L247T mutation (L247T). Acetylcholine concentrations: 1 mM for WT and M260L and 10 μM for L247T. C) Acetylcholine concentration-response data are presented for wild-type α7 nAChRs (triangles) and for α7 nAChRs containing either the M260L mutation (circles) or the L247T mutation (diamonds). Data are means ± SEM of at least three independent experiments and are normalised to the respective maximum response obtained with each nAChR variant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. The influence of a type I (NS-1738) and a type II PAM (TQS) on wild-type and mutated (L247T or M260L) α7 nAChRs. A) Representative traces illustrating responses with wild-type α7 nAChRs to acetylcholine (100 μM) and after the pre- and co-application of either NS-1738 (10 μM; Left pair of traces) or TQS (30 μM; Right). B) Representative traces illustrating responses with mutated (L247T) α7 nAChRs to acetylcholine (10 μM) and to NS-1738 (10 μM; Left) or TQS (30 μM; Right). C) Representative traces illustrating responses with mutated (M260L) α7 nAChRs to acetylcholine (100 μM) and NS-1738 (10 μM; Left) or TQS (30 μM; Right).
Fig. 7. Potentiation and agonist effects of TBS compounds on wild-type and mutated α7 nAChRs. A) Representative traces are shown illustrating responses from wild-type α7 nAChRs to acetylcholine (100 μM), together with responses to acetylcholine (100 μM) after pre- and co-application of TBS compounds (10 μM). To illustrate differences in the rate of desensitsation, all responses on wild-type α7 nAChRs have been normalised to their peak response. B) Representative traces are shown illustrating agonist responses from α7 nAChRs containing the L247T (9′) mutation to either acetylcholine (10 μM) or TBS compounds (10 μM). C) Representative traces are shown illustrating agonist responses from α7 nAChRs containing the M260L (22′) mutation to either acetylcholine (100 μM) or TBS compounds (10 μM).
Fig. 8. Antagonism of agonist responses to acetylcholine and TQS responses on M260L and L247T α7 nAChRs. A) Representative traces with M260L α7 nAChRs illustrating initial agonist responses with acetylcholine (1 mM) or TQS (10 μM) (left), antagonism by pre- and co-application of MLA (1 μM) (middle) and recovery in the absence of MLA (right). B) Representative traces with L247T α7 nAChRs illustrating initial agonist responses with acetylcholine (10 μM) or TQS (3 μM) (left), antagonism by pre- and co-application of MLA (1 μM) (middle) and recovery in the absence of MLA (right).
Fig. 9. Potentiation of acetylcholine responses by allosteric modulators on M260L α7 nAChRs. Representative traces are shown illustrating responses to acetylcholine (100 μM) (left) together with responses from the same oocyte after pre- and co-application of an allosteric modulator (10 μM) (right). Representative traces are shown for NS-1738 (A), TQS (B), TBS-346 (C), TBS-546 (D), TBS-345 (E), TBS-556 (F) and TBS-516 (G).
Fig. 10. Concentration–response curves for the wild-type and mutated α7 nAChRs. Data are shown from wild-type α7 nAChRs (A), α7 nAChRs containing the L247T (9′) mutation (B) and α7 nAChRs containing the M260L (22′) mutation (C). Data are presented for a range of concentrations of acetylcholine (circles), the allosteric agonist 4BP-TQS (hexagons), the type II PAMs, TQS (squares) and TBS-516 (inverted triangles), and the type I PAMs, NS-1738 (triangles) and TBS-346 (diamonds). Data are means ± SEM of at least three independent experiments and are normalised to the maximum acetylcholine response.
Fig. 11. Type I PAMs block agonist activity of TQS and TBS-516 on α7 nAChRs containing the M260L mutation. Representative traces are shown, obtained by two-electrode voltage-clamp recording in oocytes expressing α7 nAChRs containing the M260L mutation, in which a type I PAM (NS-1738 or TBS-346) was pre applied for 10 s and then co-applied with a type II PAM (TQS or TBS-516) (A–D). A) NS-1738 (10 μM) completely blocked responses to TQS (10 μM). B) TBS-346 (10 μM) blocked responses to TQS (10 μM) by 95.7 ± 1.1% (n = 3). C) NS-1738 (10 μM) blocked responses to TBS-516 (10 μM) by 92.7 ± 2.3% (n = 3). D) TBS-346 (10 μM) blocked responses to TBS-516 (10 μM) by 83.4 ± 4.1% (n = 3). E) The agonist concentration-response curve for TQS on α7 nAChRs containing the M260L mutation was shifted to the right in the presence of NS-1738 (2 μM, pre-applied for 10 s and then co-applied with TQS). The antagonism by NS-1738 was surmountable at high concentrations of TQS.
Albuquerque,
Mammalian nicotinic acetylcholine receptors: from structure to function.
2009, Pubmed
Albuquerque,
Mammalian nicotinic acetylcholine receptors: from structure to function.
2009,
Pubmed
Althoff,
X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors.
2014,
Pubmed
Amin,
GABAA receptor needs two homologous domains of the beta-subunit for activation by GABA but not by pentobarbital.
1993,
Pubmed
,
Xenbase
Arias,
Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors.
2000,
Pubmed
Arias,
Positive and negative modulation of nicotinic receptors.
2010,
Pubmed
Bafna,
Gating at the mouth of the acetylcholine receptor channel: energetic consequences of mutations in the alphaM2-cap.
2008,
Pubmed
Beckstein,
A hydrophobic gate in an ion channel: the closed state of the nicotinic acetylcholine receptor.
2006,
Pubmed
Bertrand,
Unconventional pharmacology of a neuronal nicotinic receptor mutated in the channel domain.
1992,
Pubmed
,
Xenbase
Bertrand,
Paradoxical allosteric effects of competitive inhibitors on neuronal alpha7 nicotinic receptor mutants.
1997,
Pubmed
,
Xenbase
Bertrand,
Allosteric modulation of nicotinic acetylcholine receptors.
2007,
Pubmed
Bertrand,
Positive allosteric modulation of the alpha7 nicotinic acetylcholine receptor: ligand interactions with distinct binding sites and evidence for a prominent role of the M2-M3 segment.
2008,
Pubmed
,
Xenbase
Broadbent,
Incorporation of the beta3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors.
2006,
Pubmed
,
Xenbase
Changeux,
The nicotinic acetylcholine receptor: the founding father of the pentameric ligand-gated ion channel superfamily.
2012,
Pubmed
Changeux,
Nicotinic receptors, allosteric proteins and medicine.
2008,
Pubmed
Collins,
Competitive binding at a nicotinic receptor transmembrane site of two α7-selective positive allosteric modulators with differing effects on agonist-evoked desensitization.
2011,
Pubmed
,
Xenbase
Cooper,
Host cell-specific folding and assembly of the neuronal nicotinic acetylcholine receptor alpha7 subunit.
1997,
Pubmed
Cooper,
Host cell-specific folding of the neuronal nicotinic receptor alpha8 subunit.
1998,
Pubmed
,
Xenbase
Couturier,
A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX.
1990,
Pubmed
,
Xenbase
Cully,
Identification of a Drosophila melanogaster glutamate-gated chloride channel sensitive to the antiparasitic agent avermectin.
1996,
Pubmed
,
Xenbase
Faghih,
Allosteric modulators of the alpha7 nicotinic acetylcholine receptor.
2008,
Pubmed
Gill,
Contrasting properties of α7-selective orthosteric and allosteric agonists examined on native nicotinic acetylcholine receptors.
2013,
Pubmed
Gill,
A series of α7 nicotinic acetylcholine receptor allosteric modulators with close chemical similarity but diverse pharmacological properties.
2012,
Pubmed
,
Xenbase
Gill,
Agonist activation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.
2011,
Pubmed
,
Xenbase
Gill-Thind,
Structurally similar allosteric modulators of α7 nicotinic acetylcholine receptors exhibit five distinct pharmacological effects.
2015,
Pubmed
,
Xenbase
Gotti,
Structural and functional diversity of native brain neuronal nicotinic receptors.
2009,
Pubmed
Grønlien,
Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes.
2007,
Pubmed
,
Xenbase
Gündisch,
Nicotinic acetylcholine receptor ligands, a patent review (2006-2011).
2011,
Pubmed
Hassaine,
X-ray structure of the mouse serotonin 5-HT3 receptor.
2014,
Pubmed
Haydar,
Neuronal nicotinic acetylcholine receptors - targets for the development of drugs to treat cognitive impairment associated with schizophrenia and Alzheimer's disease.
2010,
Pubmed
Hurst,
A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization.
2005,
Pubmed
,
Xenbase
Kalappa,
A positive allosteric modulator of α7 nAChRs augments neuroprotective effects of endogenous nicotinic agonists in cerebral ischaemia.
2013,
Pubmed
Lansdell,
Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site.
2015,
Pubmed
,
Xenbase
Lansdell,
Molecular characterization of Dalpha6 and Dalpha7 nicotinic acetylcholine receptor subunits from Drosophila: formation of a high-affinity alpha-bungarotoxin binding site revealed by expression of subunit chimeras.
2004,
Pubmed
Lansdell,
RIC-3 enhances functional expression of multiple nicotinic acetylcholine receptor subtypes in mammalian cells.
2005,
Pubmed
,
Xenbase
Lester,
Cys-loop receptors: new twists and turns.
2004,
Pubmed
Maricq,
Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel.
1991,
Pubmed
,
Xenbase
Mazurov,
Discovery and development of α7 nicotinic acetylcholine receptor modulators.
2011,
Pubmed
Millar,
Diversity of vertebrate nicotinic acetylcholine receptors.
2009,
Pubmed
Miyazawa,
Nicotinic acetylcholine receptor at 4.6 A resolution: transverse tunnels in the channel wall.
1999,
Pubmed
Moaddel,
Allosteric modifiers of neuronal nicotinic acetylcholine receptors: new methods, new opportunities.
2007,
Pubmed
Munro,
The α7 nicotinic ACh receptor agonist compound B and positive allosteric modulator PNU-120596 both alleviate inflammatory hyperalgesia and cytokine release in the rat.
2012,
Pubmed
Ng,
Nootropic alpha7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators.
2007,
Pubmed
,
Xenbase
Palma,
Threonine-for-leucine mutation within domain M2 of the neuronal alpha(7) nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist.
1996,
Pubmed
,
Xenbase
Papke,
The activity of GAT107, an allosteric activator and positive modulator of α7 nicotinic acetylcholine receptors (nAChR), is regulated by aromatic amino acids that span the subunit interface.
2014,
Pubmed
,
Xenbase
Revah,
Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.
1991,
Pubmed
,
Xenbase
Romanelli,
Cholinergic nicotinic receptors: competitive ligands, allosteric modulators, and their potential applications.
2003,
Pubmed
Targowska-Duda,
Antidepressant activity in mice elicited by 3-furan-2-yl-N-p-tolyl-acrylamide, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor.
2014,
Pubmed
Timmermann,
An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo.
2007,
Pubmed
,
Xenbase
Unwin,
Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
2005,
Pubmed
Weiland,
Neuronal nicotinic acetylcholine receptors: from the gene to the disease.
2000,
Pubmed
Williams,
Positive allosteric modulators as an approach to nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations.
2011,
Pubmed
Young,
Species selectivity of a nicotinic acetylcholine receptor agonist is conferred by two adjacent extracellular beta4 amino acids that are implicated in the coupling of binding to channel gating.
2007,
Pubmed
Young,
Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.
2008,
Pubmed
,
Xenbase
Zhu,
Potentiation of analgesic efficacy but not side effects: co-administration of an α4β2 neuronal nicotinic acetylcholine receptor agonist and its positive allosteric modulator in experimental models of pain in rats.
2011,
Pubmed
