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.
PLoS One
2015 Sep 04;109:e0137588. doi: 10.1371/journal.pone.0137588.
Show Gene links
Show Anatomy links
Functional Impact of 14 Single Nucleotide Polymorphisms Causing Missense Mutations of Human α7 Nicotinic Receptor.
Zhang Q
,
Du Y
,
Zhang J
,
Xu X
,
Xue F
,
Guo C
,
Huang Y
,
Lukas RJ
,
Chang Y
.
???displayArticle.abstract???
The α7nicotinic receptor (nAChR) is a major subtype of the nAChRs in the central nervous system, and the receptor plays an important role in brain function. In the dbSNP database, there are 55 single nucleotide polymorphisms (SNPs) that cause missense mutations of the human α7nAChR in the coding region. In this study, we tested the impact of 14 SNPs that cause missense mutations in the agonist binding site or the coupling region between binding site and channel gate on the receptor function. The wild type or mutant receptors were expressed or co-expressed in Xenopus oocytes, and the agonist-induced currents were tested using two-electrode voltage clamp. Our results demonstrated that 6 mutants were nonfunctional, 4 mutants had reduced current expression, and 1 mutants altered ACh and nicotine efficacy in the opposite direction, and one additional mutant had slightly reduced agonist sensitivity. Interestingly, the function of most of these nonfunctional mutants could be rescued by α7nAChR positive allosteric modulator PNU-120596 and agonist-PAM 4BP-TQS. Finally, when coexpressed with the wild type, the nonfunctional mutants could also influence the receptor function. These changes of the receptor properties by the mutations could potentially have an impact on the physiological function of the α7nAChR-mediated cholinergic synaptic transmission and anti-inflammatory effects in the human SNP carriers. Rescuing the nonfunctional mutants could provide a novel way to treat the related disorders.
???displayArticle.pubmedLink???
26340537
???displayArticle.pmcLink???PMC4560414 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Fig 2. Current responses of the wild type and mutant receptors to 3.16mM ACh or nicotine with non-injected oocyte controls.A. 13 mutants and wild type control along with un-injected oocyte control (for nonfunctional mutants). The same amount of cRNAs for the wild type or mutant receptors was injected. On the 3rd post-injection day, the oocytes were tested with ACh (with 1 μM atropine) or nicotine. The name of each condition is indicated at the bottom of each bar. Each group had 8–18 oocytes from two sets of experiments. Asterisk (*, **, or ****) represents that the difference between the wild type and each mutant is statistically significant (P<0.05, P<0.01, or P<0.0001) in Tukey multiple comparison test of one-way ANOVA. ♦, ♦♦ or ♦♦♦♦ represent the statistical difference with P<0.05, P<0.01, or P<0.0001 between blank and each mutant. B. W55G mutant and its wild type control in a separate experiment (10 oocytes each group). ****: P<0.0001 with 2-sided grouped t-test.
Fig 3. ACh concentration-response for most of the functional mutants.A. Representative current traces induced by ACh for the wild type and mutant receptors with the concentrations indicated. B. Averaged and normalized concentration-responses of charge (6–8 oocytes each group). Lines are nonlinear least squares fits of the normalized averages of the responses to the Hill equation. C. Bar graph of the pEC50 values (negative logEC50s) derived from B. ***: P<0.001 when compared to the WT value.
Fig 4. Concentration-responses of W55G mutant to ACh and nicotine.A. Concentration responses of the wild type and W55G to ACh. Top: raw current traces; bottom: normalized and averaged currents. Lines are least-squares fit of the data to the Hill equation. The resulting EC50 for ACh in the wild type receptor was 210.5±24.3 μM, and the EC50 for W55G mutant was 1375.3±130.5 μM (N = 5). B. Concentration responses of the wild type and W55G to nicotine. Top: raw current traces; bottom: normalized and averaged currents. Lines are least-squares fit of the data to the Hill equation. The resulting EC50 values for nicotine were 43.6±4.8 μM and 530.40±12.91 μM for the wild type and mutant receptor respectively (N = 5).
Fig 5. Agonist-responses for the nonfunctional mutants in the presence of a PAM or agonist-PAM.A. Co-application of 31.6 μM PNU-120596 with 200 μM ACh or nicotine rescued the receptor functions for some of the nonfunctional mutants. The same amount of cRNA was injected for each group, and recordings were performed after 3 days in 9–19 oocytes for each group). The bar graph represents the average currents rescued by PNU-120596. In case of the wild type, the current represents the rescued current from desensitization. B, Direct activation of nonfunctional mutants by 4BP-TQS. The same amount of cRNA was injected for each group, and recordings were performed after 3 days in 10–17 oocytes in each group). C, Co-application of 4BP-TQS with ACh or nicotine rescued more mutants. The same amount of cRNA was injected for each group, and recordings were performed after 3 days in 9–19 oocytes in each group. Asterisk (****) represents that the difference between the wild type and each mutant is statistically significant (P<0.0001) in Tukey multiple comparison test of one-way ANOVA. ♦♦, ♦♦♦, or ♦♦♦♦ represent the difference between blank and each mutant with statistical significance (P<0.01, P<0.001, or P<0.0001).
Fig 6. PNU120596 concentration-response for the rescued nonfunctional mutants with a fixed ACh or nicotine concentration.A. Representative current traces induced by increasing concentration of PNU-120596 in the presence of 200 μM ACh. B. Normalized and averaged (each group had at least 6 oocytes) current responses to ACh from A. Lines are nonlinear least squares fits of the normalized averages of the responses to the Hill equation. The derived EC50 values from individual fits are listed in Table 2. C. Representative current traces induced by increasing concentration of PNU-120596 in the presence of 200 μM nicotine. D. Normalized and averaged current responses (each group had at least 6 oocytes) to ACh from C. The derived EC50 values from individual fits are listed in Table 2.
Fig 7. Concentration response of 4BP-TQS direct activation of the wild type control and Y93C, C191Y, and Y211C mutants.A, Representative current traces induced by increasing concentration of 4BP-TQS. B. Normalized and averaged current responses to 4BP-TQS from A. Lines are nonlinear least squares fits of the normalized averages of the responses to the Hill equation. The resulting EC50 values were 5.54±0.31, 8.21±1.03, 13.73±1.73, and 4.23±0.30 μM for the wild type control and Y93C, C191Y, and Y211C mutants respectively (n = 6 for each group).
Fig 8. 4BP-TQS concentration-response for the rescued nonfunctional mutants with a fixed ACh or nicotine concentration.A. Representative current traces induced by increasing concentration of 4BP-TQS in the presence of 200 μM ACh. B. Normalized and averaged (each group had 5–6 oocytes) current responses from A. Lines are nonlinear least squares fits of the normalized averages of the responses to the Hill equation. The derived EC50 values from individual fits are listed in Table 3. C. Representative current traces induced by increasing concentration of 4BP-TQS in the presence of 200 μM nicotine. D. Normalized and averaged current responses (each group had 5–6 oocytes) from C. The derived EC50 values from individual fits are listed in Table 3.
Fig 9. Coexpression of wild type and nonfunctional mutant.The same amount of cRNAs encoding the wild type or wild type plus mutant (in 1:1 ratio) were injected into Xenopus oocytes, and recorded with two electrode voltage clamp for ACh-induced current after 3 days of injection. A. Peak current induced by 3.16mM ACh for all groups on the 3 post-injection day. "****": P<0.0001. Each group had at least 16 oocytes. B. Averaged concentration response relationships of the wild type and wild type plus mutant as indicated. Each group is the average of six oocyte data. C. EC50 values were derived by fitting individual concentration-response curves in B. pEC50 values (negative logEC50) are used to plot the data. ("**", “***”, “****”: p<0.01, P<0.001, or P<0.0001). D. Linear regression analysis for the relationship between Hill slope and LogEC50.
Fig 10. Different interactions of ACh and nicotine with the AChBP binding residues.A. The binding pocket between chain A and chain B of the AChBP co-crystalized with ACh (PBDID: 3WIP); B. The binding pocket between chain A and chain B of the AChBP co-crystalized with nicotine (PBDID: 1UW6). The residues of Cys191 and Trp55 in the human α7nAChR are labeled next to their homologous residues in the AChBP. Arrows indicate different interactions.
Fig 1. Location of 14 mutations in the α7 nAChR receptor.A: The residues with the mutations in the binding region (loops A (TYR93), and C (CYS191, LYS192, ASP197) of the principal side of a binding pocket, and D (TRP55) of the complementary side of the neighboring binding pocket); B, The residues with the mutations in the coupling region (loop 2 (ASN47), loop 9 (ASN171 and GLU173), pre-M1 (ARG205 and ARG206) and M1 (TYR211 and GLY212).
Albuquerque,
Mammalian nicotinic acetylcholine receptors: from structure to function.
2009, Pubmed
Albuquerque,
Mammalian nicotinic acetylcholine receptors: from structure to function.
2009,
Pubmed
Andersen,
Stoichiometry for activation of neuronal α7 nicotinic receptors.
2013,
Pubmed
Barnes,
The 5-HT3 receptor--the relationship between structure and function.
2009,
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
Bocquet,
X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation.
2009,
Pubmed
Brejc,
Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.
2001,
Pubmed
Broide,
The alpha7 nicotinic acetylcholine receptor in neuronal plasticity.
1999,
Pubmed
Brookes,
The essence of SNPs.
1999,
Pubmed
Celie,
Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures.
2004,
Pubmed
Chang,
Allosteric activation mechanism of the cys-loop receptors.
2009,
Pubmed
Changeux,
Allosteric receptors after 30 years.
1998,
Pubmed
Chen,
Evolutionarily conserved allosteric network in the Cys loop family of ligand-gated ion channels revealed by statistical covariance analyses.
2006,
Pubmed
Cheng,
Targeted molecular dynamics study of C-loop closure and channel gating in nicotinic receptors.
2006,
Pubmed
Chojnacka,
Synthesis and evaluation of a conditionally-silent agonist for the α7 nicotinic acetylcholine receptor.
2013,
Pubmed
,
Xenbase
Criado,
Role of loop 9 on the function of neuronal nicotinic receptors.
2010,
Pubmed
,
Xenbase
Dani,
Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system.
2007,
Pubmed
Davies,
A novel class of ligand-gated ion channel is activated by Zn2+.
2003,
Pubmed
Elmslie,
Genetic mapping of a major susceptibility locus for juvenile myoclonic epilepsy on chromosome 15q.
1997,
Pubmed
Freedman,
Linkage of a neurophysiological deficit in schizophrenia to a chromosome 15 locus.
1997,
Pubmed
Gay,
Aromatic residues at position 55 of rat alpha7 nicotinic acetylcholine receptors are critical for maintaining rapid desensitization.
2008,
Pubmed
,
Xenbase
Gill,
Agonist activation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.
2011,
Pubmed
,
Xenbase
Horenstein,
Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors.
2007,
Pubmed
,
Xenbase
Johnston,
GABA(C) receptors as drug targets.
2003,
Pubmed
Lee,
Principal pathway coupling agonist binding to channel gating in nicotinic receptors.
2005,
Pubmed
Lukas,
International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits.
1999,
Pubmed
Lynch,
Native glycine receptor subtypes and their physiological roles.
2009,
Pubmed
Martelli,
The cholinergic anti-inflammatory pathway: a critical review.
2014,
Pubmed
Mowrey,
Signal transduction pathways in the pentameric ligand-gated ion channels.
2013,
Pubmed
Mukhtasimova,
Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor.
2005,
Pubmed
Newell,
Mutation of glutamate 155 of the GABAA receptor beta2 subunit produces a spontaneously open channel: a trigger for channel activation.
2004,
Pubmed
,
Xenbase
Nordman,
An interaction between α7 nicotinic receptors and a G-protein pathway complex regulates neurite growth in neural cells.
2012,
Pubmed
Northrop,
Regulation of glutamate release by α7 nicotinic receptors: differential role in methamphetamine-induced damage to dopaminergic and serotonergic terminals.
2011,
Pubmed
Olsen,
Molecular recognition of the neurotransmitter acetylcholine by an acetylcholine binding protein reveals determinants of binding to nicotinic acetylcholine receptors.
2014,
Pubmed
Olsen,
International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update.
2008,
Pubmed
Olsen,
GABA A receptors: subtypes provide diversity of function and pharmacology.
2009,
Pubmed
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
Papke,
The analgesic-like properties of the alpha7 nAChR silent agonist NS6740 is associated with non-conducting conformations of the receptor.
2015,
Pubmed
,
Xenbase
Purohit,
Sources of energy for gating by neurotransmitters in acetylcholine receptor channels.
2012,
Pubmed
Purohit,
Functional anatomy of an allosteric protein.
2013,
Pubmed
Sadis,
Nicotine protects kidney from renal ischemia/reperfusion injury through the cholinergic anti-inflammatory pathway.
2007,
Pubmed
Sedelnikova,
Mapping the rho1 GABA(C) receptor agonist binding pocket. Constructing a complete model.
2005,
Pubmed
Sharp,
A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures.
2008,
Pubmed
Shaw,
Janus kinase 2, an early target of alpha 7 nicotinic acetylcholine receptor-mediated neuroprotection against Abeta-(1-42) amyloid.
2002,
Pubmed
Sherry,
dbSNP-database for single nucleotide polymorphisms and other classes of minor genetic variation.
1999,
Pubmed
Spurny,
Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor.
2015,
Pubmed
,
Xenbase
Thompson,
The 5-HT3 receptor as a therapeutic target.
2007,
Pubmed
Uteshev,
The therapeutic promise of positive allosteric modulation of nicotinic receptors.
2014,
Pubmed
Vicente-Agullo,
Multiple roles of the conserved key residue arginine 209 in neuronal nicotinic receptors.
2001,
Pubmed
,
Xenbase
Wang,
Stimulation of α7 nicotinic acetylcholine receptor by nicotine increases suppressive capacity of naturally occurring CD4+CD25+ regulatory T cells in mice in vitro.
2010,
Pubmed
Williams,
Investigation of the molecular mechanism of the α7 nicotinic acetylcholine receptor positive allosteric modulator PNU-120596 provides evidence for two distinct desensitized states.
2011,
Pubmed
,
Xenbase
Williams,
Differential regulation of receptor activation and agonist selectivity by highly conserved tryptophans in the nicotinic acetylcholine receptor binding site.
2009,
Pubmed
,
Xenbase
Young,
Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.
2008,
Pubmed
,
Xenbase
de Jonge,
The alpha7 nicotinic acetylcholine receptor as a pharmacological target for inflammation.
2007,
Pubmed
van Maanen,
The cholinergic anti-inflammatory pathway: towards innovative treatment of rheumatoid arthritis.
2009,
Pubmed