Nicotinic acetylcholine receptors (nAChRs) are pentameric ion channels expressed in the central nervous systems. nAChRs containing the α4, β2 and α5 subunits are specifically involved in addictive processes, but their functional architecture is poorly understood due to the intricacy of assembly of these subunits. Here we constrained the subunit assembly by designing fully concatenated human α4β2 and α4β2α5 receptors and characterized their properties by two-electrodes voltage–clamp electrophysiology in Xenopus oocytes. We found that α5-containing nAChRs are irreversibly blocked by methanethiosulfonate (MTS) reagents through a covalent reaction with a cysteine present only in α5. MTS-block experiments establish that the concatemers are expressed in intact form at the oocyte surface, but that reconstitution of nAChRs from loose subunits show inefficient and highly variable assembly of α5 with α4 and β2. Mutational analysis shows that the concatemers assemble both in clockwise and anticlockwise orientations, and that α5 does not contribute to ACh binding from its principal (+) site. Reinvestigation of suspected α5-ligands such as galantamine show no specific effect on α5-containing concatemers. Analysis of the α5-D398N mutation that is linked to smoking and lung cancer shows no significant effect on the electrophysiological function, suggesting that its effect might arise from alteration of other cellular processes. The concatemeric strategy provides a well-characterized platform for mechanistic analysis and screening of human α5-specific ligands.
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γ-Amino butyric acid
Green fluorescent protein
Nicotinic acetylcholine receptor
Substituted cysteine accessibility method
Wen L, Jiang K, Yuan W et al (2014) Contribution of variants in CHRNA5/A3/B4 gene cluster on chromosome 15 to tobacco smoking: from genetic association to mechanism. Mol Neurobiol 53:472–484. https://doi.org/10.1124/jpet.107.132977
Salas R, Orr-Urtreger A, Broide RS et al (2003) The nicotinic acetylcholine receptor subunit alpha 5 mediates short-term effects of nicotine in vivo. Mol Pharmacol 63:1059–1066. https://doi.org/10.1124/mol.63.5.1059
Morel C, Fattore L, Pons S et al (2014) Nicotine consumption is regulated by a human polymorphism in dopamine neurons. Mol Psychiatry 19:930–936. https://doi.org/10.1038/mp.2013.158
Fowler CD, Lu Q, Johnson PM et al (2011) Habenular α5 nicotinic receptor subunit signalling controls nicotine intake. Nature 471:597–601. https://doi.org/10.1038/nature09797
Dawson A, Wolstenholme JT, Roni MA et al (2018) Knockout of alpha 5 nicotinic acetylcholine receptors subunit alters ethanol-mediated behavioral effects and reward in mice. Neuropharmacology 138:341–348. https://doi.org/10.1016/j.neuropharm.2018.06.031
Besson M, Guiducci S, Granon S et al (2016) Alterations in alpha5* nicotinic acetylcholine receptors result in midbrain- and hippocampus-dependent behavioural and neural impairments. Psychopharmacology. https://doi.org/10.1007/s00213-016-4362-2
Forget B, Scholze P, Langa F et al (2018) A human polymorphism in CHRNA5 is linked to relapse to nicotine seeking in transgenic rats. Curr Biol 28:3244–3253.e7. https://doi.org/10.1016/j.cub.2018.08.044
Besson M, Forget BXT, Correia C et al (2019) Profound alteration in reward processing due to a human polymorphism in. Neuropsychopharmacology. https://doi.org/10.1038/s41386-019-0462-0
Exley R, McIntosh JM, Marks MJ et al (2012) Striatal 5 nicotinic receptor subunit regulates dopamine transmission in dorsal striatum. J Neurosci 32:2352–2356. https://doi.org/10.1523/JNEUROSCI.4985-11.2012
Grady SR, Salminen O, McIntosh JM et al (2009) Mouse striatal dopamine nerve terminals express α4α5β2 and two stoichiometric forms of α4β2*-nicotinic acetylcholine receptors. J Mol Neurosci 40:91–95. https://doi.org/10.1124/mol.54.6.1124
George AA, Lucero LM, Damaj MI et al (2012) Function of human α3β4α5 nicotinic acetylcholine receptors is reduced by the α5(D398N) variant. J Biol Chem 287:25151–25162. https://doi.org/10.1124/mol.63.5.1059
Mazzaferro S, Benallegue N, Carbone A et al (2011) Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity. J Biol Chem 286:31043–31054. https://doi.org/10.1074/jbc.M111.262014
Ahring PK, Liao VWY, Balle T (2018) Concatenated nicotinic acetylcholine receptors: a gift or a curse? J Gen Physiol 150:453–473. https://doi.org/10.1124/mol.54.6.1124
Groot-Kormelink PJ, Broadbent S, Beato M, Sivilotti LG (2006) Constraining the expression of nicotinic acetylcholine receptors by using pentameric constructs. Mol Pharmacol 69:558–563. https://doi.org/10.1124/mol.105.019356
Carbone A-L, Moroni M, Groot-Kormelink P-J, Bermudez I (2009) Pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) nicotinic acetylcholine receptors: subunit arrangement determines functional expression. Br J Pharmacol 156:970–981. https://doi.org/10.1111/j.1476-5381.2008.00104.x
Mazzaferro S, Benallegue N, Carbone A et al (2011) Additional acetylcholine (ACh) binding site at α4/α4 interface of (α4β2) 2α4 nicotinic receptor influences agonist sensitivity. J Biol Chem 286:31043–31054. https://doi.org/10.1523/JNEUROSCI.0627-09.2009
Benallegue N, Mazzaferro S, Alcaino C, Bermudez I (2013) The additional ACh binding site at the α4(+)/α4(−) interface of the (α4β2) 2α4 nicotinic ACh receptor contributes to desensitization. Br J Pharmacol 170:304–316. https://doi.org/10.1074/jbc.M111.221754
Eaton JB, Lucero LM, Stratton H et al (2013) The unique α4(+)/(−) α4 agonist binding site in (α4)3(β2)2 subtype nicotinic acetylcholine receptors permits differential agonist desensitization pharmacology versus the (α4)2(β2)3 subtype. J Pharmacol Exp Ther 348:46–58. https://doi.org/10.1124/mol.54.6.1124
Jin X, Bermudez I, Steinbach JH (2014) The nicotinic α5 subunit can replace either an acetylcholine-binding or nonbinding subunit in the α4β2* neuronal nicotinic receptor. Mol Pharmacol 85:11–17. https://doi.org/10.1124/mol.113.089979
Lucero LM, Weltzin MM, Eaton JB et al (2016) Differential α4(+)/(−)β2 agonist-binding site contributions to α4β2 nicotinic acetylcholine receptor function within and between isoforms. J Biol Chem 291:2444–2459. https://doi.org/10.1124/mol.115.098061
George AA, Bloy A, Miwa JM et al (2016) Isoform-specific mechanisms of a3b4*-nicotinic acetylcholine receptor modulation by the prototoxin lynx1. FASEB J 31:1398–1420. https://doi.org/10.1124/mol.115.098061
Mazzaferro S, Bermudez I, Sine SM (2018) Potentiation of a neuronal nicotinic receptor via pseudo-agonist site. Cell Mol Life Sci. https://doi.org/10.1007/s00018-018-2993-7
Gielen MC, Lumb MJ, Smart TG (2012) Benzodiazepines modulate GABAA receptors by regulating the preactivation step after GABA binding. J Neurosci 32:5707–5715. https://doi.org/10.1523/JNEUROSCI.5663-11.2012
Nashmi R, Dickinson ME, McKinney S et al (2003) Assembly of alpha4beta2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons. J Neurosci 23:11554–11567
Morales-Perez CL, Noviello CM, Hibbs RE (2016) Manipulation of subunit stoichiometry in heteromeric membrane proteins. Structure 24:797–805. https://doi.org/10.1016/j.str.2016.03.004
Hamouda AK, Deba F, Wang Z-J, Cohen JB (2016) Photolabeling a nicotinic acetylcholine receptor (nAChR) with an ( α4) 3( β2) 2nAChR-selective positive allosteric modulator. Mol Pharmacol 89:575–584. https://doi.org/10.1124/mol.54.6.1124
Olsen JA, Ahring PK, Kastrup JS et al (2014) Structural and functional studies of the modulator NS9283 reveal agonist-like mechanism of action at α4β2 nicotinic acetylcholine receptors. J Biol Chem 289:24911–24921. https://doi.org/10.1074/jbc.M114.568097
Jain A, Kuryatov A, Wang J et al (2016) Unorthodox acetylcholine binding sites formed by α5 and β3 accessory subunits in α4β2* nicotinic acetylcholine receptors. J Biol Chem 291:23452–23463. https://doi.org/10.1016/j.ymeth.2010.01.012
Xiu X, Puskar NL, Shanata JAP et al (2009) Nicotine binding to brain receptors requires a strong cation. Nature 458:534–537. https://doi.org/10.1038/nature07768
Zwart R, Carbone AL, Moroni M et al (2008) Sazetidine-A is a potent and selective agonist at native and recombinant α4β2 nicotinic acetylcholine receptors. Mol Pharmacol 73:1838–1843. https://doi.org/10.1124/mol.54.6.1124
Mazzaferro S, Gasparri F, New K et al (2014) Non-equivalent ligand selectivity of agonist sites in (α4β2) 2α4 nicotinic acetylcholine receptors. J Biol Chem 289:21795–21806. https://doi.org/10.1113/jphysiol.1989.sp017717
Samochocki M, Höffle A, Fehrenbacher A et al (2003) Galantamine is an allosterically potentiating ligand of neuronal nicotinic but not of muscarinic acetylcholine receptors. J Pharmacol Exp Ther 305:1024–1036. https://doi.org/10.1046/j.1471-4159.2000.0752492.x
Kuryatov A, Onksen J, Lindstrom J (2008) Roles of accessory subunits in α4β2* nicotinic receptors. Mol Pharmacol 74:132–143. https://doi.org/10.1074/jbc.273.44.28721
Kowal NM, Ahring PK, Liao VWY et al (2017) Galantamine is not a positive allosteric modulator of human α4β2 or α7 nicotinic acetylcholine receptors. Br J Pharmacol 175:2911–2925. https://doi.org/10.1124/mol.54.6.1124
Valera S, Ballivet M, Bertrand D (1992) Progesterone modulates a neuronal nicotinic acetylcholine receptor. Proc Natl Acad Sci USA 89:9949–9953. https://doi.org/10.1073/pnas.89.20.9949
Kuryatov A, Berrettini W, Lindstrom J (2011) Acetylcholine receptor (AChR) α5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (α4β2)2α5 AChR function. Mol Pharmacol 79:119–125. https://doi.org/10.1124/mol.110.066357
Tammimäki A, Herder P, Li P et al (2012) Impact of human D398N single nucleotide polymorphism on intracellular calcium response mediated by α3β4α5 nicotinic acetylcholine receptors. Neuropharmacology 63:1002–1011. https://doi.org/10.1016/j.neuropharm.2012.07.022
Li P, McCollum M, Bracamontes J et al (2011) Functional characterization of the α5(Asn398) variant associated with risk for nicotine dependence in the α3β4α5 nicotinic receptor. Mol Pharmacol 80:818–827. https://doi.org/10.1124/mol.111.073841
Ramirez-Latorre J, Yu CR, Qu X et al (1996) Functional contributions of alpha5 subunit to neuronal acetylcholine receptor channels. Nature 380:347–351. https://doi.org/10.1038/380347a0
Marotta CB, Dilworth CN, Lester HA, Dougherty DA (2014) Neuropharmacology. Neuropharmacology 77:342–349. https://doi.org/10.1016/j.neuropharm.2013.09.028
Hannan S, Smart TG (2018) Cell surface expression of homomeric GABA A receptors depends on single residues in subunit transmembrane domains. J Biol Chem 293:13427–13439. https://doi.org/10.1002/prot.22488
Gharpure A, Teng J, Zhuang Y et al (2019) Agonist selectivity and ion permeation in the α3β4 ganglionic nicotinic receptor. Neuron 104:501–511.e6. https://doi.org/10.1016/j.neuron.2019.07.030
Szabo A, Nourmahnad A, Halpin E, Forman SA (2019) Monod–Wyman–Changeux allosteric shift analysis in mutant α1 β3 γ2L GABA A receptors indicates selectivity and crosstalk among intersubunit transmembrane anesthetic sites. Mol Pharmacol 95:408–417. https://doi.org/10.1038/81800
Absalom NL, Ahring PK, Liao VW et al (2019) Functional genomics of epilepsy-associated mutations in the GABA A receptor subunits reveal that one mutation impairs function and two are catastrophic. J Biol Chem 294:6157–6171. https://doi.org/10.1371/journal.pone.0141359
This work was founded by the ERC Grant (No. 788974, Dynacotine), the ANR Grant (No. 17-CE11-0030 Nicofive), the “équipe FRM” (Fondation pour la Recherche Médicale) grant DEQ20140329497, a fellowship from the Fondation ARC to MSP and a European Commission Research Executive Agency individual fellowship to MG (Marie Sklodowska-Curie Action, Individual Fellowship 659371). The authors would like to thank Uwe Maskos, Stéphanie Pons, Morgane Besson, Akos Nemecz and Alexandre Mourot for critical reading of the manuscript.
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Prevost, M.S., Bouchenaki, H., Barilone, N. et al. Concatemers to re-investigate the role of α5 in α4β2 nicotinic receptors. Cell. Mol. Life Sci. 78, 1051–1064 (2021). https://doi.org/10.1007/s00018-020-03558-z
- Biochemical pharmacology
- Ligand-gated ion channels
- Nicotinic receptors