Abstract
Neuronal nicotinic receptors containing α4 and β2 subunits assemble in two pentameric stoichiometries, (α4)3(β2)2 and (α4)2(β2)3, each with distinct pharmacological signatures; (α4)3(β2)2 receptors are strongly potentiated by the drug NS9283, whereas (α4)2(β2)3 receptors are unaffected. Despite this stoichiometry-selective pharmacology, the molecular identity of the target for NS9283 remains elusive. Here, studying (α4)3(β2)2 receptors, we show that mutations at either the principal face of the β2 subunit or the complementary face of the α4 subunit prevent NS9283 potentiation of ACh-elicited single-channel currents, suggesting the drug targets the β2–α4 pseudo-agonist sites, the α4–α4 agonist site, or both sites. To distinguish among these possibilities, we generated concatemeric receptors with mutations at specified subunit interfaces, and monitored the ability of NS9283 to potentiate ACh-elicited single-channel currents. We find that a mutation at the principal face of the β2 subunit at either β2–α4 pseudo-agonist site suppresses potentiation, whereas mutation at the complementary face of the α4 subunit at the α4–α4 agonist site allows a significant potentiation. Thus, monitoring potentiation of single concatemeric receptor channels reveals that the β2–α4 pseudo-agonist sites are required for stoichiometry-selective drug action. Together with the recently determined structure of the (α4)3(β2)2 receptor, the findings have implications for structure-guided drug design.
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Abbreviations
- nAChR:
-
Nicotinic acetylcholine receptor
- ACh:
-
Acetylcholine
References
Millar NS, Gotti C (2009) Diversity of vertebrate nicotinic acetylcholine receptors. Neuropharmacology 56:237–246
Albuquerque EX, Pereira EFR, Alkondon M, Rogers SW (2009) Mammalian nicotinic acetylcholine receptors: from structure to function. Physiol Rev 89:73–120
Hurst R, Rollema H, Bertrand D (2013) Nicotinic acetylcholine receptors: from basic science to therapeutics. Pharmacol Ther 137:22–54
Taly A, Corringer P-J, Guedin D, Lestage P, Changeux J-P (2009) Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. Nat Rev Drug Discov 8:733–750
Laviolette SR, van der Kooy D (2004) The neurobiology of nicotine addiction: bridging the gap from molecules to behaviour. Nat Rev Neurosci 5:55–65
Picciotto MR, Kenny PJ (2013) Molecular mechanisms underlying behaviors related to nicotine addiction. Cold Spring Harb Perspect Med 3:a012112
Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J Mol Biol 346:967–989
Marubio LM, del Mar Arroyo-Jimenez M, Cordero-Erausquin M, Lena C, Le Novere N, de Kerchove d’Exaerde A, Huchet M, Damaj MI, Changeux JP (1999) Reduced antinociception in mice lacking neuronal nicotinic receptor subunits. Nature 398:805–810
Picciotto MR, Zoli M, Léna C, Bessis A, Lallemand Y, Le Novère N, Vincent P, Pich EM, Brûlet P, Changeux JP (1995) Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain. Nature 374:65–67
Ross SA, Wong JY, Clifford JJ, Kinsella A, Massalas JS, Horne MK, Scheffer IE, Kola I, Waddington JL, Berkovic SF, Drago J (2000) Phenotypic characterization of an alpha 4 neuronal nicotinic acetylcholine receptor subunit knock-out mouse. J Neurosci 20:6431–6441
Zoli M, Léna C, Picciotto MR, Changeux JP (1998) Identification of four classes of brain nicotinic receptors using beta2 mutant mice. J Neurosci 18:4461–4472
Zwart R, Vijverberg HP (1998) Four pharmacologically distinct subtypes of alpha4beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes. Mol Pharmacol 54:1124–1131
Moroni M, Zwart R, Sher E, Cassels BK, Bermudez I (2006) alpha4beta2 nicotinic receptors with high and low acetylcholine sensitivity: pharmacology, stoichiometry, and sensitivity to long-term exposure to nicotine. Mol Pharmacol 70:755–768
Mazzaferro S, Bermudez I, Sine SM (2017) α4β2 nicotinic acetylcholine receptors: relationships between subunit stoichiometry and function at the single channel level. J Biol Chem 292:2729–2740
Nelson ME, Kuryatov A, Choi CH, Zhou Y, Lindstrom J (2003) Alternate stoichiometries of alpha4beta2 nicotinic acetylcholine receptors. Mol Pharmacol 63:332–341
Grupe M, Jensen AA, Ahring PK, Christensen JK, Grunnet M (2013) Unravelling the mechanism of action of NS9283, a positive allosteric modulator of (α4)3(β2)2 nicotinic ACh receptors. Br J Pharmacol 168:2000–2010
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
Mazzaferro S, Benallegue N, Carbone A, Gasparri F, Vijayan R, Biggin PC, Moroni M, Bermudez I (2011) Additional acetylcholine (ACh) binding site at α4/α4 interface of (α4β2)2α4 nicotinic receptor influences agonist sensitivity. J Biol Chem 286:31043–31054
Jeanclos EM, Lin L, Treuil MW, Rao J, DeCoster MA, Anand R (2001) The chaperone protein 14-3-3η interacts with the nicotinic acetylcholine receptor α4 subunit. J Biol Chem 276:28281–28290
Exley R, Moroni M, Sasdelli F, Houlihan LM, Lukas RJ, Sher E, Zwart R, Bermudez I (2006) Chaperone protein 14-3-3 and protein kinase a increase the relative abundance of low agonist sensitivity human α4β2 nicotinic acetylcholine receptors in Xenopus oocytes. J Neurochem 98:876–885
Pear WS, Nolan GP, Scott ML, Baltimore D (1993) Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci USA 90:8392–8396
Sine SM (1993) Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of residues that determine curare selectivity. Proc Natl Acad Sci USA 90:9436–9440
Sine SM, Quiram P, Papanikolaou F, Kreienkamp HJ, Taylor P (1994) Conserved tyrosines in the alpha subunit of the nicotinic acetylcholine receptor stabilize quaternary ammonium groups of agonists and curariform antagonists. J Biol Chem 269:8808–8816
Bouzat C, Bren N, Sine SM (1994) Structural basis of the different gating kinetics of fetal and adult acetylcholine receptors. Neuron 13:1395–1402
New K, Del Villar SG, Mazzaferro S, Alcaino C, Bermudez I (2018) The fifth subunit of the (α4β2)2 β2 nicotinic ACh receptor modulates maximal ACh responses. Br J Pharmacol 175:1822–1837
Cooper ST, Harkness PC, Baker ER, Millar NS (1999) Up-regulation of cell-surface α4β2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits. J Biol Chem 274:27145–27152
Moroni M, Vijayan R, Carbone A, Zwart R, Biggin PC, Bermudez I (2008) Non-agonist-binding subunit interfaces confer distinct functional signatures to the alternate stoichiometries of the alpha4beta2 nicotinic receptor: an alpha4–alpha4 interface is required for Zn2+ potentiation. J Neurosci 28:6884–6894
Mazzaferro S, Gasparri F, New K, Alcaino C, Faundez M, Vasquez PI, Vijayan R, Biggin PC, Bermudez I (2014) Non-equivalent ligand selectivity of agonist sites in (α4β2)2α4 nicotinic acetylcholine receptors: a key determinant of agonist efficacy. J Biol Chem 289:21795–21806
Rayes D, Spitzmaul G, Sine SM, Bouzat C (2005) Single-channel kinetic analysis of chimeric alpha7-5HT3A receptors. Mol Pharmacol 68:1475–1483
Bouzat C, Bartos M, Corradi J, Sine SM (2008) The interface between extracellular and transmembrane domains of homomeric Cys-loop receptors governs open-channel lifetime and rate of desensitization. J Neurosci 28:7808–7819
Sine SM, Claudio T, Sigworth FJ (1990) Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts. Single channel current kinetics reveal distinct agonist binding affinities. J Gen Physiol 96:395–437
Mukhtasimova N, DaCosta CJB, Sine SM (2016) Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR. J Gen Physiol 148:43–63
Colquhoun D, Sigworth FL (1983) Fitting and statistical analysis of single channel records. Single channel recording. Springer, US, pp 483–587
Sigworth FJ, Sine SM (1987) Data transformations for improved display and fitting of single-channel dwell time histograms. Biophys J 52:1047–1054
Olsen JA, Kastrup JS, Peters D, Gajhede M, Balle T, Ahring PK (2013) Two distinct allosteric binding sites at α4β2 nicotinic acetylcholine receptors revealed by NS206 and NS9283 give unique insights to binding activity-associated linkage at Cys-loop receptors. J Biol Chem 288:35997–36006
Olsen JA, Ahring PK, Kastrup JS, Gajhede M, Balle T (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
Wang Z-J, Deba F, Mohamed TS, Chiara DC, Ramos K, Hamouda AK (2017) Unraveling amino acid residues critical for allosteric potentiation of (α4)3(β2)2-type nicotinic acetylcholine receptor responses. J Biol Chem 292:9988–10001
Maurer JJ, Sandager-Nielsen K, Schmidt HD (2017) Attenuation of nicotine taking and seeking in rats by the stoichiometry-selective alpha4beta2 nicotinic acetylcholine receptor positive allosteric modulator NS9283. Psychopharmacology 234:475–484
Zhu CZ, Chin CL, Rustay NR, Zhong C, Mikusa J, Chandran P, Salyers A, Gomez E, Simler G, Lewis LG, Gauvin D, Baker S, Pai M, Tovcimak A, Brown J, Komater V, Fox GB, Decker MW, Jacobson PB, Gopalakrishnan M, Lee CH, Honore P (2011) 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. In: Biochemical pharmacology, pp 967–976
DeDominicis KE, Sahibzada N, Olson TT, Xiao Y, Wolfe BB, Kellar KJ, Yasuda RP (2017) The (α4)3 (β2)2 stoichiometry of the nicotinic acetylcholine receptor predominates in the rat motor cortex. Mol Pharmacol 92:327–337
Morales-Perez CL, Noviello CM, Hibbs RE (2016) X-ray structure of the human α4β2 nicotinic receptor. Nature 538:411–415
Walsh RM, Roh SH, Gharpure A, Morales-Perez CL, Teng J, Hibbs RE (2018) Structural principles of distinct assemblies of the human α4β2 nicotinic receptor. Nature 557:261–265
Sigel E, Buhr A (1997) The benzodiazepine binding site of GABA(A) receptors. Trends Pharmacol Sci 18:425–429
Sigel E, Steinmann ME (2012) Structure, function, and modulation of GABAA receptors. J Biol Chem 287:40224–40231
Sigel EP, Luscher B (2011) A closer look at the high affinity benzodiazepine binding site on GABAA receptors. Curr Top Med Chem 11:241–246
Zhu S, Noviello CM, Teng J, Walsh RM, Kim JJ, Hibbs RE (2018) Structure of a human synaptic GABAA receptor. Nature 512:270
Ahring PK, Liao VWY, Balle T (2018) Concatenated nicotinic acetylcholine receptors: a gift or a curse? J Gen Physiol 150:453–473
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
Lucero LM, Weltzin MM, Eaton JB, Cooper JF, Lindstrom JM, Lukas RJ, Whiteaker P (2016) Differential α4(+)/(−)β2 agonist-binding site contributions to α4β2 nicotinic acetylcholine receptor function within and between isoforms. J Biol Chem 291:2444–2459
Eaton JB, Lucero LM, Stratton H, Chang Y, Cooper JF, Lindstrom JM, Lukas RJ, Whiteaker P (2013) The unique α4(+)/(−) α4 agonist binding site in (α4)3(β2)2 subtype nicotinic acetylcholine receptors permits differential agonist desensitization pharmacology versus the (4)2(2)3 subtype. J Pharmacol Exp Ther 348:46–58
Timmermann DB, Sandager-Nielsen K, Dyhring T, Smith M, Jacobsen AM, Nielsen E, Grunnet M, Christensen JK, Peters D, Kohlhaas K, Olsen GM, Ahring PK (2012) Augmentation of cognitive function by NS9283, a stoichiometry-dependent positive allosteric modulator of α2- and α4-containing nicotinic acetylcholine receptors. Br J Pharmacol 167:164–182
Seo S, Henry JT, Lewis AH, Wang N, Levandoski MM (2009) The positive allosteric modulator morantel binds at noncanonical subunit interfaces of neuronal nicotinic acetylcholine receptors. J Neurosci 29:8734–8742
Cesa LC, Higgins CA, Sando SR, Kuo DW, Levandoski MM (2012) Specificity determinants of allosteric modulation in the neuronal nicotinic acetylcholine receptor: a fine line between inhibition and potentiation. Mol Pharmacol 81:239–249
Weltzin MM, Schulte MK (2015) Desformylflustrabromine modulates α4β2 neuronal nicotinic acetylcholine receptor high- and low-sensitivity isoforms at allosteric clefts containing the β2 subunit. J Pharmacol Exp Ther 354:184–194
Dacosta CJB, Sine SM (2013) Stoichiometry for drug potentiation of a pentameric ion channel. Proc Natl Acad Sci USA 110:6595–6600
Young GT, Zwart R, Walker AS, Sher E, Millar NS (2008) Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site. Proc Natl Acad Sci USA 105:14686–14691
Alcaino C, Musgaard M, Minguez T, Mazzaferro S, Faundez M, Iturriaga-Vasquez P, Biggin PC, Bermudez I (2017) Role of the cys loop and transmembrane domain in the allosteric modulation of α4β2 nicotinic acetylcholine receptors. J Biol Chem 292:551–562
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This research was supported by NIH Grant NS31744 to SMS.
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SMS, SM, and IB conceived and coordinated the research; SM performed the research; SM and SMS analyzed the results; SMS and SM wrote the paper. All authors edited and approved the final version.
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Mazzaferro, S., Bermudez, I. & Sine, S.M. Potentiation of a neuronal nicotinic receptor via pseudo-agonist site. Cell. Mol. Life Sci. 76, 1151–1167 (2019). https://doi.org/10.1007/s00018-018-2993-7
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DOI: https://doi.org/10.1007/s00018-018-2993-7