Boffi, J.C.; Marcovich, I.; Gill-Thind, J.K.; Corradi, J.; Collins, T.; Lipovsek, M.M.; Moglie, M.; Plazas, P.V.; Craig, P.O.; Millar, N.S.; Bouzat, C.; Elgoyhen, A.B. "Differential contribution of subunit interfaces to α9α10 nicotinic acetylcholine receptor function" (2017) Molecular Pharmacology. 91(3):250-262
Estamos trabajando para incorporar este artículo al repositorio
Consulte el artículo en la página del editor
Consulte la política de Acceso Abierto del editor


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 non-equivalent. 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. Copyright © 2017 by The Author(s).


Documento: Artículo
Título:Differential contribution of subunit interfaces to α9α10 nicotinic acetylcholine receptor function
Autor:Boffi, J.C.; Marcovich, I.; Gill-Thind, J.K.; Corradi, J.; Collins, T.; Lipovsek, M.M.; Moglie, M.; Plazas, P.V.; Craig, P.O.; Millar, N.S.; Bouzat, C.; Elgoyhen, A.B.
Filiación:Instituto de Investigaciones en Ingeniería, Genética y Biología Molecular, Dr Héctor N Torres, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
Instituto de Química Biológica, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
Instituto de Investigaciones Bioquímicas de Bahía Blanca, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
Department of Neuroscience, Physiology and Pharmacology, University College London, United Kingdom
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
Instituto de Farmacología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Argentina
Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany
Centre for Developmental Neurobiology, King's College, London, United Kingdom
Palabras clave:nicotinic receptor; receptor subunit; acetylcholine; nicotinic receptor; protein subunit; animal cell; Article; binding site; controlled study; molecular docking; molecular model; nonhuman; priority journal; protein domain; rat; receptor binding; site directed mutagenesis; amino acid sequence; animal; chemistry; chicken; genetics; metabolism; mutation; protein secondary structure; protein subunit; structural homology; structure activity relation; Acetylcholine; Amino Acid Sequence; Animals; Binding Sites; Chickens; Molecular Docking Simulation; Mutation; Protein Structure, Secondary; Protein Subunits; Rats; Receptors, Nicotinic; Structural Homology, Protein; Structure-Activity Relationship
Página de inicio:250
Página de fin:262
Título revista:Molecular Pharmacology
Título revista abreviado:Mol. Pharmacol.
CAS:acetylcholine, 51-84-3, 60-31-1, 66-23-9; Acetylcholine; Protein Subunits; Receptors, Nicotinic


  • Akk, G., Contributions of the non-α subunit residues (loop D) to agonist binding and channel gating in the muscle nicotinic acetylcholine receptor (2002) J Physiol, 544, pp. 695-705
  • Andersen, N., Corradi, J., Sine, S.M., Bouzat, C., Stoichiometry for activation of neuronal α7 nicotinic receptors (2013) Proc Natl Acad Sci USA, 110, pp. 20819-20824
  • Arias, H.R., Topology of ligand binding sites on the nicotinic acetylcholine receptor (1997) Brain Res Brain Res Rev, 25, pp. 133-191
  • Arnold, K., Bordoli, L., Kopp, J., Schwede, T., The SWISS-MODEL workspace: A web-based environment for protein structure homology modelling (2006) Bioinformatics, 22, pp. 195-201
  • Azam, L., McIntosh, J.M., Molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to α-conotoxin RgIA (2012) J Neurochem, 122, pp. 1137-1144
  • Azam, L., Papakyriakou, A., Zouridakis, M., Giastas, P., Tzartos, S.J., McIntosh, J.M., Molecular interaction of α-conotoxin RgIA with the rat α9α10 nicotinic acetylcholine receptor (2015) Mol Pharmacol, 87, pp. 855-864
  • Baker, E.R., Zwart, R., Sher, E., Millar, N.S., Pharmacological properties of α9α10 nicotinic acetylcholine receptors revealed by heterologous expression of subunit chimeras (2004) Mol Pharmacol, 65, pp. 453-460
  • Blount, P., Merlie, J.P., Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor (1989) Neuron, 3, pp. 349-357
  • Bordoli, L., Kiefer, F., Arnold, K., Benkert, P., Battey, J., Schwede, T., Protein structure homology modeling using SWISS-MODEL workspace (2009) Nat Protoc, 4, pp. 1-13
  • Brejc, K., Van Dijk, W.J., Klaassen, R.V., Schuurmans, M., Van Der Oost, J., Smit, A.B., Sixma, T.K., Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors (2001) Nature, 411, pp. 269-276
  • Carbone, A.L., Moroni, M., Groot-Kormelink, P.J., Bermudez, I., Pentameric concatenated (α4)2(β2)3 and (α4)3(β2)2 nicotinic acetylcholine receptors: Subunit arrangement determines functional expression (2009) Br J Pharmacol, 156, pp. 970-981
  • Celie, P.H., Van Rossum-Fikkert, S.E., Van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures (2004) Neuron, 41, pp. 907-914
  • Chen, J., Zhang, Y., Akk, G., Sine, S., Auerbach, A., Activation kinetics of recombinant mouse nicotinic acetylcholine receptors: Mutations of alpha-subunit tyrosine 190 affect both binding and gating (1995) Biophys J, 69, pp. 849-859
  • Corradi, J., Spitzmaul, G., De Rosa, M.J., Costabel, M., Bouzat, C., Role of pairwise interactions between M1 and M2 domains of the nicotinic receptor in channel gating (2007) Biophys J, 92, pp. 76-86
  • Dellisanti, C.D., Yao, Y., Stroud, J.C., Wang, Z.Z., Chen, L., Crystal structure of the extracellular domain of nAChR α1 bound to α-bungarotoxin at 1.94 Å resolution (2007) Nat Neurosci, 10, pp. 953-962
  • Dougherty, D.A., Cation-π interactions involving aromatic amino acids (2007) J Nutr, 137, pp. 1504S-1508S. , discussion 1516S-1517S
  • Elgoyhen, A.B., Franchini, L.F., Prestin and the cholinergic receptor of hair cells: Positively-selected proteins in mammals (2011) Hear Res, 273, pp. 100-108
  • Elgoyhen, A.B., Johnson, D.S., Boulter, J., Vetter, D.E., Heinemann, S., α9: An acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells (1994) Cell, 79, pp. 705-715
  • Elgoyhen, A.B., Katz, E., The efferent medial olivocochlear-hair cell synapse (2012) J Physiol Paris, 106, pp. 47-56
  • Elgoyhen, A.B., Vetter, D.E., Katz, E., Rothlin, C.V., Heinemann, S.F., Boulter, J., α10: A determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells (2001) Proc Natl Acad Sci USA, 98, pp. 3501-3506
  • Ellison, M., Haberlandt, C., Gomez-Casati, M.E., Watkins, M., Elgoyhen, A.B., McIntosh, J.M., Olivera, B.M., α-RgIA: A novel conotoxin that specifically and potently blocks the α9α10 nAChR (2006) Biochemistry, 45, pp. 1511-1517
  • Franchini, L.F., Elgoyhen, A.B., Adaptive evolution in mammalian proteins involved in cochlear outer hair cell electromotility (2006) Mol Phylogenet Evol, 41, pp. 622-635
  • Gao, F., Bren, N., Burghardt, T.P., Hansen, S., Henchman, R.H., Taylor, P., McCammon, J.A., Sine, S.M., Agonist-mediated conformational changes in acetylcholine-binding protein revealed by simulation and intrinsic tryptophan fluorescence (2005) J Biol Chem, 280, pp. 8443-8451
  • Gao, F., Mer, G., Tonelli, M., Hansen, S.B., Burghardt, T.P., Taylor, P., Sine, S.M., Solution NMR of acetylcholine binding protein reveals agonist-mediated conformational change of the C-loop (2006) Mol Pharmacol, 70, pp. 1230-1235
  • Gleitsman, K.R., Shanata, J.A., Frazier, S.J., Lester, H.A., Dougherty, D.A., Long-range coupling in an allosteric receptor revealed by mutant cycle analysis (2009) Biophys J, 96, pp. 3168-3178
  • Guex, N., Peitsch, M.C., SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling (1997) Electrophoresis, 18, pp. 2714-2723
  • Hansen, S.B., Taylor, P., Galanthamine and non-competitive inhibitor binding to ACh-binding protein: Evidence for a binding site on non-α-subunit interfaces of heteromeric neuronal nicotinic receptors (2007) J Mol Biol, 369, pp. 895-901
  • Harkness, P.C., Millar, N.S., Changes in conformation and subcellular distribution of α4β2 nicotinic acetylcholine receptors revealed by chronic nicotine treatment and expression of subunit chimeras (2002) J Neurosci, 22, pp. 10172-10181
  • Harpsøe, K., Ahring, P.K., Christensen, J.K., Jensen, M.L., Peters, D., Balle, T., Unraveling the high- and low-sensitivity agonist responses of nicotinic acetylcholine receptors (2011) J Neurosci, 31, pp. 10759-10766
  • Hernando, G., Bergé, I., Rayes, D., Bouzat, C., Contribution of subunits to Caenorhabditis elegans levamisole-sensitive nicotinic receptor function (2012) Mol Pharmacol, 82, pp. 550-560
  • Hsiao, B., Mihalak, K.B., Magleby, K.L., Luetje, C.W., Zinc potentiates neuronal nicotinic receptors by increasing burst duration (2008) J Neurophysiol, 99, pp. 999-1007
  • Huang, S., Li, S.X., Bren, N., Cheng, K., Gomoto, R., Chen, L., Sine, S.M., Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera (2013) Biochem J, 454, pp. 303-310
  • Humphrey, W., Dalke, A., Schulten, K., VMD: Visual molecular dynamics (1996) J Mol Graph, 14, pp. 33-38
  • Indurthi, D.C., Pera, E., Kim, H.L., Chu, C., McLeod, M.D., McIntosh, J.M., Absalom, N.L., Chebib, M., Presence of multiple binding sites on α9α10 nAChR receptors alludes to stoichiometric-dependent action of the α-conotoxin, Vc1.1 (2014) Biochem Pharmacol, 89, pp. 131-140
  • Karlin, A., Emerging structure of the nicotinic acetylcholine receptors (2002) Nat Rev Neurosci, 3, pp. 102-114
  • Katz, E., Verbitsky, M., Rothlin, C.V., Vetter, D.E., Heinemann, S.F., Elgoyhen, A.B., High calcium permeability and calcium block of the α9 nicotinic acetylcholine receptor (2000) Hear Res, 141, pp. 117-128
  • Lansdell, S.J., Millar, N.S., The influence of nicotinic receptor subunit composition upon agonist, α-bungarotoxin and insecticide (imidacloprid) binding affinity (2000) Neuropharmacology, 39, pp. 671-679
  • Lester, H.A., Dibas, M.I., Dahan, D.S., Leite, J.F., Dougherty, D.A., Cys-loop receptors: New twists and turns (2004) Trends Neurosci, 27, pp. 329-336
  • Lipovsek, M., Fierro, A., Pérez, E.G., Boffi, J.C., Millar, N.S., Fuchs, P.A., Katz, E., Elgoyhen, A.B., Tracking the molecular evolution of calcium permeability in a nicotinic acetylcholine receptor (2014) Mol Biol Evol, 31, pp. 3250-3265
  • Lipovsek, M., Im, G.J., Franchini, L.F., Pisciottano, F., Katz, E., Fuchs, P.A., Elgoyhen, A.B., Phylogenetic differences in calcium permeability of the auditory hair cell cholinergic nicotinic receptor (2012) Proc Natl Acad Sci USA, 109, pp. 4308-4313
  • Luetje, C.W., Patrick, J., Both alpha- and beta-subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors (1991) J Neurosci, 11, pp. 837-845
  • Martin, M., Czajkowski, C., Karlin, A., The contributions of aspartyl residues in the acetylcholine receptor γ and δ subunits to the binding of agonists and competitive antagonists (1996) J Biol Chem, 271, pp. 13497-13503
  • Martinez, K.L., Corringer, P.J., Edelstein, S.J., Changeux, J.P., Mérola, F., Structural differences in the two agonist binding sites of the Torpedo nicotinic acetylcholine receptor revealed by time-resolved fluorescence spectroscopy (2000) Biochemistry, 39, pp. 6979-6990
  • Mazzaferro, S., Benallegue, N., Carbone, A., Gasparri, F., Vijayan, R., Biggin, P.C., Moroni, M., Bermudez, I., Additional acetylcholine (ACh) binding site at α4/α4 interface of (α4β2)2α4 nicotinic receptor influences agonist sensitivity (2011) J Biol Chem, 286, pp. 31043-31054
  • Millar, N.S., Gotti, C., Diversity of vertebrate nicotinic acetylcholine receptors (2009) Neuropharmacology, 56, pp. 237-246
  • Morales-Perez, C.L., Noviello, C.M., Hibbs, R.E., X-ray structure of the human α4β2 nicotinic receptor (2016) Nature, 538, pp. 411-415
  • Morris, G.M., Huey, R., Lindstrom, W., Sanner, M.F., Belew, R.K., Goodsell, D.S., Olson, A.J., AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility (2009) J Comput Chem, 30, pp. 2785-2791
  • Mukhtasimova, N., Free, C., Sine, S.M., Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor (2005) J Gen Physiol, 126, pp. 23-39
  • Nemecz, Á., Prevost, M.S., Menny, A., Corringer, P.J., Emerging molecular mechanisms of signal transduction in pentameric ligand-gated ion channels (2016) Neuron, 90, pp. 452-470
  • Olsen, J.A., Balle, T., Gajhede, M., Ahring, P.K., Kastrup, J.S., Molecular recognition of the neurotransmitter acetylcholine by an acetylcholine binding protein reveals determinants of binding to nicotinic acetylcholine receptors (2014) PLoS One, 9
  • Pérez, E.G., Cassels, B.K., Zapata-Torres, G., Molecular modeling of the α9α10 nicotinic acetylcholine receptor subtype (2009) Bioorg Med Chem Lett, 19, pp. 251-254
  • Plazas, P.V., Katz, E., Gomez-Casati, M.E., Bouzat, C., Elgoyhen, A.B., Stoichiometry of the α9α10 nicotinic cholinergic receptor (2005) J Neurosci, 25, pp. 10905-10912
  • Prince, R.J., Sine, S.M., Acetylcholine and epibatidine binding to muscle acetylcholine receptors distinguish between concerted and uncoupled models (1999) J Biol Chem, 274, pp. 19623-19629
  • Rayes, D., De Rosa, M.J., Sine, S.M., Bouzat, C., Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors (2009) J Neurosci, 29, pp. 6022-6032
  • Rothlin, C.V., Katz, E., Verbitsky, M., Elgoyhen, A.B., The α9 nicotinic acetylcholine receptor shares pharmacological properties with type A γ-aminobutyric acid, glycine, and type 3 serotonin receptors (1999) Mol Pharmacol, 55, pp. 248-254
  • Russell, R.B., Barton, G.J., Multiple protein sequence alignment from tertiary structure comparison: Assignment of global and residue confidence levels (1992) Proteins, 14, pp. 309-323
  • Schreiber, G., Fersht, A.R., Energetics of protein-protein interactions: Analysis of the barnase-barstar interface by single mutations and double mutant cycles (1995) J Mol Biol, 248, pp. 478-486
  • Schwede, T., Kopp, J., Guex, N., Peitsch, M.C., SWISS-MODEL: An automated protein homology-modeling server (2003) Nucleic Acids Res, 31, pp. 3381-3385
  • Sgard, F., Charpantier, E., Bertrand, S., Walker, N., Caput, D., Graham, D., Bertrand, D., Besnard, F., A novel human nicotinic receptor subunit, α10, that confers functionality to the α9-subunit (2002) Mol Pharmacol, 61, pp. 150-159
  • Sine, S.M., The nicotinic receptor ligand binding domain (2002) J Neurobiol, 53, pp. 431-446
  • Sine, S.M., Claudio, T., γ- and δ-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor (1991) J Biol Chem, 266, pp. 19369-19377
  • Sine, S.M., Engel, A.G., Recent advances in Cys-loop receptor structure and function (2006) Nature, 440, pp. 448-455
  • Sine, S.M., Huang, S., Li, S.X., DaCosta, C.J., Chen, L., Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors (2013) Biochem J, 454, pp. 311-321
  • Thompson, A.J., Lester, H.A., Lummis, S.C., The structural basis of function in Cys-loop receptors (2010) Q Rev Biophys, 43, pp. 449-499
  • Tomaselli, G.F., McLaughlin, J.T., Jurman, M.E., Hawrot, E., Yellen, G., Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor (1991) Biophys J, 60, pp. 721-727
  • Unwin, N., Refined structure of the nicotinic acetylcholine receptor at 4A resolution (2005) J Mol Biol, 346, pp. 967-989
  • Verbitsky, M., Rothlin, C.V., Katz, E., Elgoyhen, A.B., Mixed nicotinic-muscarinic properties of the α9 nicotinic cholinergic receptor (2000) Neuropharmacology, 39, pp. 2515-2524
  • Weisstaub, N., Vetter, D.E., Elgoyhen, A.B., Katz, E., The α9α10 nicotinic acetylcholine receptor is permeable to and is modulated by divalent cations (2002) Hear Res, 167, pp. 122-135
  • Xie, Y., Cohen, J.B., Contributions of Torpedo nicotinic acetylcholine receptor γTrp-55 and δTrp-57 to agonist and competitive antagonist function (2001) J Biol Chem, 276, pp. 2417-2426
  • Yu, R., Kompella, S.N., Adams, D.J., Craik, D.J., Kaas, Q., Determination of the α-conotoxin Vc1.1 binding site on the α9α10 nicotinic acetylcholine receptor (2013) J Med Chem, 56, pp. 3557-3567
  • Zouridakis, M., Giastas, P., Zarkadas, E., Chroni-Tzartou, D., Bregestovski, P., Tzartos, S.J., Crystal structures of free and antagonist-bound states of human α9 nicotinic receptor extracellular domain (2014) Nat Struct Mol Biol, 21, pp. 976-980


---------- APA ----------
Boffi, J.C., Marcovich, I., Gill-Thind, J.K., Corradi, J., Collins, T., Lipovsek, M.M., Moglie, M.,..., Elgoyhen, A.B. (2017) . Differential contribution of subunit interfaces to α9α10 nicotinic acetylcholine receptor function. Molecular Pharmacology, 91(3), 250-262.
---------- CHICAGO ----------
Boffi, J.C., Marcovich, I., Gill-Thind, J.K., Corradi, J., Collins, T., Lipovsek, M.M., et al. "Differential contribution of subunit interfaces to α9α10 nicotinic acetylcholine receptor function" . Molecular Pharmacology 91, no. 3 (2017) : 250-262.
---------- MLA ----------
Boffi, J.C., Marcovich, I., Gill-Thind, J.K., Corradi, J., Collins, T., Lipovsek, M.M., et al. "Differential contribution of subunit interfaces to α9α10 nicotinic acetylcholine receptor function" . Molecular Pharmacology, vol. 91, no. 3, 2017, pp. 250-262.
---------- VANCOUVER ----------
Boffi, J.C., Marcovich, I., Gill-Thind, J.K., Corradi, J., Collins, T., Lipovsek, M.M., et al. Differential contribution of subunit interfaces to α9α10 nicotinic acetylcholine receptor function. Mol. Pharmacol. 2017;91(3):250-262.