Differential Contribution of Subunit Interfaces to Α9α10 Nicotinic Acetylcholine Receptor Function
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1521-0111/91/3/250–262$25.00 http://dx.doi.org/10.1124/mol.116.107482 MOLECULAR PHARMACOLOGY Mol Pharmacol 91:250–262, March 2017 Copyright ª 2017 by The Author(s) This is an open access article distributed under the CC-BY Attribution 4.0 International license. Differential Contribution of Subunit Interfaces to a9a10 Nicotinic Acetylcholine Receptor Function Juan Carlos Boffi,1 Irina Marcovich, JasKiran K. Gill-Thind, Jeremías Corradi, Toby Collins, María Marcela Lipovsek,2 Marcelo Moglie, Paola V. Plazas, Patricio O. Craig, Neil S. Millar, Cecilia Bouzat, and Ana Belén Elgoyhen Instituto de Investigaciones en Ingeniería, Genética y Biología Molecular, Dr Héctor N Torres (J.C.B., I.M., M.M. L., M.M., P.V.P., A.B.E.), Instituto de Química Biológica (P.O.C.), and Instituto de Investigaciones Bioquímicas de Bahía Blanca (J.C., C.B), Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina; Department of Neuroscience, Physiology Downloaded from and Pharmacology, University College London, United Kingdom (J.K.G.-T., T.C., N.S.M.); Departamento de Química Biológica Facultad de Ciencias Exactas y Naturales (P.O.C.), and Instituto de Farmacología, Facultad de Medicina (P.V.P., A.B.E.), Universidad de Buenos Aires, Buenos Aires, Argentina; and Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca, Argentina (J.C., C.B). Received November 16, 2016; accepted January 4, 2017 molpharm.aspetjournals.org ABSTRACT Nicotinic acetylcholine receptors can be assembled from either Here, we have made use of site-directed mutagenesis to homomeric or heteromeric pentameric subunit combinations. At examine the contribution of subunit interface domains to the interface of the extracellular domains of adjacent subunits a9a10 receptors by a combination of electrophysiological and lies the acetylcholine binding site, composed of a principal radioligand binding studies. Characterization of receptors con- component provided by one subunit and a complementary taining Y190T mutations revealed unexpectedly that both a9 and component of the adjacent subunit. Compared with neuronal a10 subunits equally contribute to the principal components of nicotinic acetylcholine cholinergic receptors (nAChRs) assem- the a9a10 nAChR. In addition, we have shown that the in- bled from a and b subunits, the a9a10 receptor is an atypical troduction of a W55T mutation impairs receptor binding and at ASPET Journals on September 29, 2021 member of the family. It is a heteromeric receptor composed function in the rat a9 subunit but not in the a10 subunit, only of a subunits. Whereas mammalian a9 subunits can form indicating that the contribution of a9 and a10 subunits to functional homomeric a9 receptors, a10 subunits do not complementary components of the ligand-binding site is non- generate functional channels when expressed heterologously. equivalent. We conclude that this asymmetry, which is sup- Hence, it has been proposed that a10 might serve as a structural ported by molecular docking studies, results from adaptive subunit, much like a b subunit of heteromeric nAChRs, providing amino acid changes acquired only during the evolution of only complementary components to the agonist binding site. mammalian a10 subunits. Introduction each of which has a large extracellular N-terminal region, four transmembrane helices (M1–M4), and an intracellular domain Nicotinic acetylcholine (ACh) receptors (nAChRs) are mem- (Thompson et al., 2010). At the interface of the extracellular bers of the pentameric ligand-gated ion channel family (Nemecz domains of adjacent subunits lies the ACh binding site, formed – – et al., 2016). Seventeen nAChR subunits (a1 a10, b1 b4, d, g, by six noncontiguous regions (loops A–F). Each binding site is and «) have been identified in vertebrates (Nemecz et al., 2016), composed of a principal component or (1)faceprovidedbyone subunit, which contributes three loops of highly conserved This work was supported by Agencia Nacional de Promoción Científica y residues (loops A–C), and a complementary component (2)of Tecnológica, Argentina; Consejo Nacional de Investigaciones Científicas y the adjacent subunit, which contributes three loops (D–F) that Técnicas, Argentina; Universidad Nacional del Sur, Argentina; and the National Institutes of Health National Institute on Deafness and Other have lower levels of sequence conservation between subunits Communication Disorders [Grant RO1DC001508]. (Brejc et al., 2001; Unwin, 2005; Dellisanti et al., 2007). T.C. was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) doctoral training account Ph.D. studentship [BB/D526961/1]. Consequently, the components of the extracellular intersubunit J.K.G.-T. was supported by a BBSRC Collaborative Award in Science and binding sites are nonequivalent and their loops contribute Engineering Ph.D. studentship [BB/F017146/1] with additional financial differently to receptor function (Karlin, 2002). support from Eli Lilly & Co., Ltd. 1Current affiliation: Department of Functional Neuroanatomy, Institute for nAChRs can be assembled from either homomeric or Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany. heteromeric subunit combinations (Millar and Gotti, 2009). 2Current affiliation: Centre for Developmental Neurobiology, King’s College, London, United Kingdom. Homomeric receptors, such as a7, have five equivalent ACh dx.doi.org/10.1124/mol.116.107482. binding sites, each formed by the same principal and ABBREVIATIONS: 5-HT3A, serotonin type 3A; a-BTX, a-bungarotoxin; ACh, acetylcholine; ANOVA, analysis of variance; BBE, best binding energy; CC/SS, double cysteine to serine; nAChR, nicotinic acetylcholine receptor. 250 a9a10 nAChR Subunit Interface 251 complementary components. ACh occupancy of one site is Materials and Methods enough for activation of the homomeric human a7 nAChR, Expression of Recombinant Receptors in Xenopus laevis and also of a chimeric receptor containing the extracellular oocytes. cDNAs encoding Gallus gallus (chick) or Rattus norvegicus domain of a7 and the transmembrane domain of the seroto- (rat) a9 and a10 nAChR subunits were subcloned into a modified nin type 3A (5-HT3A) receptor subunit (Rayes et al., 2009; pGEMHE vector for expression studies in Xenopus laevis oocytes. Andersen et al., 2013). However, whereas occupancy of three Capped cRNAs were in vitro transcribed from linearized plasmid DNA nonconsecutive binding sites is required for maximal open- templates using RiboMAX Large Scale RNA Production System channel lifetime of the chimeric receptor, only one functional (Promega, Madison, WI). Mutant subunits were produced using Quick agonist binding site is required for maximal open-channel Change XL II kit (Stratagene, La Jolla, CA). Amino acid sequences lifetime in a7. of rat and chicken a9, a10, and Torpedo a1 subunits were aligned In contrast to homomeric nAChRs, heteromeric receptors can using ClustalW (EMBL-EBI, Wellcome Genome Campus, Hinxton, have nonequivalent ACh binding sites provided by different Cambridgeshire). Residues were numbered according to the corre- sponding Torpedo a1 subunit mature protein (Karlin, 2002). subunit interfaces. For example, the Torpedo nAChR has two 1 2 The maintenance of Xenopus laevis and the preparation and cRNA structurally different binding sites provided by the a( )-d( ) injection of stage V and VI oocytes have been described in detail 1 2 and a( )-g( ) subunit interfaces (Martinez et al., 2000), which elsewhere (Verbitsky et al., 2000). Typically, oocytes were injected bind agonists with different affinities (Blount and Merlie, 1989; with 50 nl of RNase-free water containing 0.01–1.0 ng of cRNA (at a 1:1 Downloaded from Prince and Sine, 1999). According to the known stoichiometries molar ratio when pairwise combined) and maintained in Barth’s of some neuronal nAChRs (Millar and Gotti, 2009), and by solution at 18°C. Electrophysiological recordings were performed 2– analogy to the muscle-type receptor, it was originally thought 6 days after cRNA injection under two-electrode voltage clamp with an that heteromeric nAChRs have two agonist binding sites (Sine, Oocyte Clamp OC-725B or C amplifier (Warner Instruments Corp., 2002). In the case of neuronal nAChRs such as a4b2, a subunits Hamden, CT). Recordings were filtered at a corner frequency of 10 Hz were thought to only provide the (1) site to the binding using a 900BT Tunable Active Filter (Frequency Devices Inc., Ottawa, interface, whereas b subunits were thought to provide the (2) IL). Data acquisition was performed using a Patch Panel PP-50 LAB/1 molpharm.aspetjournals.org interface (Warner Instruments Corp.) at a rate of 10 points per second. site (Arias, 1997; Luetje and Patrick, 1991). However, it was Both voltage and current electrodes were filled with 3 M KCl and had subsequently shown that the composition of binding site resistances of ∼1MV. Data were analyzed using Clampfit from the interfaces is more complex. For example, the a4b2 receptor pClamp 6.1 software (Molecular Devices, Sunnyvale, CA). During has two alternative stoichiometries, (a4)2(b2)3 and (a4)3(b2)2, electrophysiological recordings, oocytes were continuously superfused leading to different binding site configurations and resulting in (∼15 ml/min) with normal frog saline composed of: 115 mM NaCl, different functional and pharmacological properties (Carbone 2.5 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES buffer, pH 7.2. ACh et al., 2009; Harpsøe et al., 2011; Mazzaferro et al., 2011);