Molecular Pharmacology Fast Forward. Published on April 1, 2008 as DOI: 10.1124/mol.108.046789 Molecular PharTmhisa acrtoiclloe ghays nFoat sbete Fn ocorpwyeadritded. aPnud bfolrimsahtteedd. Tohne fAinpal rviel r1si,o n2 m0a0y8 d iaffse rd froomi: 1th0is. 1v1er2si4on/m. ol.108.046789 MOL 46789 Roles of Accessory Subunits in α4β2* Nicotinic Receptors Alexandre Kuryatov, Jennifer Onksen and Jon Lindstrom Primary Laboratory of Origin: D Department of Neuroscience, University of Pennsylvania Medical School, o w n lo a Philadelphia, PA 19104-6074 d e d fro m m o lp h a rm .a s p e tjo u rn a ls .o rg a t A S P E T J o u rn a ls o n F e b ru a ry 1 0 , 2 0 2 3 1 Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics. Molecular Pharmacology Fast Forward. Published on April 1, 2008 as DOI: 10.1124/mol.108.046789 This article has not been copyedited and formatted. The final version may differ from this version. MOL 46789 Running Title: α4β2α5 and α4β2β3 AChRs Corresponding Author: Jon Lindstrom, Department of Neuroscience, University of Pennsylvania Medical School, 217 Stemmler Hall 36th & Hamilton Walk, Philadelphia, Pennsylvania 19104 Fax: (215) 573-2858. Email: [email protected] D o w n lo a Number of text pages: 45 d e d fro Number of tables: 4 m m o lp Number of figures: 10 h a rm .a s Numbers of references: 39 p e tjo u Words in abstract: 240 rna ls .o rg Words in introduction: 749 a t A S P Words in discussion: 1445 E T J o u rn a ls o n ABBREVIATIONS: AChR, nicotinic acetylcholine receptor; αBgt, α bungarotoxin; F e b ru a DMPP, 1,1-dimethyl-4-phenylpiperazinium iodide; DMEM, Dulbecco’s modified ry 1 0 , 2 0 Eagle’s medium; HEK, human embryonic kidney; MIR, main immunogenic region; 2 3 mAb, monoclonal antibody; PAM, positive allosteric modulator; PBS, phosphate buffered saline; TPBS, 0.5% Triton X-100 in PBS 2 Molecular Pharmacology Fast Forward. Published on April 1, 2008 as DOI: 10.1124/mol.108.046789 This article has not been copyedited and formatted. The final version may differ from this version. MOL 46789 ABSTRACT Accessory subunits in heteromeric nicotinic receptors (AChRs) do not take part in forming ACh binding sites. α5 and β3 subunits can function only as accessory subunits. We show that both α5 and β3 efficiently assemble in human α4β2* AChRs expressed in permanently transfected HEK cell lines. Only (α4β2) α5, not (α4β2) β3 AChRs, have 2 2 been detected in brain. The α4β2α5 line expressed 40% more AChRs than the parent D o α4β2 line, and was equally sensitive to upregulation by nicotine. The α4β2β3 line w n lo a d expressed 25 fold more AChRs than the parental line, and could not be further ed fro m upregulated by nicotine. Relative sensitivity to activation by ACh depends on the m o lp h a accessory subunit, with β2 conferring the greatest sensitivity, α5 less, and β3 and α4 rm .a s p e much less. Accessory subunits form binding sites for positive allosteric modulators, as tjo u rn a illustrated by the observation that α5 conferred high sensitivity to galanthamine. In the ls.o rg a presence of α5 or β3, stable, partially degraded, dead end intermediates accumulated t A S P E T within the cells. These may have of the form α5α4β2α5. The efficiency with which α5 Jo u rn a ls and β3 assemble with α4 and β2, and the necessity of avoiding formation of potentially o n F e b toxic intermediates, may explain why α5 and β3 appear to be transcribed at low levels in ru a ry 1 0 brain. Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) can be caused by , 2 0 2 3 the α4 mutation S247F. This mutant did not produce functional AChRs unless cells were cotransfected with α5, β3, or α6 to replace α4 as accessory subunit. 3 Molecular Pharmacology Fast Forward. Published on April 1, 2008 as DOI: 10.1124/mol.108.046789 This article has not been copyedited and formatted. The final version may differ from this version. MOL 46789 INTRODUCTION Heteromeric neuronal nicotinic acetylcholine receptors (AChRs) contain two ACh binding sites formed at the interfaces of α and β subunits in two αβ subunit pairs and a fifth accessory subunit, all arranged like barrel staves to form a central cation channel (Gotti et al., 2007). α5 and β3 subunits can function only as accessory subunits, forming AChRs with stoichiometries such as (α4β2) α5 or (α4β2) β3, whereas α2−4 2 2 D and β2 or β4 can either form ACh binding sites or assemble in the accessory position to ow n lo a d produce AChRs with (αβ)2α or (αβ)2β stoichiometries (Nelson et al., 2003; Kuryatov et ed fro m al., 2005; Briggs et al., 2006; Drenan et al., 2008). m o lp h a When expressed in permanently transfected human cell lines, most human α4β2 rm .a s p e AChRs are in the (α4β2)2α4 stoichiometry which has low sensitivity to ACh and rapid tjou rn a ls desensitization relative to the (α4β2)2β2 stoichiometry (Nelson et al., 2003). Nicotine .org a binds to partially assembled AChRs, acting as a pharmacological chaperone to selectively t A S P E T increase assembly of the (α4β2)2β2 stoichiometry (Kuryatov et al., 2005; Sallete et al., Jou rn a ls 2005). This stoichiometry has high sensitivity to ACh and slow desensitization. o n F e b α4β2* AChRs are the major brain subtypes with high affinity for nicotine, and ru a ry 1 0 11-37% of these, depending on brain region, are (α4β2)2α5 AChRs (Gerzanich et al., , 2 0 2 3 1998; Gotti et al., 2007; Brown et al., 2007; Mao et al., 2008). Knock out of α5 AChRs in mice reduced activation of high sensitivity brain AChRs without reducing the total number of AChRs (Brown et al., 2007) and caused resistance to nicotine-induced seizures and hypolocomotion (Salas et al., 2003; Kedmi et al., 2004). Human (α4β2) α5 AChRs have the high sensitivity to ACh of (α4β2) β2 AChRs, 2 2 but higher permeability to Ca++ when expressed in Xenopus oocytes using linked α4 and 4 Molecular Pharmacology Fast Forward. Published on April 1, 2008 as DOI: 10.1124/mol.108.046789 This article has not been copyedited and formatted. The final version may differ from this version. MOL 46789 β2 subunits in combination with free α5 subunits to force formation of this stoichiometry (Tapia et al., 2007). In this system, (α4β2) β3 and (α4β2) α4 AChRs have low 2 2 sensitivity to ACh but high permeability to Ca++. α5 subunits in human α3* AChRs expressed in Xenopus oocytes increased the Ca++ permeability and desensitization rates of all α3 AChRs (Gerzanich et al., 1998). α5 increased the sensitivity of α3β2 but not α3β4 AChRs to activation by ACh. When D o human α3* AChRs were expressed in permanently transfected HEK cell lines, w n lo a d e expression in the α3β2α5 line was 2.8 fold greater than the α3β2 line. Both α3β2 and d fro m m α3β2α5 lines were upregulated by nicotine, but α3β4 and α3β4α5 were not (Wang et al., o lp h a 1998). rm .a s p e β3-containing AChRs are located in aminergic neurons in association with α6 tjo u rn a ls subunits, and have been found as (α6β2) β3, (α6β4) β3 and (α4β2)(α6β2)β3 AChRs .o 2 2 rg a t A (Champtiaux et al., 2003; Gotti et al., 2007; Salminen et al., 2007). Because ventral S P E T J tegmental area neurons (which are involved in addiction to nicotine) and substantia nigra o u rn a ls neurons (which are involved in Parkinson’s disease) express α4, β2, β3 and α6 subunits, o n F e b (α4β2)2β3 AChRs should have the opportunity to be formed, but have not been ruary 1 0 immunoisolated from brain (Gotti et al., 2007; Perry et al., 2007; Mao et al., 2008). , 2 0 2 3 Presynaptic (α4β2)(α6β2)β3 AChRs modulate the release of dopamine and neuroprotection by nicotine, are exceptionally sensitive to activation by nicotine, and are thought to be especially important in Parkinson’s disease and its primate models (Salminen et al., 2007; Quik et al., 2007). 5 Molecular Pharmacology Fast Forward. Published on April 1, 2008 as DOI: 10.1124/mol.108.046789 This article has not been copyedited and formatted. The final version may differ from this version. MOL 46789 β3 subunits expressed in permanently transfected HEK cell lines promote assembly of (α6β2) β3 and (α6β4) β3 AChRs with increased sensitivity to upregulation 2 2 by nicotine (Tumkosit et al., 2006). Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) can be caused by the α4 mutation S247F in which a small hydrophilic serine in the M2 sequence lining the channel is replaced by a bulky hydrophobic phenylalanine (Klaassen et al., 2006; Teper D et al., 2007). When this mutant is expressed in Xenopus oocytes at subunit mRNA ratios ow n lo a d resulting primarily in the (α4β2) β2 stoichiometry, functional AChRs are formed which e 2 d fro m lack Ca++ permeability, but co-expression with α5 restores Ca++ permeability (Kuryatov m o lp h a et al., 1997). When expressed in a cell line in which the (α4β2)2α4 stoichiometry rm .a s p e predominates, no function was observed, presumably because the presence of three tjo u rn a phenylalanines blocks the channel, but the mutant AChRs are expressed efficiently and ls.o rg a nicotine increases their assembly, as with wild type AChRs (Kuryatov et al., 2005). t A S P E T Here we report the properties of (α4β2)2α5 and (α4β2)2β3 AChRs expressed in Jo u rn a permanently transfected HEK cell lines, demonstrating effects of accessory subunits on ls o n F e ACh assembly, sensitivity to activation by agonists, and modulation by allosteric bru a ry 1 modulators. We also report that replacing the α4 accessory subunit in the ADNLFE cell 0 , 2 0 2 3 line with other AChR subunits permits ion channel function and alters sensitivity to activation. 6 Molecular Pharmacology Fast Forward. Published on April 1, 2008 as DOI: 10.1124/mol.108.046789 This article has not been copyedited and formatted. The final version may differ from this version. MOL 46789 Materials and Methods cDNAs and chemicals. Human α4 and β2 cDNAs were cloned in this lab as previously described (Wang et al., 1998; Kuryatov et al., 1997). The cDNA for human α5 was provided by Dr. F. Clementi and subcloned in pCEP4 vector (Invitrogen) (Wang et al., 1998). Human β3 was obtained from Christopher Grantham at the Janssen Research Foundation (Belgium) and subcloned into pCEP4/Hygromycin(+) for transfection using D o HindIII and XhoI restriction enzymes. All chemicals were purchased from Sigma-Aldrich w n lo a d e (St. Louis, MO), unless otherwise noted. d fro m Tissue culture and transfection: The HEK tsA201 parental cell line expressing m o lp h a human α4β2 AChRs was described previously (Nelson et al., 2003; Kuryatov et al., rm .a s p e 2005). All cell lines were maintained in Dulbecco Modified Eagle Medium (DMEM, tjo u rn a high glucose) (Life Technologies, Inc.) supplemented with 10% fetal bovine serum ls.o rg a (Hyclone), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Life t A S P E T Technologies, Inc.) at 37º C, 5% CO2 at saturating humidity. Jo u rn a For transient transfection, 100 mm dishes of 25% confluent α4β2 cells were ls o n F e b transfected with 6 µg of β3 or α5 cDNAs using the FuGENE 6 DNA transfection kit ru a ry 1 (Roche Diagnostics, Indianapolis, IN). After 48 hours, cells were collected using ice cold 0, 2 0 2 3 PBS and AChRs were extracted. For permanent transfection, 35mm dishes of 50% confluent α4β2 cells were transfected with β3 or α5 cDNAs using the FuGENE 6 DNA transfection kit (Roche Diagnostics, Indianapolis, IN). Hygromycin (Roche Diagnostics, Indianapolis, IN) was added at a 0.1mg/ml concentration for α5 and β3 selection, 0.5mg/ml of Zeocin (Invitrogen) was added for α4 selection, and 0.6 mg/ml of G418 (Life Technologies, Inc.) 7 Molecular Pharmacology Fast Forward. Published on April 1, 2008 as DOI: 10.1124/mol.108.046789 This article has not been copyedited and formatted. The final version may differ from this version. MOL 46789 was added for β2 selection. The transfected cells were passed onto 10 cm dishes before they were passed and plated on a 96-well plate for serial dilution. The 96-well plate was checked for the growth of single colonies and after the colony occupied about a quarter of the size of the well, it was plated onto a 24-well plate and then passed to three 35mm dishes. Each promising clone was then tested to determine how much AChR was present. Screening for cells and extraction of stable clones continued as described by D Tumkosit et al. (2006). Solid phase assays for β2-containing AChRs were performed with o w n lo a mAb 295 coated wells, and assays for α5 and β3 containing AChRs were performed with de d fro m mAb 210 coated wells. m o lp h Antiserum and mAbs. A rat antiserum to bacterially expressed α4 subunit arm .a s p sequences (excluding the transmembrane domains) was raised as described previously etjo u rn a (Kuryatov et al., 2000). The rat mAb 210 binds to the main immunogenic region of ls .o rg a human α1, α3, α5 (Lindstrom, 2000) and β3 (Tumkosit et al., 2006). The rat mAb 295 t A S P E T binds to the extracellular domain of native β2 subunits with high affinity only when they J o u rn a are in association with α3, α4, or α6 subunits (Lindstrom, 2000). ls o n F e AChR extracts were incubated in mAb coated microtiter wells for solid phase bru a ry 1 RIA, or with mAb-coupled to activated CH-Sepharose (Amersham Biosciences, Uppsala, 0 , 2 0 2 3 Sweden) for purifying AChRs for use in immunoblot assays, or loaded directly onto 5-ml sucrose gradients (5–20% sucrose, w/w) for sedimentation analysis (Kuryatov et al., 2005). For immunoprecipitation of AChRs with subunit-specific antibodies, the extract was incubated overnight with mAb or antiserum in the presence of [3H] epibatidine (2 nM). The AChR-antibody complexes were immunoprecipitated with sheep anti-rat IgG 8 Molecular Pharmacology Fast Forward. Published on April 1, 2008 as DOI: 10.1124/mol.108.046789 This article has not been copyedited and formatted. The final version may differ from this version. MOL 46789 for rat antibodies. [3H] Epibatidine-labeled AChRs in the pellet were quantified using liquid scintillation counting. Nonspecific precipitation was measured using either normal mouse serum or normal rat serum. AChR extraction and determining α5 and β3 incorporation. Cells from which AChRs were to be extracted were collected in ice-cold PBS (100 mM NaCl and 10 mM sodium phosphate, pH 7.4) then centrifuged at 13,000 g for 15 min in Eppendorf tubes D with 1 ml of buffer A (50 mM NaPO4, pH 7.5, 50 mM NaCl, 5 mM EDTA, 5 mM ow n lo a EGTA, 5 mM benzamidine, 15 mM iodoacetamide, and 2 mM phenylmethylsulphonyl d e d fro fluoride). The pellets were resuspended in buffer A plus 2% Triton-X-100 and incubated m m o lp for 1 hour at room temperature to solubilize AChRs. Insoluble material was removed by h a rm .a s centrifugation at 13,000 g for 15 min. Total protein concentration of solubilized AChRs p e tjo u was determined using a BCA protein assay kit (Pierce Chemical, Rockford, IL). rna ls .o rg The most stable α4β2β3 clones and α4β2α5 clones were selectively screened and a t A S P E tested for high expression of β3 and α5 based on liquid phase radioimmune assays with T J o u rn mAb 295 and mAb 210 as described previously (Kuryatov et al., 2005; Tumkosit et al., a ls o n F 2006). AChR-antibody complexes were immunoprecipitated with sheep anti-rat IgG. For e b ru a ry immunoprecipitation of AChRs with subunit-specific antibodies, the extract was 1 0 , 2 0 incubated overnight with mAb or antiserum in the presence of [3H]epibatidine (2 nM) 23 (PerkinElmer Life And Analytical Sciences, Inc, Walthman, MA). [3H]Epibatidine- labeled AChRs in the pellet were quantified using liquid scintillation counting. Nonspecific precipitation was measured using normal rat serum. Sucrose gradients. Aliquots of 150 µl of cell extract in 2% Triton-X-100 in Buffer A were layered onto 11.3 ml of linear 5 to 20% sucrose gradients (w/v) in 0.5% 9 Molecular Pharmacology Fast Forward. Published on April 1, 2008 as DOI: 10.1124/mol.108.046789 This article has not been copyedited and formatted. The final version may differ from this version. MOL 46789 Triton-X-100 solution of PBS, 5mM EDTA, 5mM EGTA and 1mM NaN at pH 7.5. The 3 gradient was centrifuged for 16 h at 40,000 rpm in a Beckman SW41 rotor. An aliquot (1 µl) of 2 mg/ml purified Torpedo californica electric organ AChR was added to the cell extract as an internal sedimentation standard. After centrifugation, 17-drop fractions were collected from the bottom. Immulon 96-well 4HBX plates (Thermo Electron Corporation, Waltman, MA) were coated with mAb 295 to detect β2 subunits, mAb 210 to detect α5 D or β3 subunits, or mAb 299 or mAb 371 to detect α4 subunits. Aliquots (20 µl) from o w n lo a d each gradient fraction were added to appropriate wells to detect epibatidine binding or α- e d fro m bungarotoxin binding. m o lp h Biotinylation. Cells from two 10-cm dishes of α4β2β3 were collected using ice- arm .a s p e cold PBS and then washed in the same buffer. The cell suspension was labeled by EZ- tjo u rn a link Sulfo-NHS-LC biotin (Pierce, Rockford, IL) at 1mg/ml at 0º C for one hour. The ls .o rg a reaction was stopped by washing in PBS + 100 mM glycine. The pellet was solubilized t A S P E T in Triton X-100 as described above. Biotinylated AChRs from the cell surface were J o u rn a immunoisolated from sucrose gradients fractions on microwells coated with streptavidin. ls o n F Binding of [3H] epibatidine. Surface expression in α4β2α5 cells was determined eb ru a ry similarly to Kuryatov et al. (2005). Surface expression of α4β2β3 cells has to be done on 10 , 2 0 2 3 collagen coated 24 well plates (Becton Dickinson Labware, Bedford, MA) due to low adhesion of this cell line. When the α4β2β3 cells reached more than 50% confluence, 0.5 nM [3H] epibatidine was added to wells to label AChRs. Binding to AChRs in the cell surface was inhibited by 1 mM butyrylcholine chloride (Sigma-Aldrich, St. Louis, MO), a membrane impermeable quaternary amine, to determine the internal pool of AChRs. Nonspecific binding was determined by addition of 100 µM nicotine. After incubation for 10