The α4β2 nicotine acetylcholine receptor agonist ispronicline induces c-Fos expression in selective regions of the rat forebrain
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Immunization with a nicotine conjugate vaccine (3′-AmNic-rEPA) significantly reduced nicotine's ability to enter the brain and bind to β2-containing nicotinic acetylcholine receptors (β2*-nAChRs) by sequestering nicotine in the blood. Healthy tobacco smokers experienced a 12.5% decrease in β2*-nAChR occupancy by nicotine after vaccination, which was associated with a 23.6% decrease in the amount of nicotine available to enter the brain, and significant reductions in cigarette use and craving.
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Nicotine withdrawal
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Objective To identify the effects of acute denervation on time-dependent change in mRNA and protein expression of adult-type(e-AChR) and embryonic-type(γ-AChR) nicotinic acetylcholine receptor in rat gastrocnemius muscle.Methods Sprague-Dawley rats were divided into the control group(Group C) and the denervated group(Group D).Rats in group D were sacrificed randomly on the 7th(Group D7),14th(Group D14),21st(Group D21),28th(Group D28) and 35th day(Group D35) after severing the left sciatic nerve.Messenger RNA(mRNA) and protein expression of e-AChR and γ-AChR in gastrocnemius muscle from both hinder limbs was measured by RT-PCR and Western blotting respectively to detect the ratio change in expression of e-AChR and γ-AChR.Results The total AChR expression increased significantly from the 14th day and maintained at a high level.e-AChR expression first decreased within 7 days after denervation and then increased significantly from the 14th day after denervation while γ-AChR increased constantly,and reached a peak percentage on the 7th day.Conclusion Time-dependent expression changes in e-AChR and γ-AChR exists obviously after denervation,which may affect the denervated muscle on its sensitivity to muscle relaxants.
Gastrocnemius muscle
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A study of the relationship between the actions of acetylcholine and nicotine on frog hearts indicates that both the excitatory and inhibitory effects of acetylcholine are antagonised by nicotine. Hearts stopped by acetylcholine and also spontaneously stopped hearts were restarted by nicotine. It has been suggested that exogenously administered acetylcholine and nicotine act through same receptors in a competitive manner.
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Water-soluble models of ligand-gated ion channels would be advantageous for structural studies. We investigated the suitability of three versions of the N-terminal extracellular domain (ECD) of the α7 subunit of the nicotinic acetylcholine receptor (AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression inXenopus oocytes. The third was truncated before the first transmembrane domain and was soluble without detergent. For all three, their equilibrium binding affinities for α-bungarotoxin, nicotine, and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the α7 ECD can form a water-soluble AChR that is a model of the ECD of the full-length α7 AChR. Water-soluble models of ligand-gated ion channels would be advantageous for structural studies. We investigated the suitability of three versions of the N-terminal extracellular domain (ECD) of the α7 subunit of the nicotinic acetylcholine receptor (AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression inXenopus oocytes. The third was truncated before the first transmembrane domain and was soluble without detergent. For all three, their equilibrium binding affinities for α-bungarotoxin, nicotine, and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the α7 ECD can form a water-soluble AChR that is a model of the ECD of the full-length α7 AChR. Nicotinic acetylcholine receptors (AChRs) 1The abbreviations used are: AChR, nicotinic acetylcholine receptor; ACh, acetylcholine; αBgt, α-bungarotoxin; ECD, extracellular domain; EK, enterokinase; ER, endoplasmic reticulum; GPI, glycophosphatidylinositol; M1–4, transmembrane domains 1–4; WS, water-soluble; mAb, monoclonal antibody; PBS, phosphate-buffered saline. 1The abbreviations used are: AChR, nicotinic acetylcholine receptor; ACh, acetylcholine; αBgt, α-bungarotoxin; ECD, extracellular domain; EK, enterokinase; ER, endoplasmic reticulum; GPI, glycophosphatidylinositol; M1–4, transmembrane domains 1–4; WS, water-soluble; mAb, monoclonal antibody; PBS, phosphate-buffered saline. are integral-membrane, pentameric ion channels in the central and peripheral nervous systems that participate in signal transmission associated with the release of acetylcholine (ACh). A considerable collection of studies of their cell biology, electrophysiology, and structure makes them the best characterized family of a superfamily of homologous neurotransmitter-gated channels that includes glycine, γ-aminobutyric acidA, and 5-hydroxytryptamine3 receptors (1Lindstrom J. Narahashi T. Ion Channels. 4. Plenum Press, New York1996: 377-450Google Scholar, 2Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (205) Google Scholar, 3Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (563) Google Scholar). Muscle-type AChRs are composed of four different subunits with the subunit composition (α1)2(β1)δ(γ or ε) and bind the snake venom toxin α-bungarotoxin (αBgt). Neuronal AChRs that do not bind αBgt are formed from combinations of α2, α3, α4, or α6 subunits with β2, β3, β4, and/or α5 subunits. Neuronal AChRs that do bind αBgt are formed from α7, α8, and α9 subunits, perhaps in combination with unknown subunits. When heterologously expressed, α7, α8, and α9 form functional homomeric AChRs that appear to contain five identical subunits. AChRs are composed of five homologous membrane-spanning subunits that are ordered around a central, cation-selective channel. The topology of AChRs that is predicted by hydrophobicity plots has received substantial experimental support (4Chavez R.A. Hall Z.W. J. Cell Biol. 1992; 116: 385-393Crossref PubMed Scopus (55) Google Scholar, 5Anand R. Bason L. Saedi M.S. Gerzanich V. Peng X. Lindstrom J. Biochemistry. 1993; 32: 9975-9984Crossref PubMed Scopus (37) Google Scholar). The approximately 200 residues at the N-terminal half of each AChR subunit are extracellular, areN-glycosylated, contain sites for agonist and antagonist binding, and form the vestibule through which cations reach the transmembrane channel. Relatively little of the remainder of the primary sequence is extracellular. Three of the four transmembrane domains (M1–M3) that form the channel are grouped together following the N-terminal extracellular domain (ECD) and are separated from M4 by a large cytoplasmic loop. For the muscle-type AChR, three distinct regions of the primary sequence around amino acid residues 86–93, 149, and 190–198 of α1 subunits and peptide loops around residues 34, 55–59, 113–119, and 174–180 of the γ or δ subunit contribute to the ACh binding sites at the interfaces between α and δ and between α and γ (or ε) subunits, based on photoaffinity labeling and site-directed mutagenesis (6Galzi J.-L. Changeux J.-P. Curr. Opin. Struct. Biol. 1994; 4: 554-565Crossref Scopus (198) Google Scholar, 7Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Because of primary sequence and topological similarity, homologous residues at subunit-subunit interfaces in other neuronal AChR subunits are expected to have similar roles in the agonist binding site. For example, residues of the α7 subunit homologous to those of α1 and to those around γ55 and δ57 have been shown to contribute to the agonist binding in homomeric α7 AChRs (8Corringer P.-J. Galzi J.-L. Eiselé J.-L. Bertrand S. Changeux J.-P. Bertrand D. J. Biol. Chem. 1995; 270: 11749-11752Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 9Galzi J.-L. Bertrand D. Thiéry-Devillers A. Revah F. Bertrand S. Changeux J.-P. FEBS Lett. 1991; 294: 198-202Crossref PubMed Scopus (137) Google Scholar). Our knowledge of the molecular structure of AChRs, however, is far from complete. Electron diffraction methods using two-dimensional, tubular arrays of AChRs from Torpedo californica have successfully yielded structural details at 9 Å resolution in three dimensions (10Beroukhim R. Unwin N. Neuron. 1995; 15: 323-331Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 11Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (715) Google Scholar, 12Unwin N. Nature. 1995; 373: 37-43Crossref PubMed Scopus (905) Google Scholar) and at 7.5 Å in two-dimensional projection (13Unwin N. J. Mol. Biol. 1996; 257: 586-596Crossref PubMed Scopus (99) Google Scholar). Achieving higher resolution, however, has been elusive with membrane-bound or detergent-solubilized AChRs. No member of this superfamily of integral-membrane receptors has been crystallized, and the intact AChRs are too large (more than 200 kDa) for nuclear magnetic resonance spectroscopy. An AChR formed from the ECD may be a suitable structural model of a full-length AChR, if the ECD can both fold and oligomerize in the absence of the remainder of the subunit. Several lines of evidence suggest that the ECD meets these requirements. First, the specific interactions between subunits that are important in assembly of the muscle-type AChR and for formation of mature acetylcholine binding sites appear to depend primarily on the ECD (14Gu Y. Camacho P. Gardner P. Hall Z.W. Neuron. 1991; 6: 879-887Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 15Verrall S. Hall Z.W. Cell. 1992; 68: 23-31Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 16Yu X.M. Hall Z.W. Nature. 1991; 352: 64-67Crossref PubMed Scopus (70) Google Scholar, 17Kreienkamp H.-J. Maeda R.K. Sine S.M. Taylor P. Neuron. 1995; 14: 1-20Abstract Full Text PDF PubMed Scopus (92) Google Scholar, 18Chavez R.A. Maloof J. Beeson D. Newsom-Davis J. Hall Z.W. J. Biol. Chem. 1992; 267: 23023-23034Abstract Full Text PDF PubMed Google Scholar, 19Sumikawa K. Nishizaki T. Mol. Brain Res. 1994; 25: 257-264Crossref PubMed Scopus (20) Google Scholar, 20Sumikawa K. Mol. Brain Res. 1992; 13: 349-353Crossref PubMed Scopus (27) Google Scholar, 21Hall Z. Trends Cell Biol. 1992; 2: 66-68Abstract Full Text PDF PubMed Scopus (18) Google Scholar). The long cytoplasmic loop of α1 participates in assembly subsequent to the formation of heterodimers (22Yu X.M. Hall Z.W. Neuron. 1994; 13: 247-255Abstract Full Text PDF PubMed Scopus (44) Google Scholar). Second, membrane-tethered ECDs of mouse muscle α1 and δ form heterodimers with ligand binding sites that reflect properties of a full-length AChR (23Wang Z.-Z. Hardy S.F. Hall Z.W. J. Biol. Chem. 1996; 271: 27575-27584Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 24Wang Z.-Z. Hardy S.F. Hall Z.W. J. Cell Biol. 1996; 135: 809-817Crossref PubMed Scopus (15) Google Scholar, 25Wang Z.-Z. Fuhrer C. Shtrom S. Sugiyama J.E. Ferns M.J. Hall Z.W. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 363-371Crossref PubMed Google Scholar). Third, sequences of α7 that affect homomeric assembly also have been localized to the first half of the ECD and an area around M1 based on chimeras of α7 and α3 (26Garcı́a-Guzmán M. Sala F. Sala S. Campos-Caro A. Criado M. Biochemistry. 1994; 33: 15198-15203Crossref PubMed Scopus (54) Google Scholar). According to this report, the long cytoplasmic loop of α7 is not essential for oligomerization. To determine whether AChRs formed from the ECD (residues 1–208) of α7 subunits are water-soluble structural models for the ECD of the full-length α7 AChR, we expressed two constructs of the ECD of the α7 subunit with M1 retained and one construct of the ECD without M1 in Xenopus oocytes. The constructs with M1 were included to explore the feasibility of removing M1 by in vitroprocessing subsequent to in vivo synthesis. We examined ligand binding properties and velocity sedimentation profiles as indicators of global structure and local structure at the agonist binding site of the resulting AChRs, which we have designated as α7 ECD AChRs. We found that each construct, including the water-soluble one without M1, assembles into an AChR with affinities for125I-labeled αBgt (125I-αBgt), nicotine, and ACh that match those of the full-length α7 AChR. These properties demonstrate that the α7 ECD forms a water-soluble α7 ECD AChR that can be a starting point for structural studies of this superfamily of ion channels. The full-length cDNA sequence of chicken α7 (27Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (407) Google Scholar) previously was cloned into a modified SP64T expression vector (28Melton D.A. Krieg P.A. Rebagliati M.R. Maniatis T. Zinn K. Green M.R. Nucleic Acids Res. 1984; 12: 7035-7056Crossref PubMed Scopus (4054) Google Scholar, 29Gerzanich V. Anand R. Lindstrom J. Mol. Pharmacol. 1994; 45: 212-220PubMed Google Scholar). For α7M1, which encodes the ECD of α7 up to the start of M2, the α7 coding sequence from the beginning of M2 to past the native stop codon was removed by digestion withBglII and BsmI. It was replaced by a double-stranded oligonucleotide cassette coding in-frame for the 19-amino acid sequence SQVTGEVIFQTPLIKNPRV and a stop signal. This sequence contained the epitope of mAb 142 from residues 2 to 17 (5Anand R. Bason L. Saedi M.S. Gerzanich V. Peng X. Lindstrom J. Biochemistry. 1993; 32: 9975-9984Crossref PubMed Scopus (37) Google Scholar), followed by a MluI restriction site in the DNA sequence. The last native residue from α7 was Ile240, according to the numbering scheme of the mature chicken α7 AChR subunit (27Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (407) Google Scholar). The mAb 142 epitope was introduced for immunoblotting and for binding the subunit protein to mAb 142-coated plastic wells for solid-phase assays (5Anand R. Bason L. Saedi M.S. Gerzanich V. Peng X. Lindstrom J. Biochemistry. 1993; 32: 9975-9984Crossref PubMed Scopus (37) Google Scholar). The MluI site was included so that the epitope insert could be extended with additional residues after digestion withMluI and BsmI. It has been shown that such extension may be necessary for accessibility of the epitope by the antibody (5Anand R. Bason L. Saedi M.S. Gerzanich V. Peng X. Lindstrom J. Biochemistry. 1993; 32: 9975-9984Crossref PubMed Scopus (37) Google Scholar). The other construct that included M1 was α7EK, which was similar to α7M1 except for the inclusion of a peptide spacer between the end of the ECD and the beginning of M1. To prepare α7EK, a double-stranded oligonucleotide cassette coding for the 38-amino acid residue sequence TMRRRTGTVSISPESDRPDLSTFTSDDDDKILERRRTL was ligated in frame between the proximal BsmAI and distal HgaI sites that are nearly juxtaposed to the 5′ side of M1 in α7M1. Residues 1–6 reconstructed the end of the N-terminal extracellular domain from residues Thr203 to Thr208; the DNA sequence for residues 7–8 introduced a KpnI site; residues 9–23 were the epitope for mAb 236 (5Anand R. Bason L. Saedi M.S. Gerzanich V. Peng X. Lindstrom J. Biochemistry. 1993; 32: 9975-9984Crossref PubMed Scopus (37) Google Scholar); the DNA sequence for residues 24–25 introduced a SpeI site; residues 26–31 (DDDDKI) were the specificity sequence for EK that is modeled after its site of proteolysis on trypsinogen (30LaVallie E.R. Rehemtulla A. Racie L.A. DiBlasio E.A. Ferenz C. Grant K.L. Light A. McCoy J.M. J. Biol. Chem. 1993; 268: 23311-23317Abstract Full Text PDF PubMed Google Scholar, 31Anderson L.E. Walsh K.A. Neurath H. Biochemistry. 1977; 16: 3354-3360Crossref PubMed Scopus (44) Google Scholar); the DNA sequence for residues 32–33 introduced a XhoI site; and residues 34–38 reconstructed the native sequence of Arg205 to Leu209 at the distal end of the insert. Beginning with Tyr210 (native numbering), the remainder of the sequence was identical to α7M1. The KpnI, XhoI, andSpeI restriction sites were included so that the mAb 236 epitope and the target for protease digestion easily could be modified readily. A total of 27 amino acid residues were inserted between the proximal copy of Thr208 and the distal copy of Arg205. The third construct, α7WS, was truncated at the end of the ECD of α7 and did not include M1. To prepare α7WS, the full-length sequence of α7 was cut at the BglII site between M1 and M2. The resulting N-terminal domain sequence was cut at theBsmAI site proximal to the start of the M1 coding sequence. A double-stranded oligonucleotide cassette coding for the 22-amino acid residue sequence TMRRRTQVTGEVIFQTPLIKNP followed by a stop codon was ligated between the BsmAI site of the N-terminal sequence and the EcoRI site of a modified SP64T expression vector. Residues 1–6 of this sequence reconstructed the native sequence Arg203 to Thr208; residues 7–22 constituted the epitope for mAb 142. The oligonucleotides were synthesized using the phosphoramidite method on a MilliGen oligonucleotide DNA synthesizer. DNA from each of the three plasmids was purified on a CsCl gradient. cRNA was synthesized using an SP6 mMessage mMachine™ kit (Ambion, Austin, TX) and linearized DNA. The cytoplasm of each oocyte was injected with approximately 50 ng of cRNA and incubated at 18 °C for 3–5 days in 50% Leibovitz's L-15 medium (Life Technologies, Inc.) in 10 mm HEPES, pH 7.5, containing 10 units/ml penicillin and 10 μg/ml streptomycin. For α7M1 and α7EK, extraction of membrane-bound subunit protein with a buffer containing Triton X-100 was accomplished with a previously reported procedure (29Gerzanich V. Anand R. Lindstrom J. Mol. Pharmacol. 1994; 45: 212-220PubMed Google Scholar). Oocytes were homogenized by hand in ice-cold buffer A (50 mm sodium phosphate, 50 mm NaCl, 5 mm EDTA, 5 mm EGTA, 5 mm benzamidine, 15 mm iodoacetamide, pH 7.5). The membrane-containing fraction was separated by centrifugation, was washed twice with buffer A, and then was extracted with buffer B (40 mm sodium phosphate, 40 mm NaCl, 4 mm EDTA, 4 mm EGTA, 4 mmbenzamidine, 12 mm iodoacetamide, 2% Triton) during gentle agitation for 2 hours at 4 °C. The soluble fraction from this detergent extraction step was separated by centrifugation and was used for both Western blotting and assays of ligand binding. For α7WS, the secreted fraction was defined as the subunit protein present in the L-15 medium incubating injected oocytes. Particulates in this fraction were removed by centrifugation. The cytoplasmic fraction of α7WS was defined as the subunit protein present in the soluble fraction following homogenization of the oocytes by hand in buffer A and centrifugation to sediment the insoluble, membranous component. The Triton-extracted fraction of α7WS was defined as the subunit protein present in the solvent following extraction in buffer B of the membranous component from the homogenization step during gentle agitation for 2 h at 4 °C. Triton-extracted α7M1 and α7EK or secreted α7WS was incubated overnight at 4 °C with mAb 142 that had been coupled to Sepharose CL-4B (Pharmacia) with CNBr (32Wilchek M. Miron T. Kohn J. Methods Enzymol. 1984; 104: 3-55Crossref PubMed Scopus (198) Google Scholar). After this concentration step, the protein was eluted at 55 °C with 2% SDS and 20 mm β-mercaptoethanol. Proteins were deglycosylated for 18 h at 37 °C with 1 unit of a mixture of endoglycosidase F and glycopeptidase F according to instructions of the manufacturer (Boehringer Mannheim). A sample without enzyme was run in parallel as the negative control. Protein was denatured and reduced at 55 °C in SDS-polyacrylamide gel electrophoresis sample buffer containing 2% SDS, separated on a 13% acrylamide SDS-polyacrylamide gel electrophoresis gel, and transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore). After being blocked in 5% powdered milk in phosphate-buffered saline (PBS, 100 mm NaCl, 10 mm sodium phosphate, pH 7.5) containing 0.5% Triton, the membrane was incubated with 2 nm125I-mAb 142. Specific activities of the labeled antibodies ranged from 1017 to 1018cpm/mol. Labeling was visualized by autoradiography. Immulon 4 plastic microwells (Dynatech Laboratories, Chantilly, VA) were coated with mAb 142 or mAb 236 for solid-phase assays (5Anand R. Bason L. Saedi M.S. Gerzanich V. Peng X. Lindstrom J. Biochemistry. 1993; 32: 9975-9984Crossref PubMed Scopus (37) Google Scholar). The wells were blocked with 3% bovine serum albumin in PBS. For α7M1 and α7EK, a volume of the Triton-solubilized protein from the equivalent of from one to three oocytes that had been injected with cRNA was added to each microwell. For the measurement of αBgt affinity, 125I-αBgt was added to the Triton extract and incubated overnight at 4 °C. Total volume in each well was 100 μl. The wells were washed three times with ice-cold PBS containing 0.5% Triton, and the amount of radioactivity was measured using a γ counter. Each data point was measured in duplicate. For the competitive inhibition assays, the wells were washed free of the Triton solution after 24 h and loaded with125I-αBgt at 4 nm in the presence ofl-nicotine or ACh. The wells were incubated 8 h at 4 °C before washing and then measuring the amount of radioactivity. Each data point was measured in duplicate. Nonspecific binding was measured with Triton-solubilized protein extracts from uninjected oocytes and generally was less than 5% of the specific binding. For α7WS, the volume of L-15 medium above about six oocytes was added to mAb 142-coated microwells and incubated overnight at 4 °C for capture of the secreted α7WS. The L-15 was washed away with PBS. For the measurement of αBgt affinity in 0% Triton,125I-αBgt was added to buffer C (same composition as buffer B, except without Triton) and incubated overnight at 4 °C. Total volume in each well was 100 μl. The wells were washed three times with ice-cold PBS, and the amount of radioactivity was measured using a γ counter. Inhibition by nicotine and ACh in 0% Triton was measured after capture of the secreted α7WS, washing of L-15, and loading of each well with 0.6 nm125I-αBgt and the appropriate amount of inhibitor in buffer C. Total volume in each well was 100 μl. The wells were incubated 8 h at 4 °C before washing and measuring the amount of radioactivity. Data points were measured in duplicate. Nonspecific binding was measured with L-15 medium that was used to incubate uninjected oocytes and generally was less than 10% of the specific binding. Buffer B was substituted for buffer C at the step of loading the wells with 125I-αBgt or with 125I-αBgt and nicotine or ACh for the ligand affinity measurements of α7WS in 2% Triton. The equilibrium dissociation constants K d for125I-αBgt were determined by least-squares, nonlinear fitting to a Hill-type equation (Equation 1) using the graphing software KaleidaGraph (Synergy Software) C=C0·11+KdLnEquation 1 where C is the measured signal (counts/min),C 0 is the maximal signal (which corresponds in this case to the maximum number of 125I-αBgt binding sites), L is the concentration of 125I-αBgt,n is the Hill coefficient. The half-maximal inhibition constants, IC50, for nicotine and ACh in the presence of125I-αBgt were determined by nonlinear fitting to Equation 2, where L is the concentration of nicotine or ACh,C 0 is the maximal signal, andC 1 is a constant that represents signal that is not displaced by high concentrations of agonist. We used the Cheng-Prusoff equation (Equation 3) to estimate equilibrium dissociation constants from IC 50 values (33Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12222) Google Scholar), C=C0·11+LIC50n+C1Equation 2 Kd=IC501+[αBgt]Kd,αBgtEquation 3 although other equations also have been described for that purpose (34Lazareno S. Birdsall N.J.M. Br. J. Pharmacol. 1993; 109: 1110-1119Crossref PubMed Scopus (143) Google Scholar). The K d values shown in Table I are the average and standard error of at least three independent assays of ligand affinity unless otherwise noted. Uncertainties shown in figures are standard errors.Table ILigand affinities for full-length α7 and α7 ECD AChRsAChRLigand affinity (K d)αBgtn 1-aHill coefficient n from Equations 1 or 2.NicotinenAChnμmα7 AChR (full-length)1-bData from α7 AChRs expressed in oocytes and solubilized in Triton X-100 (37).0.0016 ± 0.00010.54 ± 0.0225 ± 4.7α7-containing AChRs from chick brain1-bData from α7 AChRs expressed in oocytes and solubilized in Triton X-100 (37).0.0017 ± 0.00011.4 ± 0.2100 ± 10α7M1 AChR0.0016 ± 0.00010.7 ± 0.11.0 ± 0.41-cValues determined from two independent measurements of IC50. All other values were determined from three or more independent measurements.1.2 ± 0.150 ± 201-cValues determined from two independent measurements of IC50. All other values were determined from three or more independent measurements.1.2 ± 0.4α7EK AChR mAb 142 tether for assay0.0020 ± 0.00040.8 ± 0.11.0 ± 0.31-cValues determined from two independent measurements of IC50. All other values were determined from three or more independent measurements.1.7 ± 0.250 ± 201.3 ± 0.2 mAb 236 tether for assay0.0029 ± 0.00060.8 ± 0.11.4 ± 0.71.1 ± 0.260 ± 301.1 ± 0.1α7WS AChR In 0% Triton X-1000.0004 ± 0.00011.3 ± 0.10.09 ± 0.051.6 ± 0.11.3 ± 0.41.6 ± 0.3 In 2% Triton X-1000.0017 ± 0.00011-cValues determined from two independent measurements of IC50. All other values were determined from three or more independent measurements.1.1 ± 0.10.22 ± 0.011.7 ± 0.13.9 ± 0.32.0 ± 0.11-a Hill coefficient n from Equations 1 or 2.1-b Data from α7 AChRs expressed in oocytes and solubilized in Triton X-100 (37Anand R. Peng X. Lindstrom J. FEBS Lett. 1993; 327: 241-246Crossref PubMed Scopus (116) Google Scholar).1-c Values determined from two independent measurements of IC50. All other values were determined from three or more independent measurements. Open table in a new tab Membranes from 10–20 oocytes that had been injected with the α7M1, α7EK, or full-length α7 cRNA were extracted in buffer B. AChRs in membrane vesicles fromT. californica and AChRs from the TE671 cell line (35Conroy W.G. Saedi M.S. Lindstrom J. J. Biol. Chem. 1990; 265: 21642-21651Abstract Full Text PDF PubMed Google Scholar) were solubilized in buffer B. About 200-μl aliquots of solubilized protein containing from 20 to 100 fmol of 125I-αBgt binding sites were layered onto 5-ml sucrose gradients (5–20% (w/v)) in 0.5% Triton solution of 100 mm NaCl, 10 mm sodium phosphate, 5 mm EGTA, 5 mm EDTA, and 1 mm NaN3 at pH 7.5. The gradients were centrifuged 75 min at 70,000 rpm (approximately 340,000 ×g) and 4 °C in a Beckman NVT90 rotor. For determining a ligand binding profile, aliquots of 11 drops each (approximately 130 μl) from the gradient were collected into Immulon 4 plastic microwells coated with mAb 142 for α7M1, mAb 236 for α7EK, or mAb 318 (a rat monoclonal antibody against an epitope in the cytoplasmic domain) (27Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (407) Google Scholar) for the full-length α7. The entire gradient was collected in 40 fractions. After 24 h at 4 °C, the microwells were washed and filled with from 4 to 12 nm125I-αBgt in buffer B for 6 h at 4 °C, followed by washing and quantitation of bound 125I-αBgt by γ counting. Processing of α7WS was slightly more involved because of the low concentration of the secreted protein in the incubation medium. The protein was concentrated by binding overnight at 4 °C to α-toxin that had been isolated from the venom of Naja naja siamensiswith ion exchange chromatography (36Karlsson E. Arnberg H. Eaker D. Eur. J. Biochem. 1971; 21: 1-16Crossref PubMed Scopus (200) Google Scholar) and then coupled to Sepharose CL-4B with CNBr (32Wilchek M. Miron T. Kohn J. Methods Enzymol. 1984; 104: 3-55Crossref PubMed Scopus (198) Google Scholar). The bound protein was eluted from the α-toxin with 200 μl of 100 mm nicotine and was layered onto 5-ml sucrose gradients (5–20%) without Triton in centrifuge tubes. The gradient was centrifuged 75 min at 70,000 rpm (approximately 340,000 × g) and 4 °C, as was done for α7M1 and α7EK. The entire gradient was collected in 39 fractions of 6 drops each, because the drop size was larger in the absence of detergent. After 24 h at 4 °C, the microwells were washed and filled with 1 nm125I-αBgt in buffer C for 6 h at 4 °C, followed by washing and quantitation of bound125I-αBgt by γ counting. The yield was defined as the theoretical maximal amount of bound 125I-αBgt, which was the value of C 0 in Equation 1 from125I-αBgt-binding assays. The AChR yield was calculated in terms of 125I-αBgt binding sites per oocyte. The three variations of the ECD sequence of the α7 subunit were studied (Fig.1). The first construct, α7M1, includes the N-terminal ECD, M1, and the portion of α7 between M1 and M2 up to residue Ile240. It was intended to demonstrate the pharmacological properties and oligomerization behavior of the α7 subunit proximal to M2 and tethered to the membrane through M1. The second construct, designated α7EK, contains a peptide spacer of 27 amino acid residues that is spliced between Thr208 and Leu209 at the junction of the ECD and M1. α7EK also contains M1 and terminates at Ile240, like α7M1. The position of an RRR motif from 205 to 207 at the junction of the ECD with M1 suggests a structural role for these positively-charge residues; therefore, the native sequence Arg205 to Thr208 was repeated at the C-terminal end of the interposed segment before M1. The name “α7EK” is derived from the DDDDKI sequence that was included in the peptide spacer for site-specific proteolysis by EK. α7EK was intended to test the feasibility of removing M1 from the ECD by in vitro enzymatic proteolysis after synthesis. The third variation, α7WS, was intended to be a direct route to a water-soluble α7 ECD AChR. It is truncated at Thr208, just before M1 and contains no transmembrane domain. Epitopes for monoclonal antibodies mAb 142 and mAb 236 were included in the truncated constructs so that the proteins could be detected by immunoblotting and tethered through the antibodies to plastic wells for solid-phase ligand-binding assays (5Anand R. Bason L. Saedi M.S. Gerzanich V. Peng X. Lindstrom J. Biochemistry. 1993; 32: 9975-9984Crossref PubMed Scopus (37) Google Scholar). Immunoblotting of Triton X-100 extracts from oocytes injected with α7M1 and α7EK cRNA confirmed the expression of these proteins (Fig.2). α7M1 before deglycosylation migrated at an apparent mass of 37 kDa; α7EK migrated at 40 kDa. These apparent masses were about 7 kDa higher than the values of 30 kDa for α7M1 and 33.5 kDa for α7EK that were calculated from amino acid compositions. The detection of predominately a single band before deglycosylation suggested that post-translational modifications were comparatively uniform on all molecules. Deglycosylation shifted each band to approximately the molecular mass of the protein calculated without modifications. Without M1, α7WS was secreted in soluble form into the incubation medium by oocytes that had been injected with α7WS cRNA. The secreted protein displayed an apparent mass of about 41 kDa by immunoblotting, compared with a calculated mass of 26 kDa (Fig. 2). The detection of a diffuse pattern of bands before deglycosylation suggested more heterogeneous glycosylation than was present on α7M1 and α7EK. Deglycosylation shifted the apparent mass down to about 26 kDa, confirming that α7WS had the most extensive glycosylation of the three proteins. The larger shift in apparent mass compared with α7M1 and
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