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• W2005388346 abstract "The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that modulates neurotransmitter release in the central nervous system. We show here that functional, homo-oligomeric α7 nAChRs can be synthesized in vitro with a rabbit reticulocyte lysate translation system supplemented with endoplasmic reticulum microsomes, reconstituted into planar lipid bilayers, and evaluated using single-channel recording techniques. Because wild-type α7 nAChRs desensitize rapidly, we used a nondesensitizing form of the α7 receptor with mutations in the second transmembrane domain (S2′T and L9′T) to record channel activity in the continuous presence of agonist. Endoglycosidase H treatment of microsomes containing nascent α7 S2′T/L9′T nAChRs indicated that the receptors were glycosylated. A proteinase K protection assay revealed a 36-kDa fragment in the ER lumen, consistent with a large extracellular domain predicted by most topological models, indicating that the protein was folded integrally through the ER membrane. α7 S2′T/L9′T receptors reconstituted into planar lipid bilayers had a unitary conductance of ∼50 pS, were highly selective for monovalent cations over Cl−, were nonselective between K+ and Na+, and were blocked by α-bungarotoxin. This is the first demonstration that a functional ligand-gated ion channel can be synthesized using an in vitro expression system. The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that modulates neurotransmitter release in the central nervous system. We show here that functional, homo-oligomeric α7 nAChRs can be synthesized in vitro with a rabbit reticulocyte lysate translation system supplemented with endoplasmic reticulum microsomes, reconstituted into planar lipid bilayers, and evaluated using single-channel recording techniques. Because wild-type α7 nAChRs desensitize rapidly, we used a nondesensitizing form of the α7 receptor with mutations in the second transmembrane domain (S2′T and L9′T) to record channel activity in the continuous presence of agonist. Endoglycosidase H treatment of microsomes containing nascent α7 S2′T/L9′T nAChRs indicated that the receptors were glycosylated. A proteinase K protection assay revealed a 36-kDa fragment in the ER lumen, consistent with a large extracellular domain predicted by most topological models, indicating that the protein was folded integrally through the ER membrane. α7 S2′T/L9′T receptors reconstituted into planar lipid bilayers had a unitary conductance of ∼50 pS, were highly selective for monovalent cations over Cl−, were nonselective between K+ and Na+, and were blocked by α-bungarotoxin. This is the first demonstration that a functional ligand-gated ion channel can be synthesized using an in vitro expression system. nicotinic acetylcholine receptor α-bungarotoxin acetylcholine endoglycosidase H endoplasmic reticulum polyacrylamide gel electrophoresis 3-(N-morpholino)propanesulfonic acid picosiemens The nicotinic acetylcholine receptor (nAChR)1 is a member of a superfamily of ligand-gated ion channels that also includes GABAA receptors, serotonin (5-HT3) receptors, glycine receptors, and an invertebrate glutamate-gated chloride channel (1Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (564) Google Scholar). Nicotinic receptors are located at the neuromuscular junction and in the central and peripheral nervous systems. Muscle-type nAChRs are pentamers of homologous subunits in the stoichiometry of α2βγδ (or α2βδε) arranged around a central pore (2Unwin N. J. Struct. Biol. 1998; 121: 181-190Crossref PubMed Scopus (77) Google Scholar). Neuronal nAChRs also form pentameric complexes (3Anand R. Conroy W.G. Schoepfer R. Whiting P. Lindstrom J. J. Biol. Chem. 1991; 266: 11192-11198Abstract Full Text PDF PubMed Google Scholar, 4Cooper E. Couturier S. Ballivet M. Nature. 1991; 350: 235-238Crossref PubMed Scopus (400) Google Scholar) from various combinations of the 11 neuronal nAChR genes (α2-α9 and β2-β4) that have been identified to date (5McGehee D.S Role L.W. Annu. Rev. Physiol. 1995; 57: 521-546Crossref PubMed Scopus (897) Google Scholar). α7, α8, and α9 nAChRs are blocked by the snake peptide toxin α-bungarotoxin (α-BTX), which also blocks muscle and Torpedo nAChRs but not other subtypes of neuronal nAChRs (5McGehee D.S Role L.W. Annu. Rev. Physiol. 1995; 57: 521-546Crossref PubMed Scopus (897) Google Scholar). α7 nAChRs are the most abundantly expressed nicotinic receptor subunit in the central nervous system (6Boyd T. Crit. Rev. Toxicol. 1997; 27: 299-318Crossref PubMed Scopus (81) Google Scholar) and are important in neuronal development, hippocampal function, and the modulation of fast neurotransmission (7Role L.W. Berg D.K. Neuron. 1996; 16: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 8Albuquerque E.X. Alkondon M. Pereira E.F.R. Castro N.G. Schrattenholz A. Barbosa C.T.F. Bonfante-Cabarcas R. Aracava Y. Eisenberg H.M. Maelicke A. J. Pharmacol. Exp. Ther. 1997; 280: 1117-1136PubMed Google Scholar). α7 nAChRs are highly calcium-permeable (9Séguéla P. Wadiche J. Dineley-Miller K. Dani J.A. Patrick J.W. J. Neurosci. 1993; 13: 596-604Crossref PubMed Google Scholar,10Sands S.B. Costa A.C.S. Patrick J.W. Biophys. J. 1993; 65: 2614-2621Abstract Full Text PDF PubMed Scopus (78) Google Scholar), and calcium influx through presynaptic α7 nAChRs modulates the release of excitatory neurotransmitters (11McGehee D.S Heath M.J.S. Gelber S. Devay P. Role L.W. Science. 1995; 269: 1692-1696Crossref PubMed Scopus (911) Google Scholar, 12Gray R. Rajan A.S. Radcliffe K.A. Yakehiro M. Dani J.A. Nature. 1996; 383: 713-716Crossref PubMed Scopus (852) Google Scholar). During biosynthesis of nAChRs, the polypeptide is translocated into the endoplasmic reticulum (ER) membrane. The ER contains enzymes necessary for signal sequence cleavage (13Anderson D.J. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5598-5602Crossref PubMed Scopus (84) Google Scholar) and other post-translational modifications required for correct subunit folding, assembly, and ligand-binding site formation. These modifications include core glycosylation (13Anderson D.J. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5598-5602Crossref PubMed Scopus (84) Google Scholar) and disulfide bond formation (14Arias H.R. Brain Res. Brain Res. Rev. 1997; 25: 133-191Crossref PubMed Scopus (134) Google Scholar, 15Green W.N. Millar N.S. Trends Neurosci. 1995; 18: 280-287Abstract Full Text PDF PubMed Scopus (174) Google Scholar). In addition, ER and cytoplasmic chaperone proteins are thought to be involved in the maturation of muscle-type and α7 nAChRs (16Gelman M.S. Chang W. Thomas D.Y. Bergeron J.J.M. Prives J.M. J. Biol. Chem. 1995; 270: 15085-15092Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 17Keller S.H. Lindstrom J. Taylor P. J. Biol. Chem. 1996; 271: 22871-22877Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 18Blount P. Merlie J.P. J. Cell Biol. 1991; 113: 1125-1132Crossref PubMed Scopus (79) Google Scholar, 19Helekar S.A. Char D. Neff S. Patrick J.W. Neuron. 1994; 12: 179-189Abstract Full Text PDF PubMed Scopus (99) Google Scholar). Based on current topological models, nicotinic receptor subunits have four putative transmembrane domains, a large, glycosylated N-terminal extracellular domain that contains the agonist-binding site (14Arias H.R. Brain Res. Brain Res. Rev. 1997; 25: 133-191Crossref PubMed Scopus (134) Google Scholar, 20Chen D. Dang H. Patrick J.W. J. Neurochem. 1998; 70: 349-357Crossref PubMed Scopus (54) Google Scholar), and a short extracellular C terminus (1Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (564) Google Scholar). The second transmembrane domain (M2) from each of the five subunits is postulated to line the ion-conducting pore (21Hille B. Ionic Channels of Excitable Membranes. Sinauer Associates, Inc., Sunderland, MA1992Google Scholar). The subunit composition of nAChRs containing the α7 gene product is not completely clear. The injection of α7 cRNA into Xenopus oocytes results in the formation of ACh-gated ion channels without requiring the co-expression of other neuronal nAChR subunit cRNAs (22Couturier S. Bertrand D. Matter J.-M. Hernandez M.-C. Bertrand S. Millar N. Valera S. Barkas T. Ballivet M. Neuron. 1990; 5: 847-856Abstract Full Text PDF PubMed Scopus (817) Google Scholar), suggesting that α7 nAChRs are homo-oligomeric. However, Xenopus oocytes also express low levels of endogenous nAChR α subunits, which can co-assemble with β, γ, and δ muscle-type nAChR subunits to form functional nAChRs (23Buller A.L. White M.M. Mol. Pharmacol. 1990; 37: 423-428PubMed Google Scholar). These α-subunits could, potentially, co-assemble with expressed α7 receptors in Xenopus oocytes as well. Based on co-immunoprecipitation experiments, native α7 receptors in rat brain appear to be homo-oligomeric (24Chen D. Patrick J.W. J. Biol. Chem. 1997; 272: 24024-24029Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), whereas native chick α7 subunits are thought to form both homo-oligomeric and hetero-oligomeric receptors, complexing with α8 (25Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (407) Google Scholar, 26Gotti C. Moretti M. Longhi R. Briscini L. Manera E. Clementi F. J. Recept. Res. 1993; 13: 453-465Crossref PubMed Scopus (7) Google Scholar, 27Keyser K.T. Britto L.R.G. Schoepfer R. Whiting P. Cooper J. Conroy W. Brozozowska-Prechtl A. Karten H.J. Lindstrom J. J. Neurosci. 1993; 13: 442-454Crossref PubMed Google Scholar) and other neuronal nAChR subunits (28Yu C.R. Role L.W. J. Physiol. 1998; 509: 651-656Crossref PubMed Scopus (139) Google Scholar). However, it is possible that the apparent homomeric α7 nAChRs in native tissues could represent heteromeric complexes containing yet unidentified nAChR subunits. α7 nAChRs have been difficult to express in several mammalian heterologous expression systems. The folding, assembly, and subcellular localization of heterologously expressed α7 nAChRs is deficient in some cell lines, due to misfolding and trapping of proteins in the ER (29Cooper S.T. Millar N.S. J. Neurochem. 1997; 68: 2140-2151Crossref PubMed Scopus (131) Google Scholar). For example, human α7 nAChRs have been expressed in HEK-293 cells (30Gopalakrishnan M. Buisson B. Touma E. Giordano T. Campbell J.E. Hu I.C. Donnelly-Roberts D. Arneric S.P. Bertrand D. Sullivan J.P. Eur. J. Pharmacol. 1995; 290: 237-246Crossref PubMed Scopus (153) Google Scholar), but attempts to express chick or rat α7 nAChRs in HEK-293 cells have not yet been successful (29Cooper S.T. Millar N.S. J. Neurochem. 1997; 68: 2140-2151Crossref PubMed Scopus (131) Google Scholar, 31Rangwala F. Drisdel R.C. Rakhilin S. Ko E. Atluri P. Harkins A.B. Fox A.P. Salman S.B. Green W.N. J. Neurosci. 1997; 17: 8201-8212Crossref PubMed Google Scholar, 32Kassner P.D. Berg D.K. J. Neurobiol. 1997; 33: 968-982Crossref PubMed Scopus (45) Google Scholar). Another powerful approach to determine whether α7 nAChRs can form functional, homo-oligomeric receptors is to express them in a cell-free system. Muscle-type nAChRs translated in the presence of ER microsomes have been studied biochemically (13Anderson D.J. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5598-5602Crossref PubMed Scopus (84) Google Scholar, 33Shtrom S.S. Hall Z.W. J. Biol. Chem. 1996; 271: 25506-25514Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 34Chavez R.A. Hall Z. J. Biol. Chem. 1991; 266: 15532-15538Abstract Full Text PDF PubMed Google Scholar) but have not been examined for functional channel activity. Our goal was to express chick α7 nAChRs in vitro, where the co-expression of other nAChR subunits is extremely unlikely, and to study their biochemical and functional properties. Our experimental strategy was to express receptors using rabbit reticulocyte lysates in the presence of ER microsomes, reconstitute the channels into planar lipid bilayers, and record single-channel activity. This method has been used successfully to synthesize and reconstitute functional Shaker potassium channels (35Rosenberg R.L. East J.E. Nature. 1992; 360: 166-169Crossref PubMed Scopus (53) Google Scholar), amiloride-sensitive sodium channels (36Awayda M.S. Ismailov I.I. Berdiev B.K. Benos D.J. Am. J. Physiol. 1995; 268: C1450-C1459Crossref PubMed Google Scholar), and gap junction channels (37Falk M.M. Buehler L.K. Kumar N.M. Gilula N.B. EMBO J. 1997; 16: 2703-2716Crossref PubMed Scopus (135) Google Scholar). In this paper, we show that α7 S2′T/L9′T nAChRs expressed in vitro were glycosylated, were processed integrally though the membrane, and formed functional channels when reconstituted into planar lipid bilayers. These data also show that α7 nAChR subunits can form functional, homo-oligomeric channels. Chick wild-type α7 nAChR cDNA was a gift from Mark Ballivet (University of Geneva, Geneva, Switzerland). Wild-type and S2′T/L9′T α7 nAChR cDNAs cloned into the pAMV vector (38Nowak M.W. Kearney P.C. Sampson J.R. Saks M.E. Labarca C.G. Silverman S.K. Zhong W. Thorson J. Abelson J.N. Davidson N. Schultz P.G. Dougherty D.A. Lester H.A. Science. 1995; 268: 439-442Crossref PubMed Scopus (213) Google Scholar) under control of the T7 promoter were kindly provided by Purnima Deshpande, Dr. Henry Lester, and Dr. Cesar Labarca (California Institute of Technology). The numbering of the residues in the M2 domain follows the convention of Miller (39Miller C. Neuron. 1989; 2: 1195-1205Abstract Full Text PDF PubMed Scopus (150) Google Scholar), where the N-terminal (cytoplasmic) residue of the M2 domain is denoted as 1′. The Ser at the 2′ position and Leu at the 9′ position correspond to amino acids Ser240 and Leu247, respectively, in the chick α7 cDNA sequence (22Couturier S. Bertrand D. Matter J.-M. Hernandez M.-C. Bertrand S. Millar N. Valera S. Barkas T. Ballivet M. Neuron. 1990; 5: 847-856Abstract Full Text PDF PubMed Scopus (817) Google Scholar). The plasmid templates were digested with Not I. Capped cRNA transcripts were generated from the cDNA templates with T7 RNA polymerase using the mMessage mMachine kit (Ambion, Austin, TX) according to the manufacturer's instructions. The cRNA was resuspended in 100 mm KCl and stored at −70 °C. Oocytes were surgically removed from female Xenopus laevis (Nasco, Fort Atkinson, WI) and treated with collagenase as described (40Goldin A.L. Methods Enzymol. 1992; 207: 266-278Crossref PubMed Scopus (236) Google Scholar) to remove the follicular cell layer. Oocytes were injected with 20 ng of α7 cRNA and incubated at 19 °C for 2–5 days in ND96 (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2, 5 mm Na-HEPES, pH 7.5) supplemented with 50 μg/ml gentamicin and 0.55 mg/ml sodium pyruvate (41Stühmer W. Methods Enzymol. 1992; 207: 319-338Crossref PubMed Scopus (261) Google Scholar). Two-electrode voltage clamp was performed with a GeneClamp 500 amplifier controlled by pCLAMP6 software (Axon Instruments, Foster City, CA). Microelectrodes were filled with 3m KCl and had resistances of 0.5–2.1 MΩ. Oocytes were superfused with ES-EGTA (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1 mm EGTA, 10 mm Na-HEPES, pH 7.5) in the presence and absence of ACh. Unless otherwise indicated, oocytes were voltage clamped at a holding potential of −60 mV. Currents from S2′T/L9′T receptors were filtered at 50 Hz (8-pole Bessel low pass) and digitized at 100 Hz with a Digidata 1200 A/D converter. Currents from wild-type receptors were filtered at 250 Hz and sampled at 500 Hz. Additional filtering was added to the traces for display purposes. To adjust for run-down during acquisition of dose-response data, the peak currents from repeated applications of a standard dose of ACh were used to normalize the test responses. Dose-response relationships were fitted to the Hill equation in Prism (GraphPad Software, San Diego, CA). Current-voltage relationships during the sustained maximal response to ACh were generated by applying a series of voltage steps (125 ms, 10 mV intervals) from the holding potential of −60 mV. Leak currents obtained in the absence of ACh were subtracted from ACh-evoked currents. Endoplasmic reticulum microsomes were obtained from canine pancreas (Promega, Madison, WI) or were prepared from the oviducts of mature, laying Japanese quail (42Das R.C. Brinkley S.A. Heath E.C. J. Biol. Chem. 1980; 255: 7933-7940Abstract Full Text PDF PubMed Google Scholar). Briefly, the magnum portion of the oviduct was minced, suspended in 5 volumes of TKMD buffer (50 mm Tris-HCl, pH 7.7, 25 mm KCl, 2.5 mm MgCl2, 3 mm dithiothreitol) plus 0.88 m sucrose, homogenized, and centrifuged at 4000 ×g in an HB4 rotor for 10 min. The supernatant was diluted with TKMD buffer to a final sucrose concentration of 0.6 mand layered onto a sucrose step gradient of TMKD buffer containing 1.5m and 2.0 m sucrose. After centrifugation for 16–20 h at 100,000 × g in an SW28 rotor, the microsomes were harvested from the 1.5 m and 2.0m sucrose interface and resuspended in 20 mmNa-HEPES. The volume was adjusted with 20 mm Na-HEPES to obtain an A260 reading of 0.6 in a sample containing 1% SDS. One-half volume of 750 mm sucrose, 20 mm Na-HEPES, pH 7.5 was added to the resuspended membranes, which were frozen in liquid nitrogen and stored at −70 °C. Coupled in vitro transcription/translation was performed using the Promega TNT kit. For analysis of translation and processing efficiency, reaction mixtures (25 μl) contained 12.5 μl of rabbit reticulocyte lysate, 0.5 μl of 10× reaction buffer, 20 μm amino acids minus methionine, 10 units of RNasin (Promega), 0.5 μl of T7 RNA polymerase, and 10 μCi of [35S]methionine (Amersham Pharmacia Biotech) with or without 1 μg of α7 S2′T/L9′T nAChR cDNA. Reactions were also supplemented with 1 mm dithiothreitol and 1 mmoxidized glutathione. Some reactions contained microsomes from canine pancreas (1.8 μl/25-μl reaction) or quail oviduct (1.0 μl/25-μl reaction). Reactions were incubated for 90 min at 30 °C and stopped by placing on ice. After reserving 3 μl of the reaction mixture for gel analysis (labeled M in Figs. Figure 2, Figure 3, Figure 4), the membranes were pelleted by centrifugation for 25 min at 14,000 rpm in a microcentrifuge, washed twice in solution D′ (160 mm KCl, 20 mm MOPS, pH 7.4), and resuspended in water. Samples were heated for 30 s at ∼95 °C in SDS-PAGE sample buffer and electrophoresed on 10% or 12.5% SDS-polyacrylamide gels (43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206965) Google Scholar). Gels were treated with Fluoro-Hance (Research Products International, Mount Prospect, IL), dried, and exposed to Kodak X-Omat film at −70 °C.Figure 3Endo H cleavage of carbohydrate from α7 S2′T/L9′T nAChRs expressed in vitro. The sizes of 14C-labeled molecular mass standards (lane 1) are indicated in kDa at the left. An in vitro transcription/translation containing α7 S2′T/L9′T nAChR cDNA, [35S]Met, and canine pancreatic microsomes (lane 2) was centrifuged to separate the supernatant (lane 3) from the membrane fraction (lanes 4 and 5). The membrane pellet was divided into equal aliquots and treated with carrier buffer (lane 4) or carrier buffer plus 1 milliunit of Endo H (lane 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Proteinase K digestion of in vitro translated α7 S2′T/L9′T nAChRs in the presence and absence of detergent. A, lane 1,14C-labeled molecular mass standards. Lanes 2–6 show a coupled transcription/translation of α7 S2′T/L9′T nAChRs in the presence of canine pancreatic microsomes and [35S]Met. Lanes 2 and 3 show the unseparated reaction mixture and supernatant, respectively. The membrane pellet was divided into three equal parts and treated with water (lane 4) or proteinase K in the absence (−, lane 5) or presence (+, lane 6) of 1% Triton X-100. B, diagram of the putative membrane topology of an nAChR subunit showing the expected membrane orientation following translation into the endoplasmic reticulum. In this membrane orientation, digestion with proteinase K in the absence of detergent is predicted to generate a protein fragment that contains the glycosylated N-terminal extracellular domain and first transmembrane domain, which has a predicted molecular mass of 37.5 kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A coupled transcription/translation reaction (37.5 μl) was performed as described above in the presence of α7 S2′T/L9′T nAChR cDNA and canine pancreatic microsomal vesicles. The membranes were pelleted (15 min at 14,000 rpm), resuspended in 50 mm sodium acetate, 1% (w/v) β-mercaptoethanol, pH 6.0, and divided into equal fractions. Endoglycosidase H (1 milliunit; Roche Molecular Biochemicals) or carrier buffer (25 mm EDTA, 0.05% sodium azide, 0.1% SDS, 50 mm NaH2PO4, pH 7.0) was added, and the samples were incubated for 2 h at 37 °C. The reaction was stopped by chilling on ice. SDS-PAGE sample buffer was added, and the samples were heated at ∼95 °C for 30 s and analyzed by SDS-PAGE. Pelleted microsomal membranes from a 50-μl coupled transcription/translation reaction were resuspended in 30 μl of reaction buffer (160 mm NaCl, 5 mmCaCl2, 50 mm Tris-Cl, pH 7.5). Each aliquot was treated with 0.1 mg/ml proteinase K, 0.1 mg/ml proteinase K plus 1% Triton X-100, or water for 45 min on ice. The reactions were stopped by adding 2 mm phenylmethylsulfonyl fluoride and an equal volume of 2x SDS-PAGE sample buffer. The reactions were heated immediately at ∼95 °C for 30 s and analyzed by SDS-PAGE. Coupled transcription/translations containing [35S]Met were performed in the absence or presence of canine pancreatic microsomes as described above. Mixtures (25 μl) from translations performed in the absence of microsomes were diluted to 40 μl in a final concentration of 10 mm NaH2PO4, 50 mmNaCl, 1 mm EDTA, pH 7.0, and 0.5% Triton X-100. Translations performed in the presence of microsomes were scaled to a final volume of 100 μl and centrifuged to pellet the microsomes. The microsomes were washed and resuspended in 40 μl of solution D′ and solubilized in 0.5% Triton X-100 for 60 min on ice. Samples were layered on top of 5-ml linear 5–20% (w/w) sucrose gradients containing 0.2% Triton X-100, 10 mmNaH2PO4, 50 mm NaCl, and 1 mm EDTA, pH 7.0 (44Reynolds J.A. Karlin A. Biochemistry. 1978; 17: 2035-2038Crossref PubMed Scopus (242) Google Scholar). Sucrose gradients run in parallel contained the standard proteins ovalbumin (3.6 S), bovine serum albumin (4.2 S), human gamma globulin (7 S), and catalase (11 S). The gradients were centrifuged for 12 h at 300,000 × g at 4 °C in a SW50.1 rotor. Fractions were collected from the top of the gradients, and the sucrose concentration of each fraction was determined by refractometry. Proteins from each fraction were separated by SDS-PAGE and visualized by fluorimetry (35S-labeled protein) or Coomassie Blue staining (standard proteins). Gels or autoradiographs were digitized, and the relative amount of protein in each band was analyzed by densitometry using NIH Image software. Coupled transcription/translations for the incorporation of microsomal vesicles into planar lipid bilayers were performed as described above except that [35S]methionine was omitted, 20 μm amino acids minus cysteine were added to provide the full complement of all unlabeled amino acids, and all volumes were scaled up to make a final volume of 50 μl. After translation, the membranes were pelleted by centrifugation for 15 min at 9,000 rpm. The supernatant was removed immediately, and the pellet was washed twice with solution D′ and resuspended thoroughly by gentle trituration in 5 μl of 250 mm sucrose and 20 mm HEPES, pH 7.4. Bilayer experiments were generally performed on the same day as translations. Synthetic lipids (1-palmitoyl-2-oleoyl phosphatidylethanolamine and 1-palmitoyl-2-oleoyl phosphatidylserine; Avanti Polar Lipids, Alabaster, AL) were resuspended in n-decane at concentrations of 15 mg/ml and 5 mg/ml, respectively. Planar lipid bilayers were formed by applying the decane solution across a 200-μm hole in a polyvinyldifluoride partition separating two aqueous chambers denoted cis and trans. Endoplasmic reticulum-derived microsomal vesicles from in vitro translations were incorporated into planar lipid bilayers by applying them directly onto the cis face of the bilayer with a fire-polished glass probe (45Perez G. Lagrutta A. Adelman J.P. Toro L. Biophys. J. 1994; 66: 1022-1027Abstract Full Text PDF PubMed Scopus (51) Google Scholar). To promote the fusion of vesicles with the bilayer, 0.5 mm CaCl2 was present on the cis side. Acetylcholine was added to both chambers to activate all incorporated nAChRs regardless of membrane orientation. For each 50-μl translation, three to five bilayer experiments could be performed, each of which lasted about 1 h. Bilayers were voltage-clamped with a Warner Instruments patch-clamp amplifier (Hamden, CT). Voltages were assigned as cis relative to trans, with trans corresponding to the luminal side of the ER (the extracellular face). Data were filtered at 200 Hz using a 4-pole Bessel low pass filter, digitized at 1 kHz, and analyzed off-line using in-house analysis programs written in AxoBasic (Axon Instruments, Foster City, CA). The goal of these experiments was to characterize the biochemical and functional properties of α7 nAChRs synthesized in vitro and reconstituted into planar lipid bilayers. To study the activity of ligand-gated ion channels in a planar lipid bilayer, agonist is continually present to evoke channel opening events. Because wild-type α7 nAChRs desensitize very rapidly in the continued presence of ACh, we selected a nondesensitizing form of the α7 nAChR so that channel activity could be observed for extended periods of time. This receptor has a serine to threonine mutation at the 2′ position and a leucine to threonine mutation at the 9′ position (α7 S2′T/L9′T nAChR). Fig.1 A shows that wild-type α7 nAChRs activated rapidly and desensitized completely within 1 s of ACh application. In contrast, α7 S2′T/L9′T nAChRs (Fig.1 B) did not desensitize during a 30-s application of ACh, behavior similar to that of α7 L9′T nAChRs described by Revah et al. (46Revah F. Bertrand D. Galzi J.-L. Devillers-Thiéry A. Mulle C. Hussy N. Bertrand S. Ballivet M. Changeux J.-P. Nature. 1991; 353: 846-849Crossref PubMed Scopus (437) Google Scholar). Like wild-type and α7 L9′T nAChRs (47Bertrand D. Devillers-Thiéry A. Revah F. Galzi J.-L. Hussy N. Mulle C. Bertrand S. Ballivet M. Changeux J.-P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1261-1265Crossref PubMed Scopus (214) Google Scholar), α7 S2′T/L9′T nAChRs were completely inhibited by α-BTX (Fig.1 B). Fig. 1 C shows the ACh dose-response characteristics of wild-type and α7 S2′T/L9′T nAChRs. As was seen with α7 L9′T nAChRs (46Revah F. Bertrand D. Galzi J.-L. Devillers-Thiéry A. Mulle C. Hussy N. Bertrand S. Ballivet M. Changeux J.-P. Nature. 1991; 353: 846-849Crossref PubMed Scopus (437) Google Scholar), the EC50 of α7 S2′T/L9′T nAChRs (14.1 μm) was much lower than that of wild-type α7 nAChRs (345 μm). Fig. 1 D shows that Ca2+ permeates through α7 S2′T/L9′T nAChRs. In the absence of any permeant ions in the bath, no ACh-evoked currents were observed. In the presence of 10 mm Ca2+ (and no other permeant ions), robust ACh-evoked currents were recorded. Thus, α7 S2′T/L9′T nAChRs displayed the nondesensitizing kinetics necessary to sustain channel activity in planar lipid bilayers in the continued presence of agonist. In addition, α7 S2′T/L9′T nAChRs displayed sensitivity to α-BTX, high potency of ACh, and Ca2+permeability, as expected. To determine whether the S2′T/L9′T α7 nAChRs synthesized in vitro were full-length, glycosylated, and correctly folded, we evaluated a number of its biochemical properties. Fig.2 shows a fluorogram of [35S]Met-labeled proteins generated from a coupled transcription/translation performed in the presence and absence of endoplasmic reticulum microsomes derived from quail oviduct. In the absence of microsomes, a ∼41-kDa protein and a 28 kDa protein were observed (lane 3). The primary sequence of wild-type α7 nAChRs indicates a nonglycosylated molecular mass of 54 kDa (22Couturier S. Bertrand D. Matter J.-M. Hernandez M.-C. Bertrand S. Millar N. Valera S. Barkas T. Ballivet M. Neuron. 1990; 5: 847-856Abstract Full Text PDF PubMed Scopus (817) Google Scholar, 25Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (407) Google Scholar), a value substantially higher than the ∼41 kDa observed. Other nAChR α-subunits also run anomalously fast on SDS-polyacrylamide gels; nonglycosylated α1 subunits from Torpedo and mouse muscle nAChRs have calculated molecular masses of 50 kDa but migrate as 41–43-kDa proteins (33Shtrom S.S. Hall Z.W. J. Biol. Chem. 1996; 271: 25506-25514Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 34Chavez R.A. Hall Z. J. Biol. Chem. 1991; 266: 15532-15538Abstract Full Text PDF PubMed Google Scholar, 48Noda M. Takahashi H. Tanabe T. Toyosato M. Furutani Y. Hirose T. Asai M. Inayama S. Miyata T. Numa S. Nature. 1982; 299: 793-797Crossref PubMed Scopus (502) Google Scholar). Thus, the ∼41-kDa protein expressed in vitro is likely to be the full-length α7 S2′T/L9′T nAChR. The 28-kDa protein could be either a premature translation stop or a proteolyt" @default.
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• W2005388346 title "Cell-free Expression and Functional Reconstitution of Homo-oligomeric α7 Nicotinic Acetylcholine Receptors into Planar Lipid Bilayers" @default.
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