FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2103800908> ?p ?o ?g. }

• W2103800908 endingPage "37339" @default.
• W2103800908 startingPage "37333" @default.
• W2103800908 abstract "The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized withN-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in αM1, αM4, γM1, and γM4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear α-helical structure, but not so for M1, for which “classical” models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily. The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized withN-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in αM1, αM4, γM1, and γM4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear α-helical structure, but not so for M1, for which “classical” models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily. nicotinic acetylcholine receptor 3-doxyl-17β-hydroxy-5α-androstane spin label 3β-doxyl-5α-cholestane spin label 5-doxylstearic acid 7-doxylstearic acid 12-doxyl dioleoylphosphatidylcholine 12-doxylstearic acid N-(1-pyrenyl)-maleimide transmembrane high performance liquid chromatography polyacrylamide gel electrophoresis N-[2-hydroxy-1,1-bis)hydroxymethyl)ethyl]glycine 3-trifluoromethyl-3-[125I]iodophenyldiazirine Fourier transform infrared spectroscopy The muscle and electric organ nicotinic acetylcholine receptor (AChR)1 is a pentameric integral transmembrane protein of homologous α2βγδ subunits. The AChR belongs to a superfamily of ligand-gated ion channels, together with the glycine receptor, a subtype of the serotonin receptor (5-HT3), and the GABAAreceptor (1Smith G.B. Olsen R.W. Trends Pharmacol. Sci. 1995; 16: 162-168Abstract Full Text PDF PubMed Scopus (453) Google Scholar, 2Jackson M.B. Yakel J.L. Annu. Rev. Physiol. 1995; 57: 447-468Crossref PubMed Scopus (189) Google Scholar, 3Kuhse J. Betz H. Kirsch J. Curr. Opin. Neurobiol. 1995; 5: 318-323Crossref PubMed Scopus (189) Google Scholar, 4Changeux J.P. Edelstein S.J. Neuron. 1998; 21: 959-980Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Each AChR subunit contains a relatively large amino-terminal extracellular domain of ∼200 amino acids followed by four hydrophobic domains of 20–30 amino acids in length (M1-M4) connected by hydrophilic loops of varying length and ending with a very short extracellular carboxyl terminus (reviewed in Ref. 5Barrantes F.J. The Nicotinic Acetylcholine Receptor: Current Views and Future Trends. Springer Verlag, Berlin/Heidelberg and Landes Publishing Co., Georgetown, TX1998Crossref Google Scholar). Although the exact topology of the AChR relative to the membrane has not yet been determined unambiguously, it is usually accepted that the four hydrophobic segments M1-M4 correspond to transmembrane (TM) domains (6Blanton M.P. Cohen J.B. Biochemistry. 1992; 31: 3738-3750Crossref PubMed Scopus (138) Google Scholar, 7Chavez R.A. Hall Z.W. J. Cell Biol. 1992; 116: 385-393Crossref PubMed Scopus (55) Google Scholar). There is still contradictory evidence on their secondary structure. The original postulation of a four-helix bundle with an all-helical secondary structure (8Noda M. Takahashi H. Tanaba T. Toyosato M. Kikyotani S. Furutani Y. Hirose T. Takashima H. Inayama S. Miyata T. Numa S. Nature. 1983; 302: 528-532Crossref PubMed Scopus (533) Google Scholar) has been challenged by the results of cryoelectron microscopy of frozen AChR tubules (9Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (717) Google Scholar, 10Unwin N. Nature. 1995; 373: 37-43Crossref PubMed Scopus (913) Google Scholar) and computer-aided molecular modeling indicating that the dimensions of the AChR TM region are not compatible with a pentameric four-helix bundle (11Ortells, M. O., Barrantes, G. E. & Barrantes, F. J. (1998) in The Nicotinic Acetylcholine Receptor: Current Views and Future Trends, Barrantes, F. J., ed, pp. 85–108, Springer Verlag, Berlin/Heidelberg and Landes Publishing Co., Georgetown.Google Scholar). Site-directed mutagenesis data combined with patch clamp electrophysiology, and results from photoaffinity labeling with noncompetitive channel blockers, support the notion that the M2 domain lines the walls of the ion channel proper and are indicative of α-helical periodicity in the residues exposed to the lumen of the channel (4Changeux J.P. Edelstein S.J. Neuron. 1998; 21: 959-980Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Recent NMR spectroscopy studies of the δM2 segment (12Opella S.J. Marassi F.M. Gesell J.J. Valente A.P. Kim Y. Oblatt-Montal M. Montal M. Nature Struct. Biol. 1999; 6: 374-379Crossref PubMed Scopus (301) Google Scholar), indicate that this domain is inserted in the bilayer at an angle of 12° relative to the membrane normal, with no kinks, and in totally α-helical configuration. A synthetic peptide corresponding to theTorpedo αM2 segment in chloroform:methanol containing LiClO4 also adopts a totally α-helical configuration (13Pashkov V.S. Maslennikov I.V. Tchikin L.D. Efremov R.G. Ivanov V.T. Arseniev A.S. FEBS Lett. 1999; 457: 117-121Crossref PubMed Scopus (29) Google Scholar). The cryoelectron microscopy studies are further interpreted to indicate that the other putative TM domains (M1, M3, and M4) are relatively featureless, with a large portion of the polypeptide chain in an extended (or unresolved) β-sheet configuration, arranged in the form of a large β-barrel outside the central rim of M2 channel-forming rods (9Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (717) Google Scholar, 10Unwin N. Nature. 1995; 373: 37-43Crossref PubMed Scopus (913) Google Scholar). This interpretation is in contrast to the studies using photoactivatable hydrophobic probes, in which the observed periodicity of the lipid-exposed residues in M4 and M3 is consistent with an α-helical pattern (6Blanton M.P. Cohen J.B. Biochemistry. 1992; 31: 3738-3750Crossref PubMed Scopus (138) Google Scholar, 14Blanton M.P. Cohen J.B. Biochemistry. 1994; 33: 2859-2872Crossref PubMed Scopus (212) Google Scholar, 15Blanton M.P. McCardy E.A. Huggins A. Parikh D. Biochemistry. 1998; 37: 14545-14555Crossref PubMed Scopus (37) Google Scholar) and with deuterium-exchange FTIR studies which indicate a predominantly α-helical structure in the AChR TM region (16Baenziger J.E. Méthot N. J. Biol. Chem. 1996; 270: 29129-29137Abstract Full Text Full Text PDF Scopus (82) Google Scholar). In addition, secondary structure analysis (CD and FTIR spectroscopy) of isolated and lipid-reconstituted TM AChR peptides indicate α-helical structure for M2, M3, and M4 segments (17Corbin J. Méthot N. Wang H.H. Baenziger J.E. Blanton M.P. J. Biol. Chem. 1998; 273: 771-777Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Furthermore, two-dimensional 1H NMR spectroscopy of a synthetic peptide corresponding to the αM3 segment of Torpedo AChR showed a totally α-helical structure (18Lugovskoy A.A. Maslennikov I.V. Utkin Y.N. Tsetlin V.I. Cohen J.B. Arseniev A.S. Eur. J. Biochem. 1998; 255: 455-461Crossref PubMed Scopus (45) Google Scholar), and a recent NMR study of a synthetic γM4 peptide is also compatible with an α-helical secondary structure (19Williamson P.F. Bonev B. Barrantes F.J. Watts A. Biophys. J. 2000; 78: 147AGoogle Scholar). In the present work the spatial relationship between membrane-embedded domains of the Torpedo californica AChR and the membrane bilayer was studied using fluorescence spectroscopy. The location of the pyrene-labeled cysteine residues with respect to the membrane was determined by differential quenching with spin-labeled lipids. Both in intact AChR and in reconstituted individual TM peptides, Cys residues were located close to the membrane-water interface, as predicted for the segments αM4 and γM4 (6Blanton M.P. Cohen J.B. Biochemistry. 1992; 31: 3738-3750Crossref PubMed Scopus (138) Google Scholar, 14Blanton M.P. Cohen J.B. Biochemistry. 1994; 33: 2859-2872Crossref PubMed Scopus (212) Google Scholar, 15Blanton M.P. McCardy E.A. Huggins A. Parikh D. Biochemistry. 1998; 37: 14545-14555Crossref PubMed Scopus (37) Google Scholar). In the case of M1 segments, our fluorescence studies indicate that these membrane-embedded domains are not a straight α-helix and/or possess pronounced kinks. Our observations lend support to the hypothesis that M1 membrane-embedded domains in general depart from linear structures, thus extending the conclusion of cysteine-scanning mutagenesis (20Akabas M.H. Karlin A. Biochemistry. 1995; 34: 12496-12500Crossref PubMed Scopus (159) Google Scholar), fluorescence (21Kim J. McNamee M.G. Biochemistry. 1998; 37: 4680-4686Crossref PubMed Scopus (16) Google Scholar), and CD and FTIR spectroscopy (17Corbin J. Méthot N. Wang H.H. Baenziger J.E. Blanton M.P. J. Biol. Chem. 1998; 273: 771-777Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) studies on αM1. The spin-labeled fatty acids, derived from the stearic acid substituted on positions 5 (5-SASL, 5-doxylstearic acid), 7 (7-SASL, 7-doxylstearic acid), and 12 (12-SASL, 12-doxylstearic acid), and the equivalent phosphatidylcholine derivative 12-PCSL, were obtained from Avanti Polar Lipids, Birmingham, AL. Nitroxide-labeled CSL (3β-doxyl-5α-cholestane spin label) and 3-doxyl-17β-hydroxy-5α-androstane spin label (ASL) were from Aldrich Chemical Co., and used as received. The formula of the spin-labeled lipid analogs are shown in SchemeFSI. Pyrene maleimide (PM) was obtained from Molecular Probes and [125I]TID (10 Ci/mmol) from Amersham Pharmacia Biotech. Affi-Gel 10 was purchased from Bio-Rad, Genapol C-100 (10%) from Calbiochem, and sodium cholate from Sigma. Asolectin, a crude soybean lipid extract, came from Avanti Polar Lipids and Spectra/Por Dispo Dialyzers (molecular mass cutoff: 2,000 Da) from Spectrum. AChR-rich membranes were isolated from the electric organ of T. californica (Aquatic Research Consultants, San Pedro, CA) according to the procedure of Sobel et al. (22Sobel A. Weber M. Changeux J.P. Eur. J. Biochem. 1977; 80: 215-224Crossref PubMed Scopus (278) Google Scholar), with the modifications described previously (23Pedersen S.E. Dreyer E.B. Cohen J.B. J. Biol. Chem. 1986; 261: 13735-13743Abstract Full Text PDF PubMed Google Scholar). The final membrane suspensions in ∼36% sucrose/0.02% NaN3 were stored at −80 °C. TheTorpedo AChR was solubilized in 1% sodium cholate, and PM (2 mm) was allowed to react with available cysteine residues (1 h incubation). The labeled AChR (PM-labeled AChR) was purified by affinity chromatography in the presence of asolectin lipids. Affinity column purification was performed using an acetylcholine affinity matrix (24Ellena J.F. Blazing M.A. McNamee M. Biochemistry. 1983; 22: 5523-5535Crossref PubMed Scopus (163) Google Scholar) with a number of modifications (25Blanton M.P. Wang H.H. Biochim. Biophys. Acta. 1991; 1067: 1-8Crossref PubMed Scopus (16) Google Scholar). Briefly, the affinity column matrix was prepared by coupling cystamine to Affi-Gel 10 (Bio-Rad), reduction with dithiothreitol, and final modification with bromoacetylcholine bromide. Affinity purified PM-AChRs were reconstituted with asolectin at a lipid:protein ratio of 800:1 on a mol/mol basis and stored at −80 °C. The ability of PM-AChR to undergo agonist-induced conformational transitions was verified by examining the extent of [125I]TID photoincorporation into PM-AChR subunits in the absence and presence of agonist. The amount of [125I]TID photoincorporation into AChR subunits is ∼10-fold greater in the resting state AChR than in the desensitized state and therefore [125I]TID subunit labeling is an extremely sensitive indicator of the conformational state of the AChR as well as of agonist-induced state transitions (26White B.H. Howard S. Cohen S.G. Cohen J.B. J. Biol. Chem. 1991; 266: 21595-21607Abstract Full Text PDF PubMed Google Scholar,27Blanton M.P. McCardy E.A. Gallagher M.J. J. Biol. Chem. 2000; 275: 3469-3478Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Proteolytic fragments of AChR subunits which contain the M1 or M4 transmembrane segment of the α- and γ-subunit were prepared as described in Corbin et al. (17Corbin J. Méthot N. Wang H.H. Baenziger J.E. Blanton M.P. J. Biol. Chem. 1998; 273: 771-777Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Each TM peptide contains either a single cysteine residue (αM1, γM1, and γM4) or two cysteine residues (αM4), as shown in Scheme FSII. The isolated fragments in 0.1% sodium dodecyl sulfate were reacted with pyrene maleimide (3 mm) overnight. The PM-labeled peptides were purified by reverse-phase HPLC using a Brownlee Aquapore C4 column (100 × 2.1 mm). Solvent A was 0.08% trifluoroacetic acid in water, and solvent B was 0.05% trifluoroacetic acid in 60% acetonitrile, 40% 2-propanol. The flow rate was maintained at 0.2 ml/min and 0.5-ml fractions were collected. Peptides were eluted with a nonlinear gradient from 25 to 100% solvent B in 80 min. The elution of peptides was monitored by the absorbance at 210 nm as well as by fluorescence emission (350 nm excitation, 400 nm emission). Peptide-containing HPLC fractions were pooled and dried by vacuum centrifugation. Peptides were resuspended in 1 ml of 2% octyl-β-glucoside and asolectin lipid in 2% sodium cholate was added to achieve an estimated lipid to peptide molar ratio of ∼200:1. The octyl-β-glucoside and sodium cholate were removed by dialysis using Spectra/Por CE Dispo Dialyzers with a 2000 M rcutoff, for 2 days against phosphate buffer (10 mmphosphate, 5 mm NaCl, pH 7.0). Each sample was concentrated to 200 μl using a Centricon-3 and stored at −80 °C. A small aliquot of each sample was subjected to NH2-terminal amino acid sequence analysis in order to confirm the identity of the peptide and estimate its concentration (αM1, Ile210-Lys242; αM4, Val401-Arg428, γM1, Lys218-Lys251; γM4, Val446-Arg485). An additional aliquot was labeled with the hydrophobic photoreagent [125I]TID. The labeled peptide was resolved on a 1.0-mm thick Tricine gel (28Schagger H. von-Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10502) Google Scholar) and the dried gel subjected to autoradiography. Labeling with [125I]TID served as an additional test of peptide purity and to confirm that no significant peptide aggregation (irreversible) had occurred. Amino-terminal sequence analysis was performed on a Beckman Instruments (Porton) Model 20/20 protein sequencer using gas phase cycles (Texas Tech Biotechnology Core Facility). Peptide aliquots (∼10 μl) were immobilized on chemically modified glass fiber discs (Beckman Instruments), which were used to improve the sequencing yields of hydrophobic peptides. Peptides were subjected to 10 sequencing cycles and initial yield (I o) and repetitive yield (R) were calculated by nonlinear least square regression of the observed release (M) for each cycle (n): M =I o R n . These were carried out using an SLM model 4800 spectrofluorimeter (SLM Instruments, Urbana, IL) with its cuvette holder thermostated at 20 °C. Whole PM-labeled AChR reconstituted in asolectin was made to a final concentration of 0.05 μm in 5 × 5-mm quartz cuvettes. Fluorescence quenching was carried out with nitroxide spin-labeled phosphatidylcholine (12-PCSL), spin-labeled cholestane (CSL), and androstane (ASL), and the spin-labeled stearic acid analogs 5-SASL, 7-SASL, and 12-SASL. The highest nitroxide concentration employed was 24 μm. Lipid concentration was kept at 37 μm. In the case of PM-labeled AChR peptide fragments, they were resuspended at a final concentration of 0.05 μm. Lipid concentration was 8 μm. Titration with spin-labeled probes was made up to a final concentration of 8 μm. Pyrene was excited at λexc = 345 nm and its emission was monitored at λem = 382 nm. Slits of 5 nm were used in both monochromators. Relevant corrections were taken into account, as follows: (i) inner filter effect due to the quencher absorption. The nitroxide labels used have a weak absorption in the UV, λ430 nm = 14m−1 cm−1 (29Green S.A. Simpson D.J. Zhou G. Ho P.S. Blough N.V. J. Am. Chem. Soc. 1990; 112: 7337-7346Crossref Scopus (233) Google Scholar). From the absorption tail a value of E = 100m−1 at the pyrene excitation wavelength (λexc = 345 nm) is determined. The molar absorptivity of pyrene is 38,000 (30Narayanaswami V. Kim J. McNamee M.G. Biochemistry. 1993; 32: 12413-12419Crossref PubMed Scopus (29) Google Scholar). From Equation 1 the correction factor C for the fluorescence intensity is obtained as,C=(Atotal/Af)×((1−10f−A)/(1−10total−A))Equation 1 where A f is the pyrenyl absorption andA total the absorption of both PM-labeled AChR (or peptide) and nitroxide spin label. Under the experimental conditions used, this factor amounts to only 0.2% for the highest concentration of nitroxide used. (ii) Effective quencher concentration in the membrane. The determination of effective spin label concentrations in the membrane volume [Q]L, should take into account the different incorporation of the various fatty acid probes in the bilayer described by its partition constantK p. This was carried out using the relationship (31Castanho M. Prieto M. Acuña A.U. Biochim. Biophys. Acta. 1996; 1279: 164-168Crossref PubMed Scopus (34) Google Scholar),[Q] L=(Kp/(1+(Kp−1)*γL*[L])*[Q] TEquation 2 where γL is the lipid molar volume and [Q]Tthe analytical quencher concentration. The partition constants used for the spin-labeled fatty acids are: 9.6 × 104 (5-SASL), 9.8 × 104 (7-SASL), and 2.9 × 104(12-SASL) (32Blatt E. Chatelier R.C. Saywer W.H. Photochem. Photobiol. 1984; 39: 477-483Crossref Scopus (44) Google Scholar). These values are reported for a lipid system also in the fluid phase (egg phosphatidylcholine), and at 25 °C, a temperature close to the one used in this work. It should be stressed that since the partition constants depend on the mean occupation number of the quencher in the lipid structure, the ones corresponding to the lower occupation numbers were used. Incorporation of the ASL, CSL, and 12-PCSL was considered to be quantitative. For the lipid molar volume the value γL = 0.95 dm3 mol−1was used, as estimated from published data (33Cornell B.A. Separovic F. Biochim. Biophys. Acta. 1983; 733: 189-193Crossref PubMed Scopus (99) Google Scholar, 34Lewis B.A. Engelman D.M. J. Mol. Biol. 1983; 166: 211-217Crossref PubMed Scopus (689) Google Scholar). The values for [Q]L were also corrected for the dilution effect due to the quencher incorporation. (iii) Blank intensity corrections. Fluorescence intensities in the absence (F o) and the presence (F) of quencher were corrected by subtracting intensities measured upon injection of an equivalent amount of the ethanol solution according to Equation 3,(Fo/F) corr=1/(1−(F/Fo) ethanol+(F/Fo) Q)Equation 3 From the fluorescence quenching data, Stern-Volmer plots were obtained according to Equation 4,(Fo/F)=1+Ksv[Q] LEquation 4 where F o and F correspond to the fluorescence emission of pyrene-labeled AChR or transmembrane fragments in the absence and presence of spin-labeled lipids and [Q] is the concentration of the quencher. Plots ofF o/F versus [Q] yield a slope equal to K sv, the Stern-Volmer constant,i.e. the product of the bimolecular rate constantk q and the fluorophore lifetime (τ) (dynamic mechanism), which was considered invariant for all the fragments.K sv is a measure of the quencher concentration in the fluorophore vicinity, which allowed us to obtain topological information on the labeled cysteine transverse location. Any contribution from static quenching does not hamper the conclusions regarding the topography of the cysteine with respect to the membrane bilayer, as dealt with in a previous publication (31Castanho M. Prieto M. Acuña A.U. Biochim. Biophys. Acta. 1996; 1279: 164-168Crossref PubMed Scopus (34) Google Scholar). For labeling of the intact AChR at free cysteine residues, the AChR was solubilized from T. californica AChR-rich membranes in 1% sodium cholate and allowed to react with PM (2 mm) for 1 h. The PM-labeled AChR was then affinity purified and reconstituted into asolectin lipid vesicles (see “Experimental Procedures”). SDS-PAGE of PM-labeled AChRs showed that the majority of the fluorescence is localized in the α- and γ-subunits. The fluorescence was further mapped to large proteolytic fragments of the α- and γ-subunits: α-V8-20 (Ser173-Glu338), α-V8-10 (Asn339-Gly437), γ-V8-24 (Ala167-Glu372), and γ-V8-14 (Leu373-Pro489) (Fig.1 A). In the fragment α-V8-10 there are only two cysteine residues, Cys412 and Cys418, both of which are located in the transmembrane segment M4. In α-V8-20 there are three cysteine residue, Cys222 in the transmembrane segment M1 and Cys192 and Cys193 which are located extracellularly and contribute to the agonist recognition site. In the intact AChR, Cys192 and Cys193 form a vicinal disulfide bond leaving Cys222 as the site of reaction with PM. The ability of PM-labeled AChRs to undergo agonist-induced state transitions was tested by measuring the agonist sensitivity of the AChR with the hydrophobic photoreactive probe [125I]TID. The extent of [125I]TID photoincorporation into AChR subunits is ∼10-fold greater in the resting state AChR than in the desensitized state, and in the resting state the extent of [125I]TID incorporation into the γ-subunit is ∼4-fold greater than that into the α-, β-, or δ-subunit (22Sobel A. Weber M. Changeux J.P. Eur. J. Biochem. 1977; 80: 215-224Crossref PubMed Scopus (278) Google Scholar, 23Pedersen S.E. Dreyer E.B. Cohen J.B. J. Biol. Chem. 1986; 261: 13735-13743Abstract Full Text PDF PubMed Google Scholar). For PM-labeled AChRs we also found a greater extent of [125I]TID incorporation into the γ-subunit relative to the other subunits and the addition of agonist results in a substantial reduction of subunit labeling, indicating that labeled AChRs retain the ability to undergo agonist-induced state transitions (data not shown). Peptides containing the TM segments αM1 (Ile210-Lys242), αM4 (Tyr401-Arg429), γM1 (Lys218-Lys251), and γM4 (Val446-Arg485) were isolated from proteolytic digests and receptor subunits and their identity confirmed by amino-terminal sequence analysis (15Blanton M.P. McCardy E.A. Huggins A. Parikh D. Biochemistry. 1998; 37: 14545-14555Crossref PubMed Scopus (37) Google Scholar) (see Scheme FSII). Peptides were labeled with PM and purified by reverse-phase HPLC. Fig.1 B shows the reverse-phase HPLC elution profile of the fragment αIle210-Lys242, which contains the transmembrane segment αM1. Peak HPLC fractions (arrow) were pooled, the solvent removed, the peptide resuspended in detergent (octyl-β-glucoside) and reconstituted into asolectin lipid vesicles. The fluorescence spectrum of whole PM-labeled AChR reconstituted into asolectin vesicles consisted of only pyrenyl monomer emission (Fig.2 a). The extrinsic fluorescence of pyrene-labeled AChR was quenched with three different types of nitroxide spin-labeled lipid analogs: (a) 12-PCSL; (b) the CSL and ASL, with the nitroxide group at carbon 3; and (c) spin-labeled stearic acid analogs, with the nitroxide group at positions 5, 7, and 12 along the acyl chain (5-SASL, 7-SASL, and 12-SASL). Fig. 2 b depicts the Stern-Volmer plot of whole AChR quenching by spin-labeled lipid analogs. No deviation from linearity is apparent, and the data were therefore not fitted to a model assuming a fraction of non-accessible fluorophores (e.g. Ref. 35Lehrer S.S. Biochemistry. 1971; 10: 3254-3263Crossref PubMed Scopus (1686) Google Scholar). The Stern-Volmer constants K SV are given in TableI.Table IStern-Volmer constants (KSV), for the quenching of pyrene labeled transmembrane fragments and whole AChR by spin-labeled positional isomers of fatty acids, phosphatidylcholine, and the steroids cholestane and androstaneK SVWhole AChRα-M1γ-M1α-M4γ-M4m −15-SASL9.87 ± 1.096.59 ± 0.487.75 ± 0.2511.44 ± 1.2912.79 ± 1.457-SASL1.82 ± 0.071.46 ± 0.092.07 ± 0.141.38 ± 0.081.83 ± 0.0712-SASL1.88 ± 0.041.14 ± 0.332.09 ± 0.121.45 ± 0.022.46 ± 0.2512-PCSL0.78 ± 0.05CSL0.21 ± 0.070.39 ± 0.06ASL3.16 ± 1.002.75 ± 0.413.43 ± 0.382.55 ± 0.273.59 ± 0.58 Open table in a new tab It is apparent that 5-SASL and ASL were the most effective quenchers of the pyrene fluorescence of whole AChR reconstituted into asolectin vesicles. In the case of spin-labeled stearic acid derivatives, the 5-SASL isomer quenched more effectively than the 7-SASL and the 12-SASL analogs (Table I), indicating a superficial location of the cysteine-bound pyrenes. PM-labeled AChR TM peptides (see Scheme FSII) were reconstituted into asolectin vesicles and studied by fluorescence spectroscopy. Upon excitation at 345 nm, pyrenyl monomer emission with maxima at 382 and 402 nm was observed in all cases (Fig.3). For the γM4 fragment, an additional weak emission from the single tryptophan residue (Trp453) was observed upon excitation at a wavelength corresponding to intrinsic fluorescence (λexc = 280 nm, not shown). Although αM4 possesses two cysteine residues, and hence two potential pyrene tags, pyrenyl emission was not observed to be any stronger with this TM fragment, nor was there any indication of pyrenyl excimer emission (λem = 480 nm). Upon addition of the quenchers, a decrease in intensity was observed in all cases (Fig. 4 and Table I). As is observed for αM4 in Fig. 4, the Stern-Volmer plot shows a linear dependence. The two cysteines in αM4 are several residues apart, and are thus likely to exhibit different accessibility to the quenchers. If the two Cys residues had reacted with PM, a deviation from linearity would result; this is not the case (Fig. 4). Thus, of the two potential tags only a single cysteine in αM4 appears to have reacted to any significant extent (Cys412 or Cys418). Furthermore, and on the basis of previous studies showing that Cys412 exhibits a preferential reactivity to structurally diverse hydrophobic probes (15Blanton M.P. McCardy E.A. Huggins A. Parikh D. Biochemistry. 1998; 37: 14545-14555Crossref PubMed Scopus (37) Google Scholar), it is likely that Cys412is the residue labeled with pyrene in αM4. Quenching of the pyrenyl fluorescence in the other TM fragments followed the same pattern as that of αM4. The use of a families of nitroxide spin-labeled analogs having known graded series of depths in the membrane has set the experimental basis (30Narayanaswami V. Kim J. McNamee M.G. Biochemistry. 1993; 32: 12413-12419Crossref PubMed Scopus (29) Google Scholar, 32Blatt E. Chatelier R.C. Saywer W.H. Photochem. Photobiol. 1984; 39: 477-483Crossref Scopus (44) Google Scholar, 36Chattopadhyay A. Gaber B.P. Easwaran K.R.K. Biomembrane Structure and Function: The State of the Art. Adenine Press, Schenectady, NY1992: 153-163Google Scholar, 37Abrams F.S. Chattopadhyay A. London E. Biochemistry. 1992; 31: 5322-5327Crossref PubMed Scopus (93) Google Scholar, 38Chattopadhyay A. London E. Biochemistry. 1987; 26: 39-45Crossref PubMed Scopus (599) Google Scholar, 39Ladokhin A.S. Biophys. J. 1999; 76: 946-955Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) for the well established differential quenching methodologies. From inspection of Table I it can be concluded that the fluorophore is in all cases very close to the membrane-water interface: quenching is highest for 5-SASL, much lower, similar values are obtained for 7-SASL and 12-SASL. The very high value of 5-SASL compared with the nearby located 7-SASL points to a very specific location of the former at a shallow position in the membrane. The quenching efficiency for CSL was determined for the γM4 fragment (Table I). The absolute value of" @default.
• W2103800908 created "2016-06-24" @default.
• W2103800908 creator A5006767226 @default.
• W2103800908 creator A5048872989 @default.
• W2103800908 creator A5052760661 @default.
• W2103800908 creator A5079635755 @default.
• W2103800908 date "2000-12-01" @default.
• W2103800908 modified "2023-10-13" @default.
• W2103800908 title "Topography of Nicotinic Acetylcholine Receptor Membrane-embedded Domains" @default.
• W2103800908 cites W1481524180 @default.
• W2103800908 cites W1505990424 @default.
• W2103800908 cites W1538053966 @default.
• W2103800908 cites W1643706501 @default.
• W2103800908 cites W1665596665 @default.
• W2103800908 cites W1857017278 @default.
• W2103800908 cites W192634894 @default.
• W2103800908 cites W1972994417 @default.
• W2103800908 cites W1975085160 @default.
• W2103800908 cites W1976067224 @default.
• W2103800908 cites W1976642662 @default.
• W2103800908 cites W1983329775 @default.
• W2103800908 cites W1995725157 @default.
• W2103800908 cites W1996489992 @default.
• W2103800908 cites W1996555147 @default.
• W2103800908 cites W1998441265 @default.
• W2103800908 cites W2004349398 @default.
• W2103800908 cites W2009484451 @default.
• W2103800908 cites W2016343967 @default.
• W2103800908 cites W2018531782 @default.
• W2103800908 cites W2022377078 @default.
• W2103800908 cites W2027535192 @default.
• W2103800908 cites W2028747294 @default.
• W2103800908 cites W2030671064 @default.
• W2103800908 cites W2030888952 @default.
• W2103800908 cites W2031368977 @default.
• W2103800908 cites W2038482077 @default.
• W2103800908 cites W2038697935 @default.
• W2103800908 cites W2041004635 @default.
• W2103800908 cites W2045961778 @default.
• W2103800908 cites W2047572594 @default.
• W2103800908 cites W2048353380 @default.
• W2103800908 cites W2056778461 @default.
• W2103800908 cites W2067237230 @default.
• W2103800908 cites W2070986555 @default.
• W2103800908 cites W2077167494 @default.
• W2103800908 cites W2081556250 @default.
• W2103800908 cites W2090270307 @default.
• W2103800908 cites W2093844357 @default.
• W2103800908 cites W2099559490 @default.
• W2103800908 cites W2100868096 @default.
• W2103800908 cites W2105069140 @default.
• W2103800908 cites W2115029196 @default.
• W2103800908 cites W2135439310 @default.
• W2103800908 cites W2145137650 @default.
• W2103800908 cites W2156839406 @default.
• W2103800908 cites W2322069423 @default.
• W2103800908 cites W2951839447 @default.
• W2103800908 doi "https://doi.org/10.1074/jbc.m005246200" @default.
• W2103800908 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10967108" @default.
• W2103800908 hasPublicationYear "2000" @default.
• W2103800908 type Work @default.
• W2103800908 sameAs 2103800908 @default.
• W2103800908 citedByCount "71" @default.
• W2103800908 countsByYear W21038009082012 @default.
• W2103800908 countsByYear W21038009082013 @default.
• W2103800908 countsByYear W21038009082014 @default.
• W2103800908 countsByYear W21038009082015 @default.
• W2103800908 countsByYear W21038009082016 @default.
• W2103800908 countsByYear W21038009082017 @default.
• W2103800908 countsByYear W21038009082018 @default.
• W2103800908 countsByYear W21038009082019 @default.
• W2103800908 countsByYear W21038009082022 @default.
• W2103800908 countsByYear W21038009082023 @default.
• W2103800908 crossrefType "journal-article" @default.
• W2103800908 hasAuthorship W2103800908A5006767226 @default.
• W2103800908 hasAuthorship W2103800908A5048872989 @default.
• W2103800908 hasAuthorship W2103800908A5052760661 @default.
• W2103800908 hasAuthorship W2103800908A5079635755 @default.
• W2103800908 hasBestOaLocation W21038009081 @default.
• W2103800908 hasConcept C116289061 @default.
• W2103800908 hasConcept C12554922 @default.
• W2103800908 hasConcept C170493617 @default.
• W2103800908 hasConcept C185592680 @default.
• W2103800908 hasConcept C188987157 @default.
• W2103800908 hasConcept C2775910092 @default.
• W2103800908 hasConcept C2779570518 @default.
• W2103800908 hasConcept C45855136 @default.
• W2103800908 hasConcept C51927110 @default.
• W2103800908 hasConcept C55493867 @default.
• W2103800908 hasConcept C64615621 @default.
• W2103800908 hasConcept C80161118 @default.
• W2103800908 hasConcept C86803240 @default.
• W2103800908 hasConcept C98274493 @default.
• W2103800908 hasConceptScore W2103800908C116289061 @default.
• W2103800908 hasConceptScore W2103800908C12554922 @default.
• W2103800908 hasConceptScore W2103800908C170493617 @default.
• W2103800908 hasConceptScore W2103800908C185592680 @default.
• W2103800908 hasConceptScore W2103800908C188987157 @default.

• next