Abstract
The acetylcholine receptor (AchR) was the first neurotransmitter receptor to be identified and purified in an active form. It is a complex transmembrane glycoprotein present in the postsynaptic side of the neuromuscular junctions. When an action potential reaches the motor nerve terminals, acetylcholine is released into the synaptic cleft, where its local concentration can rise transiently to 10-4 to 10-3 M. Binding of Ach to specific sites located on the extracellular domains of the AchR molecules triggers the opening of short-lived cation channels, thus increasing the permeability of the postsynaptic membrane and causing the muscle fiber membrane to be depolarized beyond a critical threshold. The final result of this chain of events is muscle contraction. The AchR is present in high amounts in the electric organ of certain fishes. Using Torpedo (electric ray) electroplax as the starting material, one can purify milligram quantities of active protein, as well as substantial amounts of its constituent subunits. Moreover, a group of closely related protein toxins (α-neurotoxins) have been isolated from the venom of several Elapid snakes, which bind to the AchR with dissociation constants in the nanomolar to subnanomolar range [for review see Karlsson (1979) and Low (1979)]. The high affinity of α-neurotoxins for AchR, combined with their extreme specificity, has greatly facilitated the purification and characterization of AchR from different sources.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
Abbreviations
- ADS:
-
anthracene-1,5-disulfonic acid
- AchR:
-
acetylcholine receptor
- ANTS:
-
8-amino-l,3,6-naphthalene trisulfonate
- α-Bgt:
-
α-bungarotoxin
- Carb:
-
carbamylcholine
- DPH:
-
l,6-diphenyl-l,3,5-hexatriene
- MBTA:
-
4-(N-maleimide)benzyltrimethylammonium
- NEM:
-
N-ethylmaleimide
- OG:
-
ß-D-octylglucopyranoside
- PM:
-
N-(1-pyrene)maleimide
- PTSA:
-
1,3,6,8-pyrene tetrasulfonate
- PySA:
-
pyrene-1-sulfonyl azide
- PySAH:
-
N-(l-pyrene-sulfonyl)hexadecylamine
- TMA-DPH:
-
l-[4-(trimethylamino)phenyl]-6-phenylhexa-l,3,5-triene
References
Anderson, D. J., Walter, P., and Blobel, G., 1982, Signal-recognition protein is required for the integration of acetylcholine receptor δ subunit, a transmembrane glycoprotein, into the endoplasmic reticulum membrane, J. Cell Biol. 93: 501–506.
Andreasen, T. J., and McNamee, M. G., 1977, Phospholipase A inhibition of acetylcholine receptor function in Torpedo californica membrane vesicles, Biochem. Biophys. Res. Commun. 79: 958–965.
Andreasen, T. J., Doerge, D. R., and McNamee, M. G., 1979, Effects of phospholipase A2 on the binding and ion permeability control properties of the acetylcholine receptor, Arch. Biochem. Biophys. 194: 468–480.
Andrich, M. P., and Vanderkooi, J. M., 1976, Temperature dependence of l,6-diphenyl-l,3,5- hexatriene fluorescence in phospholipid artifical membranes, Biochemistry 15: 1257–1261.
Anholt, R., Lindstrom, J., and Montai, M., 1981, Stabilization of acetylcholine receptor channels by lipids on cholate solution and during reconstitution in vesicles, J. Biol Chem. 256: 4377–4387.
Barrantes, F. J., 1983, Recent developments in the structure and function of the acetylcholine receptor, Int. Rev. Neurobiol. 24: 259–341.
Brisson, A., and Unwin, P. M. T., 1985, Quaternary structure of the acetylcholine receptor, Nature 315: 474–477.
Cash, D. J., and Hess, G. P., 1981, Quenched-flow technique with plasma membrane vesicles: Acetylcholine receptor mediated transmembrane ion flux, Anal. Biochem. 112: 39–51.
Changeux, J. P., Devillers-Thiery, A., and Chenouilli, P., 1984, Acetylcholine receptor: An al-losteric protein, Science 225: 1335–1345.
Clarke, J. H., and Martinez-Carrion, M., 1986, Labeling of functionally sensitive sulfhydryl containing domains of acetylcholine receptor from Torpedo californica membranes, J. Biol. Chem. 261: 10063–10072.
Clarke, J. H., Garcia-Borron, J. C., and Martinez-Carrion, M., 1987, (l-Pyrene)sulfonyl azide is a fluorescent probe for measuring the transmembrane topology of acetylcholine receptor subunits, Arch. Biochem. Biophys. 256: 101–109.
Claudio, T., Ballivet, M., Patrick, J., and Heinemann, S., 1983, Nucleotide and deduced amino acid sequences of Torpedo californica acetylcholine receptor 7 subunit, Proc. Natl. Acad. Sci. U.S.A. 80: 1111–1115.
Conti-Tronconi, B. M., and Raftery, M. A., 1982, The nicotinic acetylcholine receptor: Correlation of molecular structure with functional properties, Annu. Rev. Biochem. 51: 491–530.
Criado, M., Eibl, H., and Barrantes, F. J., 1982, Effects of lipids on acetylcholine receptor. Essential need of cholesterol for maintenance of agonist induced state transitions in lipid vesicles, Biochemistry 21: 3622–3629.
Criado, M., Eibl, H., and Barrantes, F. J., 1984, Functional properties of the acetylcholine receptor incorporated in model lipid membranes, J. Biol. Chem. 259: 9188–9198.
Dalziel, A. W., Rollins, E. S., and McNamee, M. G., 1980, The effect of cholesterol on agonist induced flux in reconstituted acetylcholine receptor vesicles, FEBS Lett. 122: 193–196.
Damle, V. N., McLaughlin, M., and Karlin, A., 1978, Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica, Biochem. Biophys. Res. Commun. 84: 845–851.
Devillers-Thiery, A., Giraudat, J., Bentaboulet, M., and Changeux, J. P., 1983, Complete mRNA sequence of the acetylcholine binding subunit from Torpedo marmorata acetylcholine receptor: A model for the transmembrane organization of the polypeptide chain, Proc. Natl. Acad. Sci. U.S.A. 80: 2067–2071.
Donnelly, D., Mihovilovic, M., Gonzalez-Ros, J. M., Ferragut, J. A., Richman, D., and Martinez-Carrion, M., 1984, A noncholinergic site directed monoclonal antibody can impair agonist induced ion flux in Torpedo californica acetylcholine receptor, Proc. Natl. Acad. Sci. U. S.A. 81: 7999–8003.
Ellena, J. F., Blazing, M. A., and McNamee, M. G., 1983, Lipid-protein interactions in reconstituted membranes containing acetylcholine receptor, Biochemistry 22: 5523–5535.
Farach, M. C., and Martinéz-Carrion, M., 1983, A differential scanning calorimetry study of acetylcholine receptor-rich membranes from Torpedo californica, J. Biol. Chem. 258: 4166–4170.
Finer-Moore, J., and Stroud, R., 1984, Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor, Proc. Natl. Acad. Sci. U.S.A. 81: 155–159.
Fong, T. M., and McNamee, M. G., 1986, Correlation between acetylcholine receptor function and structural properties of membranes, Biochemistry 25: 830–840.
Giraudat, J., Montecucco, C., Bisson, R., and Changeux, J. P., 1985, Transmembrane topology of acetylcholine receptor subunits probed with photoreactive phospholipids, Biochemistry 24: 3121–3127.
Gonzalez-Ros, J. M., Calvo-Fernandez, P., Sator, V., and Martinez-Carrion, M., 1979a, Pyrene-sulfonyl azide as a fluorescent label for the study of protein-lipid boundaries of acetylcholine receptor in membranes, J. Supramol. Struct. 11: 327–338.
Gonzalez-Ros, J. M., Sator, V., Calvo-Fernandez, P., and Martinez-Carrion, M., 1979b, Pyrenesulfonyl azide: A covalent probe permitting in vitro desensitization of labeled acetylcholine receptor rich membrane fragments from Torpedo californica, Biochem. Biophys. Res. Commun. 87: 214–220.
Gonzalez-Ros, J. M., Llanillo, M., Paraschos, A., and Martinez-Carrion, M., 1982, Lipid environment of acetylcholine receptor from Torpedo californica, Biochemistry 21: 3467–3474.
Gonzalez-Ros, J. M., Farach, M. C., and Martinez-Carrion, M., 1983, Ligand induced effects at regions of acetylcholine receptor accessible to membrane lipids, Biochemistry 22: 3807–3811.
Gonzalez-Ros, J. M., Ferragut, J. A., and Martinez-Carrion, M., 1984, Binding of anti-acetylcho-line receptor antibodies inhibits the acetylcholine receptor mediated cation flux, Biochem. Biophys. Res. Commun. 120: 368–375.
Guy, R., 1984, A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations, Biophys. J. 45: 249–261.
Heidmann, T., Sobel, A., Popot, J. L., and Changeux, J. P., 1980, Reconstitution of a functional acetylcholine receptor. Conservation of the conformational and allosteric transitions and recovery of the permeability response; role of lipids, Eur. J. Biochem. 110: 35–55.
Hess, G. P., Cash, D. J., and Aoshima, H., 1979, Acetylcholine receptor controlled ion fluxes in membrane vesicles investigated by fast reaction techniques, Nature 282: 329–331.
Hess, G. P., Aoshima, H., Cash, D. J., and Lenchitz, B., 1981, Specific reaction rate of acetylcholine receptor controlled ion translocation: A comparison of measurements with membrane vesicles and with muscle cells, Proc. Natl. Acad. Sci. U.S.A. 78: 1361–1365.
Hess, G. P., Pasquale, F. B., Walker, J. W., and McNamee, M. G., 1982, Comparison of acetylcholine receptor controlled cation flux in membrane vesicles from Torpedo californica and Electrophorus electricus: Chemical kinetics measurements in the millisecond region, Proc. Natl. Acad. Sci. U.S.A. 79: 963–967.
Horn, R., and Stevens, C. F., 1980, Relation between structure and function of on channels, Comments Mol. Cell. Biophys. 1: 57–68.
Huang, L. Y. M., Catterall, W. A., and Ehrenstein, G., 1978, Selectivity of cations and nonelec-trolytes for acetylcholine activated channels in cultured muscle cells, J. Gen. Physiol. 71: 397–410.
Kanaoka, Y., Machida, M., Kokubun, H., and Sekine, T., 1968, Fluorescence and structure of proteins as measured by incorporation of fluorophore. III. Fluorescence characteristics of N-[p(2-benzoxazolyl)phenyl] maleimide and the derivatives, Chem. Pharm. Bull. (Tokyo) 16: 1747–1753.
Kao, P. N., and Karlin, A., 1986, Acetylcholine receptor binding site contains a disulfide crosslink between adjacent half-cystinyl residues, J. Biol. Chem. 261: 8085–8088.
Karlsson, E., 1979, Chemistry of protein toxins in snake venoms, in Handbook of Experimental Pharmacology (Ch.-Y. Lee, ed.), Vol. 52, pp. 159–212 (Springer-Verlag, Berlin).
Karpen, J. W., Sachs, A. B., Cash, D. J., Pasquale, E. B., and Hess, G. P., 1983, Direct spectrophotometric detection of cation flux in membrane vesicles: Stopped-flow measurements of acetylcholine receptor mediated ion flux, Anal. Biochem. 135: 83–94.
Kasai, M., and Changeux, J. P., 1971, In vitro excitation of purified membrane fragments by cholinergic agonists, J. Membr. Biol. 6: 1–23.
Kistler, J., Stroud, R. M., Klymkowsky, M. W., Lalancette, R. A., and Fairclough, R. H., 1982, Structure and function of an acetylcholine receptor, Biophys. J. 37: 371–383.
Klymkowsky, M. W., and Stroud, R. M., 1979, Immunospecific identification and three dimensional structure of a membrane bound acetylcholine receptor from Torpedo californica, J. Mol. Biol. 128: 319–334.
Klymkowsky, M. W., Heuser, J. E., and Stroud, R. M., 1980, Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica, J. Cell Biol. 85: 823–838.
Lewis, C. A., 1979, Ion concentration dependence of the reversal potential and the single channel conductance of ion channels at the frog neuromuscular junction, J. Physiol. 286: 417–445.
Lindstrom, J., Gullick, W., Conti-Tronconi, B., and Ellisman, M., 1980, Proteolytic nicking of the acetylcholine receptor, Biochemistry 19: 4791–4795.
Lindstrom, J., Criado, M., Hochschwender, S., Fox, J. L., and Sarin, V., 1984, Immunochemical tests of acetylcholine receptor subunit models, Nature 311: 573–575.
Low, B. W., 1979, The three dimensional structure of postsynaptic snake neurotoxins: Consideration of structure and function, in Handbook of Experimental Pharmacology (Ch.-Y. Lee, ed.), Vol. 52, pp. 213–257, Springer-Verlag, Berlin.
Martinez-Carrion, M., Sator, V., and Raftery, M. A., 1975, The molecular weight of an acetylcholine receptor isolated from Torpedo californica, Biochem. Biophys. Res. Commun. 65: 129–137.
Martinez-Carrion, M., Gonzalez-Ros, J. M., Llanillo, M., and Paraschos, A., 1982, Acetylcholine receptors from electroplax membranes: In vitro and in situ properties, in Advances in Experimental Medicine and Biology (F. Bossa, E. Chiancone, A. Finnazi-Agro, and R. Strom, eds.), Vol. 148, pp. 209–224, Plenum Press, New York.
Martinez-Carrion, M., Gonzalez-Ros, J. M., Mattingly, J. R., Ferragut, J. A., Farach, M. C., and Donnelly, D., 1984, Fluorescent probes for the study of acetylcholine receptor function, Biophys. J. 45: 141–143.
Middlemas, D. S., and Raftery, M. A., 1983, Exposure of the acetylcholine receptor to the lipid bilayer, Biochem. Biophys. Res. Commun. 115: 1075–1082.
Mihovilovic, M., and Richman, D. P., 1984, Modification of α-bungarotoxin and cholinergic ligand binding properties of Torpedo acetylcholine receptor by a monoclonal anti-acetylcholine receptor antibody, J. Biol. Chem. 259: 15051–15059.
Moore, H. P., and Raftery, M. A., 1979, Reversible and irreversible interactions of an alkylating agonist with Torpedo californica acetylcholine receptor, Biochemistry 10: 1862–1867.
Moore, H. P., and Raftery, M. A., 1980, Direct spectroscopic studies of cation translocation by Torpedo acetylcholine receptor on a time scale of physiological relevance, Proc. Natl. Acad. Sci. U.S.A. 77: 4509–4513.
Neubig, R. R., and Cohen, J. B., 1980, Permeability control by cholinergic receptors in Torpedo postsynaptic membranes: Agonist dose response relations measured at second and millisecond times, Biochemistry 19: 2770–2779.
Neumann, D., Barchan, D., Safran, A., Gershoni, J. M., and Fuchs, S., 1986, Mapping of the α-bungarotoxin binding site within the subunit of acetylcholine receptor, Proc. Natl. Acad. Sci. U.S.A. 83: 3008–3011.
Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., Asai, M., Inayama, S., Miyata, T., and Numa, S., 1982, Primary structure of a subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence, Nature 299: 793–797.
Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S., Hirose, T., Asai, M., Takashima, H., Inayama, S., Miyata, T., and Numa, S., 1983a, Primary structure of the β and δ subunit precursors of Torpedo californica acetylcholine receptor deduced from cDNA sequences, Nature 301: 251–255.
Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S., Furutani, Y., Hirose, T., Takashima, H., Inayama, S., Miyata, T., and Numa, S., 1983b, Structural homology of Torpedo californica acetylcholine receptor subunits, Nature 302: 528–532.
Paraschos, A., Gonzalez-Ros, J. M., and Martinez-Carrion, M., 1983, Absorption filtration: A tool for the measurement of ion tracer flux in native membranes and reconstituted lipid vesicles, Biochem. Biophys. Acta. 733: 223–233.
Pick, U., 1981, Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures, Arch. Biochem. Biophys. 212: 186–194.
Popot, J. L., and Changeux, J. P., 1984, Nicotinic receptor of acetylcholine: Structure of an oligomeric integral membrane protein, Physiol. Rev. 64: 1162–1239.
Prendergast, F. G., Haugland, R. P., Callahan, P. J., and Bodeau, D., 1981, l-[4-(Trimethylamino)phenyl]-6-phenylhexa-l,3,5-triene: Synthesis fluorescence properties, and use as a fluorescence probe of lipid bilayers, Biochemistry 20: 7333–7338.
Quast, U., Schimerlik, M., Lee, T., Witzemann, V., Blanchard, S., and Raftery, M. A., 1978,
Ligand induced conformation changes in Torpedo californica membrane bound acetylcholine receptor, Biochemistry 17: 2405–2414.
Raftery, M. A., Schmidt, J., Clark, D. G., and Wolcott, R. G., 1971, Demonstration of a specific α-bungarotoxin binding component in Electrophorus electricus electroplax membranes, Biochem. Biophys. Res. Commun. 65: 1622–1629.
Ratnam, M., and Lindstrom, J., 1984, Structural features of the nicotinic acetylcholine receptor revealed by antibodies to synthetic peptides, Biochem. Biophys. Res. Commun. 122: 1225–1233.
Ratnam, M., Nguyen, D., Rivier, J., Sargent, P., and Lindstrom, J., 1986, Transmembrane topography of nicotinic acetylcholine receptor: Immunochemical tests contradict theoretical predictions based on hydrophobicity profiles, Biochemistry 25: 2633–2643.
Reiter, M. J., Cowburn, D., Prives, J. M., and Karlin, A., 1972, Affinity labeling of the acetylcholine receptor in the electroplax: Electrophoretic separation in sodium dodecylsulfate, Proc. Natl. Acad. Sci. U.S.A. 69: 1168–1172.
Reynolds, J., and Karlin, A., 1978, Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica, Biochemistry 17: 2035–2038.
Sator, V., Raftery, M. A., and Martinez-Carrion, M., 1978, AT-(3-pyrene)maleimide: A fluorescent probe for acetylcholine receptor-Triton X-100 aggregates, Arch. Biochem. Biophys. 190: 57–66.
Sator, V., Gonzalez-Ros, J. M., Calvo-Fernandez, P., and Martinez-Carrion, M., 1979a, Pyrene sulfonyl azide. A marker of acetylcholine receptor subunits in contact with membrane hydrophobic environment, Biochemistry 18: 1200–1206.
Sator, V., Raftery, M. A., Thomas, J. K., and Martinez-Carrion, M., 1979b, Effect of cholinergic ligands and local anesthetics on acetylcholine receptor enriched preparations from Torpedo californica electroplax, Arch. Biochem. Biophys. 192: 250–259.
Shinitzky, M., and Barenholz, Y., 1974, Dynamics of the hydrocarbon layer in liposomes of lecithin and sphingomyelin containing deacetylphosphate, J. Biol. Chem. 249: 2652–2657.
Soler, G., Mattingly, J. R., and Martinez-Carrion, M., 1984, Effects of heating on the ion gating function and structural domains of the acetylcholine receptor, Biochemistry 23: 4630–4636.
Strader, C. D., and Raftery, M. A., 1980, Topographical studies of Torpedo acetylcholine receptor subunits as a transmembrane complex, Proc. Natl. Acad. Sci. U.S.A. 77: 5807–5811.
Sugiyama, H., and Changeux, J. P., 1975, Interconversion between different states of affinity for acetylcholine of the cholinergic protein from Torpedo marmorata, Eur. J. Biochem. 55: 505–515.
Watters, D., and Maelicke, A., 1983, Organization of ligand binding sites at the acetylcholine receptor. A study with monoclonal antibodies, Biochemistry 22: 1811–1819.
Weber, M., and Changeux, J. P., 1974a, Binding of Naja nigricollis [3H]α-toxin to membrane fragments from Electrophorus and Torpedo electric organs. I. Binding of the tritiated α-neu-rotoxin in the absence of effector, Mol. Pharmacol. 10: 1–14.
Weber, M., and Changeux, J. P., 1974b, Binding of Naja nigricollis [3H]α-toxin to membrane fragments from Electrophorus and Torpedo electric organs. II. Effect of cholinergic agonists and antagonists on the binding of the tritiated α-neurotoxin, Mol. Pharmacol. 10: 15–34.
Weiland, G., and Taylor, P., 1979, Ligand specificity of state transitions in the cholinergic receptor: Behavior of agonists and antagonists, Mol. Pharmacol. 15: 197–212.
Weltman, J. K., Szaro, R. P., Frackelton, A. R., Jr., Dowben, R. M., Bunting, J. R., and Cathou, R. E., 1973, N-(3-pyrene) maleimide: A long lifetime fluorescent sulfhydryl reagent, J. Biol. Chem. 248: 3173–3177.
Wilson, P. T., Lentz, T. L., and Hawrot, E., 1985, Determination of the primary amino-acid sequence specifying the α-bungarotoxin binding site on the a subunit of the acetylcholine receptor from Torpedo californica, Proc. Natl. Acad. Sci. U.S.A. 82: 8790–8794.
Wu, C. W., Yarbrough, L. Z., and Hsiuch, Y., 1976, N-(l-pyrene) maleimide: A fluorescent crosslinking reagent, Biochemistry 15: 2863–2868.
Young, A. P., Brown, F. F., Halsey, M. J., and Sigman, D. S., 1978, Volatile anesthetic facilitation of in vitro desensitization of membrane bound acetylcholine receptor from Torpedo californica, Proc. Natl. Acad. Sci. U.S.A. 75: 4563–4567.
Young, E. F., Ralston, E., Blake, J., Ramachandran, J., Hall, Z. W., and Stroud, R. M., 1985, Topological mapping of acetylcholine receptor: Evidence for a model with five transmembrane segments and a cytoplasmic COOH-terminal peptide, Proc. Natl. Acad. Sci. U.S.A. 82: 626–630.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1988 Plenum Press, New York
About this chapter
Cite this chapter
Martinez-Carrion, M., Clarke, J., Gonzalez-Ros, JM., Garcia-Borron, JC. (1988). Fluorescent Probes for the Acetylcholine Receptor Surface Environments. In: Hilderson, H.J. (eds) Fluorescence Studies on Biological Membranes. Subcellular Biochemistry, vol 13. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-9359-7_11
Download citation
DOI: https://doi.org/10.1007/978-1-4613-9359-7_11
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4613-9361-0
Online ISBN: 978-1-4613-9359-7
eBook Packages: Springer Book Archive