Skip to main content
SpringerLink
Log in

Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

  • Articles
  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Summary

Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclindine. 4-phenyl-4-(1-piperidinyl)-2.2.6.6.-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits86Rb flux (IC50=6.6μm), and [3H] phencyclidine binding to both resting and desensitized acetylcholine receptor (IC50=17 μm and 0.22 μm, respectively). From an indirect Hill plot of the inhibition of [3H]phencyclidine binding by PPT. a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in α-bungarotoxin-treated preparations. Taken together, these results indicate that PPt mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2–3 units.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adams P.R. 1981. Acetylcholine receptor kinetics.J. Membrane Biol. 58:161–174

    Google Scholar 

  • Albuquerque, E.X., Tsai, M.-C., Aronstam, R.S., Witkop, B., Eldefrawi, A., Eldefrawi, M.E. 1980. Phencyclidine interaction with the ionic channel of acetylcholine receptor and electrogenic membrane.Proc. Natl. Acad. Sci. USA 77:1224–1228

    Google Scholar 

  • Aronstam, R.S., Eldefrawi, A.T., Pessah, I.N., Daly, J.W., Albuquerque, E.X., Eldefrawi, M.E. 1981. Regulation of tritiated perhydro histrionicotoxin binding toTorpedo ocellata electroplax by effectors of the acetylcholine receptors.J. Biol. Chem. 256:2843–2850

    Google Scholar 

  • Bennett, J.P., Jr., Yamamura, H.I. 1985. Neurotransmitter, hormone, or drug receptor binding methods.In: Neurotransmitter Receptor Binding (2nd ed). H.I. Yamamura, S.J. Enna, and M.J. Kuhar, editor, pp. 61–89. Raven. New York

    Google Scholar 

  • Berliner, L.J. 1981. Using the spin-label method in enzymology.In: Spectroscopy in Biochemistry. J.E. Bell, editor, Vol. 11, pp. 1–56. CRC, Boca Raton, FL

    Google Scholar 

  • Blanton, M., McCardy, E., Gallaher, T.,Wang, H.H. 1988. Noncompetitive inhibitors reach their binding site in the acetylcholine receptor by two different paths.Mol. Pharmacol. 33:634–642

    Google Scholar 

  • Burt, D.R. 1985. Criteria for receptor identification.In: Neurotransmitter Receptor Binding. (2nd ed.). H.I. Yamamura, S.J. Enna, and M.J. Kuhar, editor, pp. 41–60. Raven, New York

    Google Scholar 

  • Cartaud, J., Benedetti, E., Sobel, A., Chageux, J.P. 1978. A morphological study of the cholinergic receptor protein fromTorpedo marmorata in its membrane environment and in its detergent extracted purified form.J. Cell. Sci. 29:313–337

    Google Scholar 

  • Changeux, J.-P., Pinset, C., Ribera, A.B. 1986. Effects of chlorpromazine and phencyclidine on mouse C2 acetylcholine receptor kinetics.J. Physiol. 378:497–513

    Google Scholar 

  • Charnet, P. Larbaca, C., Leonard, R.J., Vogeliu, N.J., Czyzyk, L., Gouin, A., Davidson, N., Lester, H.A. 1990. An open channel blocker interacts with adjacent turns of α-helices in the nicotinic acetylcholine receptor.Neuron 4:87–95

    Google Scholar 

  • Cheng, Y.C., Prusoff, W.H. 1973. Relationship between the inhibition constant (K l) and the concentration of inhibitor which causes 50 percent inhibition (I 50) of an enzymatic reaction.Biochem. Pharmacol. 22:3099–3108

    Google Scholar 

  • Cornish-Bowden, A., Koshland, D.E., Jr. 1975. Diagnostic uses of the Hill (Logit and Nernst) plots.J. Mol. Biol. 95:201–212

    Google Scholar 

  • Damle, V.N., Karlin, A. 1978. Affinity labeling of one of two α-neurotoxin binding sites in acetylcholine receptor fromTorpedo californica.Biochemistry 17:2039

    Google Scholar 

  • Eldefrawi, A.T., Miller, E.R., Murphy, D.L., Eldefrawi, M.E., 1982. [3H]Phencyclidine interactions with the nicotinic acetylcholine receptor channel and its inhibition by psychotropic antipsychotic, opiate antidepressant, antibiotic, antiviral and antiarrhythmic drugs.Mol. Pharmacol. 22:72–81

    Google Scholar 

  • Eldefrawi, M.E., Eldefrawi, A.T., Aronstam, R.S., Maleque, M.A., Warnick, J.E., Albuquerque, E.X. 1980. [3H]Phencyclidine: A probe for the ionic channel of the nicotinic receptor.Proc. Natl. Acad. Sci. USA 77:7458–7462

    Google Scholar 

  • Elliot, J., Blanchard, S.G., Wu, W., Miller, J., Strader, C.D., Hartig, P., Moore, H.-P., Racs, J., Raftery, M.A. 1980. Purification ofTorpedo californica post-synaptic membranes and fractionation of their constituent proteins.Biochem. J. 185:667–677

    Google Scholar 

  • Griffith, O.H., Dehlinger, P.J., Van, S.P. 1974. Shape of the hydrophobic barrier of phospholipid bilayers. Evidence for water penetration in biological membranesJ. Membrane Biol. 15:159–192

    Google Scholar 

  • Griffith, O.H., Libertini, L.J., Birrell, G.B. 1971. The role of lipid spin labels in membrane biophysics.J. Phys. Chem. 75:3417–3425

    Google Scholar 

  • Grünhagen, H.-H., Changeux, J.-P. 1976. Studies on the electrogenic action of acetylcholine withTorpedo marmorata electric organ: IV. Quinacrine: A fluorescent, probe for the conformational transitions of the cholinergic receptor protein in the membrane bound state.J. Mol. Biol. 106:497–516

    Google Scholar 

  • Guy, H.R., Hucho, I. 1987. The ion channel of the nicotinic acetylcholine receptors.Trends Neurosci 10:318–321

    Google Scholar 

  • Hampton, R.V., Medzihradsky, F., Woods, J.H., Dahlstrom, P.J. 1982. Sterospecific binding of3H-phencyclidine in brain membranes.Life Sci. 30:2147–2154

    Google Scholar 

  • Haring, R., Kloog, Y. 1984. Multiple binding sites for phencyclidine on the nicotinic acetylcholine receptor fromTorpedo ocellata electric organ.Life Sci. 34:1047–1055

    Google Scholar 

  • Heidman, T., Oswald, R.E., Changeux, J.P. 1983. Multiple sites of action for non-competitive blockers on acetylcholine receptor-rich membrane fragments fromTorpedo marmorata.Biochemistry 22:3112–3127

    Google Scholar 

  • Herz, J.M., Johnson, D.A., Taylor, P. 1987. Interaction of noncompetitive inhibitors with the acetylcholine receptor. The site specificity and spectrose properties of ethidium binding.J. Biol. Chem. 262:7238–7247

    Google Scholar 

  • Johnson, D.A., Brown, R.D., Herz, J.M., Berman, H.A., Andreason, G.L., Taylor, P. 1987. Decidium. A novel fluorescent probe of the agonist/antagonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor.J. Biol. Chem. 262:14022–14029

    Google Scholar 

  • Jones, O.T., McNamee, M.G. 1988. Annular and nonannular binding sites for cholesterol associated with the nicotinic acetylcholine receptor.Biochemistry 27:2364–2374

    Google Scholar 

  • Kalir, A., Edery, H., Pelah, Z., Balderman, D., Porath, G. 1969. 1-Phencycloalkylamine derivatives. II. Synthesis and pharmacological activity.J. Med. Chem. 12:473–477

    Google Scholar 

  • Katz, B., Thesleff, S. 1957. A study of the “desensitization” produced by acetylcholine at the motor end-plate.J. Physiol. 138:63–80

    Google Scholar 

  • Keith, A.D., Bulfield, G., Snipes, W. 1970. Spin-labeledNeurospora mitochondria.Biophys. J. 10:618

    Google Scholar 

  • Kistler, J., Stroud, R.M., Klymkowsky, W.W. Lalancette, R.A., Fairclough, R.H. 1982. Structure and function of the acetylcholine receptor.Biophys. J. 37:371–383

    Google Scholar 

  • Kolarvic, L., Fournier, N.L. 1986. A comparison of extraction methods for the isolation of phospholipids from biological sources.Anal. Biochem. 156:244–250

    Google Scholar 

  • Lambert, J.L., Durant, N.N., Henderson, E.G. 1983. Drug-induced modification of ionic conductance at the neuromuscular junction.Annu. Rev. Pharmacol. Toxicol. 23:505–539

    Google Scholar 

  • Lantz, O., Burgun, C., Cremel, G., Hubert, P., Darcy, F., Waksman, A. 1985. A rapid Percoll gradient procedure for the preparation of acetylcholine-rich vesicles fromTorpedo marmorata electric organ.Neurochem. Int. 7:331–339

    Google Scholar 

  • Lassmann, G., Ebert, B., Kuznetsov, A.N., Damerau, W. 1973. Characterization of hydrophobic regions in proteins by spin labeling techniques.Biochim. Biophys. Acta 310:298–304

    Google Scholar 

  • Lindstrom, J., Anholt, R., Einarson, B., Engel, A., Osame, M., Montal, M. 1980. Purification of acetylcholine receptors reconstitution into lipid vesciles, and study of agonist-induced cation channel regulation.J. Biol. Chem. 255:8340–8350

    Google Scholar 

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193:265–275

    Google Scholar 

  • Magleby, K.L., Stevens, C.F. 1972. The effects of voltage on the time course of end-plate currents.J. Physiol. 223:151–171

    Google Scholar 

  • McLafferty, F.W. 1980. Interpretation of Mass Spectra. University Science Books, Mill Valley, CA

    Google Scholar 

  • Morrison, A., Davies, A.P. 1970. Mass spectrometry of piperidine nitroxides—a class of stable free radicals.Org. Mass Spectrom. 3:353–366

    Google Scholar 

  • Nagy, A., Delgado-Escueta, A.V. 1984. Rapid preparation of synaptic somes from mammalian brain using nontoxic isosmotic gradient material (Percoll)J. Neurochem. 43:1114–1123

    Google Scholar 

  • Neubig, R.R., Boyd, N.D., Cohen, J.B. 1982. Conformations ofTorpedo acetylcholine receptor associated with ion transport and desensitization.Biochemistry 21:3460–3467

    Google Scholar 

  • Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose T., Asai, M., Inayama, S., Miyata, T., Numa, S. 1982. Primary structure of the α-subunit precursor ofTorpedo californica acetylcholine receptor deduced from the cDNA sequence.Nature 299:793–797

    Google Scholar 

  • Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S., Furutani, Y., Hirose, T., Takashima, H., Inayama, S., Miyata, T., Numa, S. 1983a. Structural homology ofTorpedo californica acetylcholine receptor subunits.Nature 302:528–532

    Google Scholar 

  • Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S., Hirose, T., Asai, M., Takashima, H., Inayama, S., Miyata, T., Numa, S. 1983. Primary structure of β-and δ-subunit precursors ofTorpedo californica acetylcholine receptor deduced from cDNA sequences.Nature 301251–255

    Google Scholar 

  • Ochoa, E.L.M., Chattopadhyay, A., McNamee, M.G. 1989. Desensitization of the nicotinic acetylcholine receptor: Molecular mechanisms and effect of modulators.Cell. Mol. Neurobiol. 9:141–178

    Google Scholar 

  • Ondrias, K., Stasko, A., Balgavy, P. 1987. Spin label study of the perturbation effect of the local anesthetics tetracaine and dibucaine on synaptosomes at pharmacological concentrations.Biochem. Pharmacol. 36:3999–4005

    Google Scholar 

  • Oswald, R., Heidmann, T., Changeux, J.P. 1983. Multiple affinity states for non-competitive blocks revealed by [3H] phencyclidine.Biochemistry 22:3128–3136

    Google Scholar 

  • Papke, R.L., Oswald, R.E. 1989. Mechanisms of noncompetitive inhibition of acetylcholine-induced single-channel currents.J. Gen. Physiol. 93:785–811

    Google Scholar 

  • Pavia, D.L., Lampman, P.L., Kriz, G.S. Jr 1979. Introduction to Spectroscopy, Saunders, Philadelphia

    Google Scholar 

  • Rang, H.P., Ritter, J.M. 1970. On the mechanism of desensitization at cholinergic receptors.Mol. Pharmacol. 6:357–382

    Google Scholar 

  • Richter, W.J., Schwarz, H. 1978. Chemical ionization—a massspectrometric analytical procedure of rapidly increasing importance.Angew. Chem. Int. Ed. Engl. 17:424–439

    Google Scholar 

  • Rodbard, D., Frazier, G.R. 1975. Statistical analysis of radioligand assay data.Meth. Enzymol. 37:1–22

    Google Scholar 

  • Rosen, G.M. 1974. Use of sodium cyanoborohydride in the preparation of biologically active membranesJ. Med. Chem. 17:358–360

    Google Scholar 

  • Saitoh, T., Oswald, R., Wennogle, L.P., Changeux, J.-P. 1980. Conditions for the selective labeling of the 66,000 dalton chain of the acetylcholine receptor by the covalent noncompetitive blocker 5-azido-[3H]trimethisoquin.FEBS Lett. 116: 30–36

    Google Scholar 

  • Sobel, A., Heidmann, T., Cartaud, J., Changeux, J.-P. 1980. Reconstitution of a functional acetylcholine receptor: Polypeptide chains, ultrastructure, and binding sites for acetylcholine and local anesthetics.Eur. J. Biochem. 110:13–33

    Google Scholar 

  • Spivak, C.E., Albuquerque, E.X. 1982. Dynamic properties of the nicotinic acetylcholine receptor ionic channel complex: Activation and blockade.In: Progress in Cholinergic Biology: Models of Cholinergic Synapses. I. Hanin and A. Goldberg, editors, pp. 323–357, Raven, New York

    Google Scholar 

  • Szoka, R., Papahadjopoulos D. 1978. A new procedure for preparation of liposomes with large internal aqueous space and high capture by reverse phase evaporation.Proc. Natl. Acad. Sci. USA 75:4194–4198

    Google Scholar 

  • Yee, A.S., Corley, D.E., McNamee, M.G. 1986. Thiol-group modification ofTorpedo californica acetylcholine receptor: Subunit localization and effects on function.Biochemistry 25:2110–2119

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departments of Biology and Chemistry, University of California, 95064, Santa Cruz, California

    Andrew L. Palma & Howard H. Wang

Authors
  1. Andrew L. Palma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Howard H. Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Palma, A.L., Wang, H.H. Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane. J. Membrain Biol. 122, 143–153 (1991). https://doi.org/10.1007/BF01872637

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01872637

Key Words

Search

Navigation