Liposomes pp 291-318 | Cite as

Interaction of Lipids and Ligands with Nicotinic Acetylcholine Receptor Vesicles Assessed by Electron Paramagnetic Resonance Spectroscopy

  • Hugo Rubén AriasEmail author
Part of the Methods in Molecular Biology™ book series (MIMB, volume 606)


Electron paramagnetic resonance (EPR) spectroscopy is a powerful technique that permits the study of membrane-embedded proteins in its lipid environment by assessing the interaction of spin labels with the protein in its natural environment (i.e., native membranes) or in reconstituted systems prepared with exogenous lipid species. Nicotinic acetylcholine receptors (AChRs) contain a large surface in intimate contact with the lipid membrane. AChRs, members of the Cys-loop receptor superfamily, have essential functional roles in the nervous system and its malfunctioning has been considered as the origin of several neurological diseases including Alzheimer’s disease, drug addiction, depression, and schizophrenia. In this regard, these receptors have been extensively studied as therapeutic targets for the action of several drugs. The majority of the marketed medications bind to the neurotransmitter sites, the so-called agonists. However, several drugs, some of them still in clinical trials, interact with non-competitive antagonist (NCA) binding sites. A potential location for these binding sites is the proper ion channel, blocking ion flux and thus, inhibiting membrane depolarization. However, several NCAs also bind to the lipid-protein interface, modulating the AChR functional properties. The best known examples of these NCAs are local and general anesthetics. Several endogenous molecules such as free fatty acids and neurosteroids also bind to the lipid-protein interface, probably mediating important physiological functions. Phospholipids, natural components of lipid membranes interacting with the AChR, are also essential to maintain the structural and functional properties of the AChR. EPR studies showed that local anesthetics bind to the lipid-protein interface by essentially the same dynamic mechanisms found in lipids, and that local and general anesthetics preferably decrease the phospholipid but not the fatty acid interactions with the AChR. This is consistent with the existence of annular and non-annular lipid domains on the AChR.

Key words

Nicotinic acetylcholine receptors Lipid-protein interface Native and reconstituted vesicles Electron paramagnetic resonance spectroscopy Spin labels Local anesthetics General anesthetics Fatty acids Phospholipids Steroids Gangliosides 



This research was supported by grants from the Science Foundation Arizona and Stardust Foundation and from the College of Pharmacy, Midwestern University. The author thanks the comments by Dr. Blanton (Texas Tech University Health Sciences Center, Lubbok, TX, USA) on AChR purification procedures.


  1. 1.
    Arias HR (2006) Ligand-gated ion channel receptor superfamilies. In: Arias HR (ed) Biological and biophysical aspects of ligand-gated ion channel receptor superfamilies. Research Signpost, Kerala, India, pp 1-25Google Scholar
  2. 2.
    Ortells MO, Lunt GG (1995) Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends Neurosci 18:121-127CrossRefPubMedGoogle Scholar
  3. 3.
    Changeux J-P, Taly A (2008) Nicotinic receptors, allosteric proteins and medicine. Trends Mol Med 14:93-102PubMedGoogle Scholar
  4. 4.
    Albuquerque EX, Pereira EF, Alkondon M, Rogers SW (2009) Mammalian nicotinic acetylcholine receptors: from structure to function. Physiol Rev 89:73-120CrossRefPubMedGoogle Scholar
  5. 5.
    Arias HR, Richards V, Ng D, Ghafoori ME, Le V, Mousa S (2009) Role of non-neuronal nicotinic acetylcholine receptors in angiogenesis. Int J Biochem Cell Biol 41:1441-1451Google Scholar
  6. 6.
    Arias HR (2001) Thermodynamics of nicotinic receptor interactions. In: Raffa RB (ed) Drug-receptor thermodynamics: introduction and applications. Wiley, USA, pp 293-358Google Scholar
  7. 7.
    Miyazawa A, Fujiyoshi Y, Unwin N (2003) Structure and gating mechanism of the acetylcholine receptor pore. Nature 423:949-955CrossRefPubMedGoogle Scholar
  8. 8.
    Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J Mol Biol 346:967-989CrossRefPubMedGoogle Scholar
  9. 9.
    Arias HR, Bouzat CB (2006) Modulation of nicotinic acetylcholine receptors by noncompetitive antagonists. In: Arias HR (ed) Biological and biophysical aspects of ligand-gated ion channel receptor superfamilies. Research Signpost, Kerala, India, pp 61-107Google Scholar
  10. 10.
    Arias HR, Bhumireddy P, Bouzat C (2006) Molecular mechanisms and binding site locations for noncompetitive antagonists of nicotinic acetylcholine receptors. Int J Biochem Cell Biol 38:1254-1276CrossRefPubMedGoogle Scholar
  11. 11.
    Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van der Oost J, Smit AB, Sixma TS (2001) Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411:269-276CrossRefPubMedGoogle Scholar
  12. 12.
    Swope SL, Moss SJ, Blackstone CD, Huganir RL (1992) Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity. FASEB J 6:2514-2523PubMedGoogle Scholar
  13. 13.
    Blanton MP, Cohen JB (1994) Identifying the lipid-protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications. Biochemistry 33:2859-2872CrossRefPubMedGoogle Scholar
  14. 14.
    Hamouda AK, Sanghvi M, Chiara DC, Cohen JB, Blanton MP (2007) Identifying the lipid-protein interface of the α4β2 neuronal nicotinic acetylcholine receptor: hydrophobic photolabeling studies with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Biochemistry 46:13837-13846CrossRefPubMedGoogle Scholar
  15. 15.
    Hamouda AK, Chiara DC, Sauls D, Cohen JB, Blanton MP (2006) Cholesterol interacts with transmembrane αhelices M1, M3, and M4 of the Torpedo nicotinic acetylcholine receptor: Photolabeling studies using [3H]azidocholesterol. Biochemistry 45:976-986CrossRefPubMedGoogle Scholar
  16. 16.
    Jones OT, McNamee MG (1988) Annular and nonannular binding sites for cholesterol associated with the nicotinic acetylcholine receptor. Biochemistry 27:2364-2374CrossRefPubMedGoogle Scholar
  17. 17.
    Brannigan G, Hénin J, Law R, Eckenhoff R, Klein ML (2007) Embedded cholesterol in the nicotinic acetylcholine receptor. Proc Natl Acad Sci USA 105:14418-14423CrossRefGoogle Scholar
  18. 18.
    Arias HR (1998) Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholine receptor. Biochim Biophys Acta 1376:173-220PubMedGoogle Scholar
  19. 19.
    Arias HR (1999) Role of local anesthetics on both cholinergic and serotonergic ionotropic receptors. Neurosci Biobehav Rev 23:817-843CrossRefPubMedGoogle Scholar
  20. 20.
    Arias HR, Blanton MP (2002) Molecular and physicochemical aspects of local anesthetics acting on nicotinic acetylcholine receptor-containing membranes. Mini Rev Med Chem 2:385-410CrossRefPubMedGoogle Scholar
  21. 21.
    Arias HR, Alonso-Romanowski S, Disalvo EA, Barrantes FJ (1994) Interaction of merocyanine 540 with nicotinic acetylcholine receptor membranes from Discopyge tschudii electric organ. Biochim Biophys Acta 1190:393-401CrossRefPubMedGoogle Scholar
  22. 22.
    Pedersen SE, Dreyer EB, Cohen JB (1986) Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit. J Biol Chem 261:13735-13743PubMedGoogle Scholar
  23. 23.
    Lowry OH, Rosebrough NJ, Farr L, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275PubMedGoogle Scholar
  24. 24.
    Arias HR, Bhumireddy P, Spitzmaul G, Trudell JR, Bouzat C (2006) Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors. Biochemistry 45:2014-2026CrossRefPubMedGoogle Scholar
  25. 25.
    Schmidt J, Raftery MA (1973) A simple assay for the study of solubilized acetylcholine receptors. Anal Biochem 52:349-354CrossRefPubMedGoogle Scholar
  26. 26.
    Sanghvi M, Hamouda AK, Jozwiak K, Trudell JR, Blanton MP, Arias HR (2008) Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-Azidoimipramine photolabeling and molecular dynamics studies. Biochem Biophys Acta 1778:2690-2699CrossRefPubMedGoogle Scholar
  27. 27.
    Keana JFW, Shimizu M, Jernstedt KK (1986) A short, flexible route to symmetrically and unsymetrically substituted diphosphatidylglycerol (cardiolipins). J Org Chem 51:2297-2299CrossRefGoogle Scholar
  28. 28.
    Schwarzmann G, Sandhoff K (1987) Lysogangliosides: synthesis and use in preparing labeled gangliosides. Methods Enzymol 138:319-341CrossRefPubMedGoogle Scholar
  29. 29.
    Hideg K, Lex L, Hankovszky HO, Tigyi J (1979) Synthesis of spin-labelled procaine and its derivatives. Synth Commun 9:781-788CrossRefGoogle Scholar
  30. 30.
    Mantipragada SB, Horváth LI, Arias HR, Schwarzmann G, Sandhoff K, Barrantes FJ, Marsh D (2003) Lipid-protein interactions and the effect of local anesthetics in acetylcholine receptor-rich membranes from Torpedo marmorata electric organ. Biochemistry 42:9167-9175CrossRefPubMedGoogle Scholar
  31. 31.
    Horváth LI, Arias HR, Hankovszky HO, Hideg K, Barrantes FJ, Marsh D (1990) Association of spin-labeled anesthetics at the hydrophobic surface of acetylcholine receptor in native membranes from Torpedo marmorata. Biochemistry 29:8707-8713CrossRefPubMedGoogle Scholar
  32. 32.
    Marsh D (2008) Electron spin resonance in membrane research: protein-lipid interactions. Methods 46:83-96CrossRefPubMedGoogle Scholar
  33. 33.
    Marsh D (2008) Protein modulation of lipids, and vice-versa, in membranes. Biochim Biophys Acta 1778:1545-1575CrossRefPubMedGoogle Scholar
  34. 34.
    Marsh D, Páli T (2004) The protein-lipid interface: perspectives from magnetic resonance and crystal structures. Biochim Biophys Acta 1666:118-141PubMedGoogle Scholar
  35. 35.
    Arias HR, Sankaram MB, Marsh D, Barrantes FJ (1990) Effect of local anaesthetics on steroid-nicotinic acetylcholine receptor interactions in native membranes of Torpedo marmorata electric organ. Biochim Biophys Acta 1027:287-294CrossRefPubMedGoogle Scholar
  36. 36.
    Earnest JP, Wang HH, McNamee MG (1984) Multiple binding sites for local anesthetics on reconstituted acetylcholine receptor membranes. Biochem Biophys Res Commun 123:862-866CrossRefPubMedGoogle Scholar
  37. 37.
    Earnest JP, Limbacher HP, McNamee MG, Wang HH (1986) Binding of local anesthetics to reconstituted acetylcholine receptors: the effect of protein surface potential. Biochemistry 25:5809-5818CrossRefPubMedGoogle Scholar
  38. 38.
    Ellena JF, Blazing MA, McNamee MG (1983) Lipid-protein interactions in reconstituted membranes containing acetylcholine receptor. Biochemistry 22:5523-5535CrossRefPubMedGoogle Scholar
  39. 39.
    Raines DE, Miller KW (1993) The role of charge in lipid selectivity for the nicotinic acetylcholine receptor. Biophys J 64:632-641CrossRefPubMedGoogle Scholar
  40. 40.
    Rotstein NP, Arias HR, Barrantes FJ, Aveldaño MI (1987) Composition of lipids in elasmobranch electric organ and acetylcholine receptor membranes. J Neurochem 49:1333-1340CrossRefPubMedGoogle Scholar
  41. 41.
    Abadji VC, Raines DE, Dalton LA, Miller KW (1994) Lipid-protein interactions and protein dynamics in vesicles containing the nicotinic acetylcholine receptor: a study with ethanol. Biochim Biophys Acta 1194:25-34CrossRefPubMedGoogle Scholar
  42. 42.
    Unwin N (1995) Acetylcholine receptor channel imaged in the open state. Nature 373:37-43CrossRefPubMedGoogle Scholar
  43. 43.
    Jones OT, Eubanks JH, Earnest JP, McNamee MG (1988) A minimum number of of lipids are required to support the functional properties of the nicotinic acetylcholine receptor. Biochemistry 27:3733-3742CrossRefPubMedGoogle Scholar
  44. 44.
    McCarthy MP, Moore MA (1992) Effects of lipids and detergents on the conformation of the nicotinic acetylcholine receptor from Torpedo californica. J Biol Chem 267:7655-7663PubMedGoogle Scholar
  45. 45.
    Hamouda AK, Chiara DC, Blanton MP, Cohen JB (2008) Probing the structure of the affinity-purified and lipid-reconstituted Torpedo nicotinic acetylcholine receptor. Biochemistry 47:12787-12794CrossRefPubMedGoogle Scholar
  46. 46.
    Fong TM, McNamee MG (1986) Correlation between acetylcholine receptor function and structural properties of membranes. Biochemistry 25:830-840CrossRefPubMedGoogle Scholar
  47. 47.
    Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497-509PubMedGoogle Scholar
  48. 48.
    Dreger M, Krauss M, Herrmann A, Hucho F (1997) Interactions of the nicotinic acetylcholine receptor transmembrane segments with the lipid bilayer in native receptor-rich membranes. Biochemistry 36:839-847CrossRefPubMedGoogle Scholar
  49. 49.
    Fraser DM, Louro SRW, Horváth LI, Miller KW, Watts A (1990) A study of the effect of general anesthetics on lipid-protein interactions in acetylcholine receptor enriched membranes from Torpedo nobiliana using nitroxide spin-labels. Biochemistry 29:2664-2669CrossRefPubMedGoogle Scholar
  50. 50.
    Rousselet A, Devaux PF, Wirtz KW (1979) Free fatty acids and esters can be immobilized by receptor rich membranes from Torpedo marmorata but not phospholipids acyl chains. Biochem Biophys Res Commun 90:871-877CrossRefPubMedGoogle Scholar
  51. 51.
    Raines DE, Wu G, Dalton LA, Miller KW (1995) Elestron spin resonance studies of acyl chain motion in reconstituted nictonic acetylcholine receptor membranes. Biophys J 69:498-505CrossRefPubMedGoogle Scholar
  52. 52.
    Marsh D, Watts A, Barrantes FJ (1981) Phospholipid chain immobilization and steroid rotational immobilization in acetylcholine receptor-rich membranes from Torpedo marmorata. Biochim Biophys Acta 645:97-101CrossRefPubMedGoogle Scholar
  53. 53.
    Seto T, Firestone LL (2000) Effects of normal alcohols and isoflurane on lipid headgroup dynamics in nicotinic acetylcholine receptor-rich lipid vesicles. Biochim Biophys Acta 1509:111-122CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Pharmaceutical Sciences, College of PharmacyMidwestern UniversityGlendaleUSA

Personalised recommendations