The Journal of Membrane Biology

, Volume 249, Issue 4, pp 539–549 | Cite as

Heterogeneous Inhibition in Macroscopic Current Responses of Four Nicotinic Acetylcholine Receptor Subtypes by Cholesterol Enrichment

  • Carlos A. Báez-PagánEmail author
  • Natalie del Hoyo-Rivera
  • Orestes Quesada
  • José David Otero-Cruz
  • José A. Lasalde-DominicciEmail author


The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. In addition, the nAChR is also found in the electric organs of electric rays (e.g., the genus Torpedo). Cholesterol, which is a key lipid for maintaining the correct functionality of membrane proteins, has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs expressed in a model membrane with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P ratio in Xenopus laevis oocytes expressing the neuronal α7, α4β2, muscle-type, and Torpedo californica nAChRs in their macroscopic current responses. Using the two-electrode voltage clamp technique, it was found that the neuronal α7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle-type nAChR. The peak current versus C/P profiles during enrichment display different behaviors; α7 and Torpedo nAChRs display a hyperbolic decay with two clear components, whereas muscle-type and α4β2 nAChRs display simple monophasic decays with different slopes. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration produces a remarkable reduction in the macroscopic current responses of the neuronal α7 and Torpedo nAChRs functionality, whereas the muscle nAChR appears to be the most resistant to cholesterol inhibition among all four nAChR subtypes. Overall, the present study demonstrates differential profiles for cholesterol inhibition among the different types of nAChR to physiological cholesterol increments in the plasmatic membrane. This is the first study to report a cross-correlation analysis of cholesterol sensitivity among different nAChR subtypes in a model membrane.


Cholesterol Nicotinic acetylcholine receptor Ion channels Regulation Membrane proteins 



Nicotinic acetylcholine receptor


Wild type







This research was supported by the National Institutes of Health NIGMS grants 1R01GM098343 (JALD) and 1P20GM103642 (J.R. and JALD). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Natalie del Hoyo-Rivera was supported by the UPR-RP MARC Program (Grant Number: 5T34GM07821) (OQ) and by the NIH-MBRS Research Initiative for Scientific Enhancement Grant R25GM61151 (OQ).


  1. Abi-Char J, Maguy A, Coulombe A et al (2007) Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes. J Physiol 582:1205–1217. doi: 10.1113/jphysiol.2007.134809 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Addona GH, Sandermann H, Kloczewiak MA et al (1998) Where does cholesterol act during activation of the nicotinic acetylcholine receptor? Biochim Biophys Acta 1370:299–309CrossRefPubMedGoogle Scholar
  3. Antollini SS, Barrantes FJ (1998) Disclosure of discrete sites for phospholipid and sterols at the protein-lipid interface in native acetylcholine receptor-rich membrane. Biochemistry (Mosc) 37:16653–16662. doi: 10.1021/bi9808215 CrossRefGoogle Scholar
  4. Baenziger JE, Morris ML, Darsaut TE, Ryan SE (2000) Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor. J Biol Chem 275:777–784CrossRefPubMedGoogle Scholar
  5. Báez-Pagán CA, Martínez-Ortiz Y, Otero-Cruz JD et al (2008) Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor’s activatable pool: implications in the pathogenesis of a novel congenital myasthenic syndrome. Channels Austin Tex 2:180–190CrossRefGoogle Scholar
  6. Barbuti A, Gravante B, Riolfo M et al (2004) Localization of pacemaker channels in lipid rafts regulates channel kinetics. Circ Res 94:1325–1331. doi: 10.1161/01.RES.0000127621.54132.AE CrossRefPubMedGoogle Scholar
  7. Barrantes FJ (2002) Lipid matters: nicotinic acetylcholine receptor-lipid interactions (Review). Mol Membr Biol 19:277–284. doi: 10.1080/09687680210166226 CrossRefPubMedGoogle Scholar
  8. Barrantes FJ (2004) Structural basis for lipid modulation of nicotinic acetylcholine receptor function. Brain Res Brain Res Rev 47:71–95. doi: 10.1016/j.brainresrev.2004.06.008 CrossRefPubMedGoogle Scholar
  9. Barrantes FJ, Borroni V, Vallés S (2010) Neuronal nicotinic acetylcholine receptor-cholesterol crosstalk in Alzheimer’s disease. FEBS Lett 584:1856–1863. doi: 10.1016/j.febslet.2009.11.036 CrossRefPubMedGoogle Scholar
  10. Brannigan G, Hénin J, Law R et al (2008) Embedded cholesterol in the nicotinic acetylcholine receptor. Proc Natl Acad Sci USA 105:14418–14423. doi: 10.1073/pnas.0803029105 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Brusés JL, Chauvet N, Rutishauser U (2001) Membrane lipid rafts are necessary for the maintenance of the (alpha)7 nicotinic acetylcholine receptor in somatic spines of ciliary neurons. J Neurosci Off J Soc Neurosci 21:504–512Google Scholar
  12. Burger K, Gimpl G, Fahrenholz F (2000) Regulation of receptor function by cholesterol. Cell Mol Life Sci CMLS 57:1577–1592CrossRefPubMedGoogle Scholar
  13. Cockcroft VB, Osguthorpe DJ, Barnard EA et al (1990) Ligand-gated ion channels. Homology and diversity. Mol Neurobiol 4:129–169. doi: 10.1007/BF02780338 CrossRefPubMedGoogle Scholar
  14. Cohen BM, Zubenko GS (1985) Aging and the biophysical properties of cell membranes. Life Sci 37:1403–1409CrossRefPubMedGoogle Scholar
  15. Cooper E, Couturier S, Ballivet M (1991) Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature 350:235–238. doi: 10.1038/350235a0 CrossRefPubMedGoogle Scholar
  16. Corbin J, Wang HH, Blanton MP (1998) Identifying the cholesterol binding domain in the nicotinic acetylcholine receptor with [125I]azido-cholesterol. Biochim Biophys Acta 1414:65–74CrossRefPubMedGoogle Scholar
  17. Corringer PJ, Le Novère N, Changeux JP (2000) Nicotinic receptors at the amino acid level. Annu Rev Pharmacol Toxicol 40:431–458. doi: 10.1146/annurev.pharmtox.40.1.431 CrossRefPubMedGoogle Scholar
  18. Dalziel AW, Rollins ES, McNamee MG (1980) The effect of cholesterol on agonist-induced flux in reconstituted acetylcholine receptor vesicles. FEBS Lett 122:193–196CrossRefPubMedGoogle Scholar
  19. Demel RA, Bruckdorfer KR, van Deenen LL (1972) The effect of sterol structure on the permeability of lipomes to glucose, glycerol and Rb +. Biochim Biophys Acta 255:321–330CrossRefPubMedGoogle Scholar
  20. Fernandez-Ballester G, Castresana J, Fernandez AM et al (1994) Role of cholesterol as a structural and functional effector of the nicotinic acetylcholine receptor. Biochem Soc Trans 22:776–780CrossRefPubMedGoogle Scholar
  21. Fong TM, McNamee MG (1986) Correlation between acetylcholine receptor function and structural properties of membranes. Biochem (Mosc) 25:830–840CrossRefGoogle Scholar
  22. Gally HU, Seelig A, Seelig J (1976) Cholesterol-induced rod-like motion of fatty acyl chains in lipid bilayers a deuterium magnetic resonance study. Hoppe-Seylers Z Für Physiol Chem 357:1447–1450Google Scholar
  23. Galzi JL, Revah F, Bessis A, Changeux JP (1991) Functional architecture of the nicotinic acetylcholine receptor: from electric organ to brain. Annu Rev Pharmacol Toxicol 31:37–72. doi: 10.1146/ CrossRefPubMedGoogle Scholar
  24. Gonzalez-Ros JM, Llanillo M, Paraschos A, Martinez-Carrion M (1982) Lipid environment of acetylcholine receptor from Torpedo californica. Biochem (Mosc) 21:3467–3474CrossRefGoogle Scholar
  25. Grajales-Reyes GE, Báez-Pagán CA, Zhu H et al (2013) Transgenic mouse model reveals an unsuspected role of the acetylcholine receptor in statin-induced neuromuscular adverse drug reactions. Pharmacogenomics J 13:362–368. doi: 10.1038/tpj.2012.21 CrossRefPubMedGoogle Scholar
  26. Guzmán GR, Ortiz-Acevedo A, Ricardo A et al (2006) The polarity of lipid-exposed residues contributes to the functional differences between Torpedo and muscle-type nicotinic receptors. J Membr Biol 214:131–138. doi: 10.1007/s00232-006-0051-0 CrossRefPubMedGoogle Scholar
  27. Hamouda AK, Sanghvi M, Sauls D et al (2006) Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor. Biochem (Mosc) 45:4327–4337. doi: 10.1021/bi052281z CrossRefGoogle Scholar
  28. Ikonen E, Heino S, Lusa S (2004) Caveolins and membrane cholesterol. Biochem Soc Trans 32:121–123. doi: 10.1042/bst0320121 CrossRefPubMedGoogle Scholar
  29. Itier V, Bertrand D (2001) Neuronal nicotinic receptors: from protein structure to function. FEBS Lett 504:118–125CrossRefPubMedGoogle Scholar
  30. Jensen MØ, Mouritsen OG (2004) Lipids do influence protein function-the hydrophobic matching hypothesis revisited. Biochim Biophys Acta 1666:205–226. doi: 10.1016/j.bbamem.2004.06.009 CrossRefPubMedGoogle Scholar
  31. Jones OT, McNamee MG (1988) Annular and nonannular binding sites for cholesterol associated with the nicotinic acetylcholine receptor. Biochem (Mosc) 27:2364–2374CrossRefGoogle Scholar
  32. Kalamida D, Poulas K, Avramopoulou V et al (2007) Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity. FEBS J 274:3799–3845. doi: 10.1111/j.1742-4658.2007.05935.x CrossRefPubMedGoogle Scholar
  33. Karlin A (2002) Emerging structure of the nicotinic acetylcholine receptors. Nat Rev Neurosci 3:102–114. doi: 10.1038/nrn731 CrossRefPubMedGoogle Scholar
  34. Krainev AG, Ferrington DA, Williams TD et al (1995) Adaptive changes in lipid composition of skeletal sarcoplasmic reticulum membranes associated with aging. Biochim Biophys Acta 1235:406–418CrossRefPubMedGoogle Scholar
  35. Lasalde JA, Colom A, Resto E, Zuazaga C (1995) Heterogeneous distribution of acetylcholine receptors in chick myocytes induced by cholesterol enrichment. Biochim Biophys Acta 1235:361–368CrossRefPubMedGoogle Scholar
  36. Le Novère N, Corringer P-J, Changeux J-P (2002) The diversity of subunit composition in nAChRs: evolutionary origins, physiologic and pharmacologic consequences. J Neurobiol 53:447–456. doi: 10.1002/neu.10153 CrossRefPubMedGoogle Scholar
  37. Lechleiter J, Wells M, Gruener R (1986) Halothane-induced changes in acetylcholine receptor channel kinetics are attenuated by cholesterol. Biochim Biophys Acta 856:640–645CrossRefPubMedGoogle Scholar
  38. Lee AG (2003) Lipid-protein interactions in biological membranes: a structural perspective. Biochim Biophys Acta 1612:1–40CrossRefPubMedGoogle Scholar
  39. Lee YH, Li L, Lasalde J et al (1994) Mutations in the M4 domain of Torpedo californica acetylcholine receptor dramatically alter ion channel function. Biophys J 66:646–653CrossRefPubMedPubMedCentralGoogle Scholar
  40. Leibel WS, Firestone LL, Legler DC et al (1987) Two pools of cholesterol in acetylcholine receptor-rich membranes from Torpedo. Biochim Biophys Acta 897:249–260CrossRefPubMedGoogle Scholar
  41. Marchand S, Devillers-Thiéry A, Pons S et al (2002) Rapsyn escorts the nicotinic acetylcholine receptor along the exocytic pathway via association with lipid rafts. J Neurosci Off J Soc Neurosci 22:8891–8901Google Scholar
  42. McConnell HM, Radhakrishnan A (2003) Condensed complexes of cholesterol and phospholipids. Biochim Biophys Acta 1610:159–173CrossRefPubMedGoogle Scholar
  43. Middlemas DS, Raftery MA (1987) Identification of subunits of acetylcholine receptor that interact with a cholesterol photoaffinity probe. Biochem (Mosc) 26:1219–1223CrossRefGoogle Scholar
  44. Narayanaswami V, McNamee MG (1993) Protein-lipid interactions and Torpedo californica nicotinic acetylcholine receptor function. 2. Membrane fluidity and ligand-mediated alteration in the accessibility of gamma subunit cysteine residues to cholesterol. Biochem (Mosc) 32:12420–12427CrossRefGoogle Scholar
  45. O’Neill MJ, Murray TK, Lakics V et al (2002) The role of neuronal nicotinic acetylcholine receptors in acute and chronic neurodegeneration. Curr Drug Targets CNS Neurol Disord 1:399–411CrossRefPubMedGoogle Scholar
  46. Ohlsson RI, Lane CD, Guengerich FP (1981) Synthesis and insertion, both in vivo and in vitro, of rat-liver cytochrome P-450 and epoxide hydratase into Xenopus laevis membranes. Eur J Biochem FEBS 115:367–373CrossRefGoogle Scholar
  47. Oshikawa J, Toya Y, Fujita T et al (2003) Nicotinic acetylcholine receptor alpha 7 regulates cAMP signal within lipid rafts. Am J Physiol Cell Physiol 285:C567–C574. doi: 10.1152/ajpcell.00422.2002 CrossRefPubMedGoogle Scholar
  48. Paterson D, Nordberg A (2000) Neuronal nicotinic receptors in the human brain. Prog Neurobiol 61:75–111CrossRefPubMedGoogle Scholar
  49. Pike LJ (2006) Rafts defined: a report on the Keystone Symposium on Lipid Rafts and Cell Function. J Lipid Res 47:1597–1598. doi: 10.1194/jlr.E600002-JLR200 CrossRefPubMedGoogle Scholar
  50. Radhakrishnan A, McConnell HM (1999) Condensed complexes of cholesterol and phospholipids. Biophys J 77:1507–1517. doi: 10.1016/S0006-3495(99)76998-5 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Roher AE, Kuo YM, Kokjohn KM et al (1999) Amyloid and lipids in the pathology of Alzheimer disease. Amyloid Int J Exp Clin Investig Off J Int Soc Amyloidosis 6:136–145CrossRefGoogle Scholar
  52. Santiago J, Guzmàn GR, Rojas LV et al (2001) Probing the effects of membrane cholesterol in the Torpedo californica acetylcholine receptor and the novel lipid-exposed mutation alpha C418W in Xenopus oocytes. J Biol Chem 276:46523–46532. doi: 10.1074/jbc.M104563200 CrossRefPubMedGoogle Scholar
  53. Schiebler W, Hucho F (1978) Membranes rich in acetylcholine receptor: characterization and reconstitution to excitable membranes from exogenous lipids. Eur J Biochem FEBS 85:55–63CrossRefGoogle Scholar
  54. Sumikawa K, Houghton M, Emtage JS et al (1981) Active multi-subunit ACh receptor assembled by translation of heterologous mRNA in Xenopus oocytes. Nature 292:862–864CrossRefPubMedGoogle Scholar
  55. Sunshine C, McNamee MG (1992) Lipid modulation of nicotinic acetylcholine receptor function: the role of neutral and negatively charged lipids. Biochim Biophys Acta 1108:240–246CrossRefPubMedGoogle Scholar
  56. Tamamizu S, Lee Y, Hung B et al (1999) Alteration in ion channel function of mouse nicotinic acetylcholine receptor by mutations in the M4 transmembrane domain. J Membr Biol 170:157–164CrossRefPubMedGoogle Scholar
  57. Tamamizu S, Guzmán GR, Santiago J et al (2000) Functional effects of periodic tryptophan substitutions in the alpha M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor. Biochem (Mosc) 39:4666–4673CrossRefGoogle Scholar
  58. Wood WG, Schroeder F, Igbavboa U et al (2002) Brain membrane cholesterol domains, aging and amyloid beta-peptides. Neurobiol Aging 23:685–694CrossRefPubMedGoogle Scholar
  59. Yeagle PL (1985) Cholesterol and the cell membrane. Biochim Biophys Acta 822:267–287CrossRefPubMedGoogle Scholar
  60. Zhu D, Xiong WC, Mei L (2006) Lipid rafts serve as a signaling platform for nicotinic acetylcholine receptor clustering. J Neurosci Off J Soc Neurosci 26:4841–4851. doi: 10.1523/JNEUROSCI.2807-05.2006 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Carlos A. Báez-Pagán
    • 1
    • 2
    • 3
    Email author
  • Natalie del Hoyo-Rivera
    • 4
  • Orestes Quesada
    • 3
  • José David Otero-Cruz
    • 1
  • José A. Lasalde-Dominicci
    • 1
    • 2
    Email author
  1. 1.Department of BiologyUniversity of Puerto RicoSan JuanUSA
  2. 2.Molecular Sciences Research CenterUniversity of Puerto RicoSan JuanUSA
  3. 3.Department of Physical SciencesUniversity of Puerto RicoSan JuanUSA
  4. 4.School of Pharmacy, Medical Sciences CampusUniversity of Puerto RicoSan JuanUSA

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