Abstract
Aim:
Studies of the α7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of α7-type AChRs1. The current work aims at obtaining and characterizing a cell line with high functional expression of the human α7 AChR.
Methods:
Ric-3 cDNA was incorporated into SHE-P1-hα7 cells expressing the α7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [125I]α-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent α-bungarotoxin, anti-α7 antibody, and GFP-α7 were performed on the new clone.
Results:
The human α7-type AChR was stably expressed in a new cell line, which we coined SHE-P1-hα7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [125I]αBTX saturable binding with an apparent KD of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-P1-hα7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-α7 antibodies. In contrast, chaperone-less SHE-P1-hα7 cells expressed only intracellular α7 AChRs and failed to produce detectable single-channel currents.
Conclusion:
The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal α7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells.
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References
Millar NS, Harkness PC . Assembly and trafficking of nicotinic acetylcholine receptors (Review). Mol Membr Biol 2008; 25: 279–92.
Lester HA, Dibas MI, Dahan DS, Leite JF, Dougherty DA . Cys-loop receptors: new twists and turns. Trends Neurosci 2004; 27: 329–36.
Role LW, Berg DK . Nicotinic receptors in the development and modulation of CNS synapses. Neuron 1996; 16: 1077–85.
Jones S, Sudweeks S, Yakel JL . Nicotinic receptors in the brain: correlating physiology with function. Trends Neurosci 1999; 22: 555–6.
Seguela P, Wadiche J, Neley-Miller K, Dani JA, Patrick JW . Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. J Neurosci 1993; 13: 596–604.
Gotti C, Clementi F . Neuronal nicotinic receptors: from structure to pathology. Prog Neurobiol 2004; 74: 363–96.
Puchacz E, Buisson B, Bertrand D, Lukas RJ . Functional expression of nicotinic acetylcholine receptors containing rat alpha 7 subunits in human SH-SY5Y neuroblastoma cells. FEBS Lett 1994; 354: 155–9.
Zhao L, Kuo YP, George AA, Peng JH, Purandare MS, Schroeder KM, et al. Functional properties of homomeric, human alpha 7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line. J Pharmacol Exp Ther 2003; 305: 1132–41.
Cooper ST, Millar NS . Host cell-specific folding of the neuronal nicotinic receptor alpha8 subunit. J Neurochem 1998; 70: 2585–93.
Cooper ST, Millar NS . Host cell-specific folding and assembly of the neuronal nicotinic acetylcholine receptor alpha7 subunit. J Neurochem 1997; 68: 2140–51.
Kassner PD, Berg DK . Differences in the fate of neuronal acetylcholine receptor protein expressed in neurons and stably transfected cells. J Neurobiol 1997; 33: 968–82.
Rangwala F, Drisdel RC, Rakhilin S, Ko E, Atluri P, Harkins AB, et al. Neuronal alpha-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors. J Neurosci 1997; 17: 8201–12.
Williams ME, Burton B, Urrutia A, Shcherbatko A, Chavez-Noriega LE, Cohen CJ, et al. Ric-3 promotes functional expression of the nicotinic acetylcholine receptor alpha7 subunit in mammalian cells. J Biol Chem 2005; 280: 1257–63.
Castillo M, Mulet J, Gutierrez LM, Ortiz JA, Cautelan F, Gerber S, et al. Dual role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors. J Biol Chem 2005; 280: 27062–8.
Drisdel RC, Manzana E, Green WN . The role of palmitoylation in functional expression of nicotinic alpha7 receptors. J Neurosci 2004; 24: 10502–10.
Lansdell SJ, Gee VJ, Harkness PC, Doward AI, Baker ER, Gibb AJ, et al. RIC-3 enhances functional expression of multiple nicotinic acetylcholine receptor subtypes in mammalian cells. Mol Pharmacol 2005; 68: 1431–8.
Lansdell SJ, Collins T, Yabe A, Gee VJ, Gibb AJ, Millar NS . Host-cell specific effects of the nicotinic acetylcholine receptor chaperone RIC-3 revealed by a comparison of human and Drosophila RIC-3 homologues. J Neurochem 2008; 105: 1573–81.
Palma E, Maggi L, Barabino B, Eusebi F, Ballivet M . Nicotinic acetylcholine receptors assembled from the alpha7 and beta3 subunits. J Biol Chem 1999; 274: 18335–40.
Fucile S, Palma E, Martinez-Torres A, Miledi R, Eusebi F . The single-channel properties of human acetylcholine alpha 7 receptors are altered by fusing alpha 7 to the green fluorescent protein. Proc Natl Acad Sci U S A 2002; 99: 3956–61.
Bouzat C, Bartos M, Corradi J, Sine SM . The interface between extracellular and transmembrane domains of homomeric Cys-loop receptors governs open-channel lifetime and rate of desensitization. J Neurosci 2008; 28: 7808–19.
Roncarati R, Seredenina T, Jow B, Jow F, Papini S, Kramer A, et al. Functional properties of alpha7 nicotinic acetylcholine receptors co-expressed with RIC-3 in a stable recombinant CHO-K1 cell line. Assay Drug Dev Technol 2008; 6: 181–93
Peng JH, Lucero L, Fryer J, Herl J, Leonard SS, Lukas RJ . Inducible, heterologous expression of human alpha7-nicotinic acetylcholine receptors in a native nicotinic receptor-null human clonal line. Brain Res 1999; 825: 172–9.
Cheng Y, Prusoff WH . Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 1973; 22: 3099–08.
Ross RA, Spengler BA, Biedler JL . Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J Natl Cancer Inst 1983; 71: 741–7.
Barrantes FJ . Cholesterol effects on nicotinic acetylcholine receptor. J Neurochem 2007; 103: 72–80.
Yamanaka K, Kigoshi S, Muramatsu I . Muscarinic receptor subtypes in bovine adrenal medulla. Biochem Pharmacol 1986; 35: 3151–7.
Couturier S, Bertrand D, Matter JM, Hernandez MC, Bertrand S, Millar N, et al. A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX. Neuron 1990; 5: 847–56.
Gerzanich V, Anand R, Lindstrom J . Homomers of alpha 8 and alpha 7 subunits of nicotinic receptors exhibit similar channel but contrasting binding site properties. Mol Pharmacol 1994; 45: 212–20.
Gotti C, Hanke W, Maury K, Moretti M, Ballivet M, Clementi F, et al. Pharmacology and biophysical properties of alpha 7 and alpha 7-alpha 8 alpha-bungarotoxin receptor subtypes immunopurified from the chick optic lobe. Eur J Neurosci 1994; 6: 1281–91.
Cooper ST, Millar NS . Host cell-specific folding of the neuronal nicotinic receptor alpha8 subunit. J Neurochem 1998; 70: 2585–93.
Green WN, Millar NS . Ion-channel assembly. Trends Neurosci 1995; 18: 280–7.
Baier CJ, Barrantes FJ . Sphingolipids are necessary for nicotinic acetylcholine receptor export in the early secretory pathway. J Neurochem 2007; 101: 1072–84.
Pediconi MF, Gallegos CE, De Los Santos EB, Barrantes FJ . Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor. Neuroscience 2004; 128: 239–49.
Roccamo AM, Pediconi MF, Aztiria E, Zanello L, Wolstenholme A, Barrantes FJ . Cells defective in sphingolipids biosynthesis express low amounts of muscle nicotinic acetylcholine receptor. Eur J Neurosci 1999; 11: 1615–23.
Kopta C, Steinbach JH . Comparison of mammalian adult and fetal nicotinic acetylcholine receptors stably expressed in fibroblasts. J Neurosci 1994; 14: 3922–33.
Gu Y, Franco A Jr, Gardner PD, Lansman JB, Forsayeth JR, Hall ZW . Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells. Neuron 1990; 5: 147–57.
Gopalakrishnan M, Buisson B, Touma E, Giordano T, Campbell JE, Hu IC, et al. Stable expression and pharmacological properties of the human alpha 7 nicotinic acetylcholine receptor. Eur J Pharmacol 1995; 290: 237–46.
Anand R, Peng X, Lindstrom J . Homomeric and native alpha 7 acetylcholine receptors exhibit remarkably similar but non-identical pharmacological properties, suggesting that the native receptor is a heteromeric protein complex. FEBS Lett 1993; 327: 241–6.
Peng X, Katz M, Gerzanich V, Anand R, Lindstrom J . Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes. Mol Pharmacol 1994; 45: 546–54.
Acknowledgements
Research described in this article was supported in part by PICT 5-20155 from the Ministry of Science and Technology; PIP No 6367 from the Argentinian Scientific Research Council (CONICET); Philip Morris USA Inc and Philip Morris International; and PGI No 24/B135 from Universidad Nacional del Sur, Argentina, to Francisco J BARRANTES. Thanks are due to Prof Manuel CRIADO for providing the chaperone protein Ric-3, the α7 AChR and its GFP derivative.
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Vallés, A., Roccamo, A. & Barrantes, F. Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells. Acta Pharmacol Sin 30, 818–827 (2009). https://doi.org/10.1038/aps.2009.54
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DOI: https://doi.org/10.1038/aps.2009.54
