Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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A Role for Leu247 Residue within Transmembrane Domain 2 in Ginsenoside-Mediated α7 Nicotinic Acetylcholine Receptor Regulation

  • Lee, Byung-Hwan (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Choi, Sun-Hye (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Pyo, Mi Kyung (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Shin, Tae-Joon (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Hwang, Sung-Hee (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Kim, Bo-Ra (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Lee, Sang-MoK (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Lee, Jun-Ho (Department of Physiology, College of Oriental Medicine, Kyung-Hee University) ;
  • Lee, Joon-Hee (Department of Physical Therapy, Daebul University) ;
  • Lee, Hui Sun (Department of Physiology and Research Institute for Biomacromolecules, University of Ulsan, College of Medicine) ;
  • Choe, Han (Department of Physiology and Research Institute for Biomacromolecules, University of Ulsan, College of Medicine) ;
  • Han, Kyou-Hoon (Protein Analysis and Design Section, Molecular Cancer Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Hyoung-Chun (Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University) ;
  • Rhim, Hyewhon (Life Science Division, Korea Institute of Science and Technology) ;
  • Yong, Joon-Hwan (Department of Occupational Therapy, Dongnam Health College) ;
  • Nah, Seung-Yeol (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University)
  • Received : 2009.02.20
  • Accepted : 2009.03.19
  • Published : 2009.05.31
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Abstract

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not ${\alpha}7$, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of ${\alpha}7$ nAChR induces alterations in channel gating properties and converts ${\alpha}7$ nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside $Rg_3$ ($Rg_3$) activity against the ${\alpha}7$ nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to $Rg_3$. We further characterized $Rg_3$ regulation of L247T receptors. We found that $Rg_3$ inhibition of mutant ${\alpha}7$ nAChR channel currents was reversible and concentration-dependent. $Rg_3$ inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between $Rg_3$ and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in $Rg_3$ interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that $Rg_3$ forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas $Rg_3$ localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for $Rg_3$ at the channel pore.

Keywords

Acknowledgement

Supported by : Korea Science and Engineering Foundation, Asan Institute for Life Sciences, Ministry of Education, Science and Technology

References

  1. Arias, H.R. (1996). Luminal and non-luminal non-competitive inhibitor binding sites on the nicotinic acetylcholine receptor. Mol. Membr. Biol. 13, 1-17 https://doi.org/10.3109/09687689609160569
  2. Bertrand, D., Devillers-Thiery, A., Revah, F., Galzi, J.L., Hussy, N., Mulle, C., Bertrand, S., Ballivet, M., and Changeux, J.P. (1992). Unconventional pharmacology of a neuronal nicotinic receptor mutated in the channel domain. Proc. Natl. Acad. Sci. USA 89, 1261-1265 https://doi.org/10.1073/pnas.89.4.1261
  3. Boulter, J., Evans, K., Goldman, D., Martin, G., Treco, D., Heinemann, S., and Patrick, J. (1986). Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor alphasubunit. Nature 319, 368-374 https://doi.org/10.1038/319368a0
  4. Boulter, J., Connolly, J., Deneris, E., Goldman, D., Heinemann, S., and Patrick, J. (1987). Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family. Proc. Natl. Acad. Sci. USA 84, 7763-7767 https://doi.org/10.1073/pnas.84.21.7763
  5. Broide, R.S., Salas, R., Ji, D., Paylor, R., Patrick, J.W., Dani, J.A., and De Biasi, M. (2002). Increased sensitivity to nicotineinduced seizures in mice expressing the L250T alpha7 nicotinic acetylcholine receptor mutation. Mol. Pharmacol. 61, 695-705 https://doi.org/10.1124/mol.61.3.695
  6. Bryson, K., McGuffin, L.J., Marsden, R.L., Ward, J.J., Sodhi, J.S., and Jones, D.T. (2005). Protein structure prediction servers at University College London. Nucleic Acids Res. 33, W36-38 https://doi.org/10.1093/nar/gki410
  7. Castro, N.G., and Albuquerque, E.X. (1995). alpha-Bungarotoxinsensitive hippocampal nicotinic receptor channel has a high calcium permeability. Biophys. J. 68, 516-24 https://doi.org/10.1016/S0006-3495(95)80213-4
  8. Changeux, J.P., and Edelstein, S.J. (2001). Allosteric mechanisms in normal and pathological nicotinic acetylcholine receptors. Curr. Opin. Neurobiol. 11, 369-377 https://doi.org/10.1016/S0959-4388(00)00221-X
  9. Chini, B., Raimond, E., Elgoyhen, A.B., Moralli, D., Balzaretti M., and Heinemann, M. (1994). Molecular cloning and chromosomal localization of the human alpha 7-nicotinic receptor subunit gene (CHRNA7). Genomics 19, 379-381 https://doi.org/10.1006/geno.1994.1075
  10. Choi, S., Jung, S.Y., Lee, J.H., Sala, F., Criado, M., Mulet, J., Valor, L.M., Sala, S., Engel, A.G., and Nah, S.Y. (2002). Effects of ginsenosides, active components of ginseng, on nicotinic acetylcholine receptors expressed in Xenopus oocytes. Eur. J. Pharmacol. 442, 37-45 https://doi.org/10.1016/S0014-2999(02)01508-X
  11. Couturier, S., Bertrand, D., Matter, J.M., Hernandez, M.C., Bertrand, S., Millar, N., Valera, S., Barkas, T., and Ballivet, M. (1990). A neuronal nicotinic acetylcholine receptor subunit ($\alpha$7) is developmentally regulated and forms a homooligomeric channel blocked by $\alpha$-BTX. Neuron 5, 847-856 https://doi.org/10.1016/0896-6273(90)90344-F
  12. Elgoyhen, A.B., Johnson, D.S., Boulter, J., Vetter, D.E., and Heinemann, S. (1994). $\alpha$9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell 79, 705-715 https://doi.org/10.1016/0092-8674(94)90555-X
  13. Gotti, C., and Clementi, F. (2004) Neuronal nicotinic receptors: from structure to pathology. Prog. Neurobiol. 74, 363-396 https://doi.org/10.1016/j.pneurobio.2004.09.006
  14. Gotti, C., Hanke, W., Maury, K., Moretti, M., Ballivet, M., Clementi, F., and Bertrand, D. (1994). 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. 6, 1281-1291 https://doi.org/10.1111/j.1460-9568.1994.tb00318.x
  15. Gotti, C., Carbonnelle, E., Moretti, M., Zwart, R., and Clementi, F. (2000). Drugs selective for nicotinic receptor subtypes: a real possibility or a dream? Behav. Brain Res. 113, 183-192 https://doi.org/10.1016/S0166-4328(00)00212-6
  16. Heidmann, T., Oswald, R.E., and Changeux, J.P. (1983). Multiple sites of action for noncompetitive blockers on acetylcholine receptor rich membrane fragments from torpedo marmorata. Biochemistry 22, 3112-3127 https://doi.org/10.1021/bi00282a014
  17. Jensen, M.L., Schousboe, A., and Ahring, P.K. (2005). Charge selectivity of the Cys-loop family of ligand-gated ion channels. J. Neurochem. 92, 217-225 https://doi.org/10.1111/j.1471-4159.2004.02883.x
  18. Karlin, A. (2002). Emerging structure of the nicotinic acetylcholine receptors. Nat. Rev. Neurosci. 3, 102-114 https://doi.org/10.1038/nrn731
  19. Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E.L. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567-580 https://doi.org/10.1006/jmbi.2000.4315
  20. Le Novere, N., and Changeux, J.P. (1995). Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells. J. Mol. Evol. 40, 155-172 https://doi.org/10.1007/BF00167110
  21. Lee, J.H., Jeong, S.M., Kim, J.H., Lee, B.H., Yoon, I.S., Lee, J.H., Choi, S.H., Kim, D.H., Rhim, H., Kim, S.S., et al. (2005). Characteristics of ginsenoside Rg3-mediated brain $Na^{+}$ current inhibition. Mol. Pharmacol. 68, 1114-1126 https://doi.org/10.1124/mol.105.015115
  22. Lee, B.H., Lee, J.H., Lee, S.M., Jeong, S.M., Yoon, I.S., Lee, J.H., Choi, S.H., Pyo, M.K., Rhim, H., Kim, H.C., et al. (2007). Identification of ginsenoside interaction sites in 5-$HT_{3A}$ receptors. Neuropharmacology 52, 1139-1150 https://doi.org/10.1016/j.neuropharm.2006.12.001
  23. Lena, C., and Changeux, J.P. (1997). Pathological mutations of nicotinic receptors and nicotine-based therapies for brain disorders. Curr. Opin. Neurobiol. 7, 674-682 https://doi.org/10.1016/S0959-4388(97)80088-8
  24. Lester, H.A., Dibas, M.I., Dahan, D.S., Leite, J.F., and Dougherty, D.A. (2004). Cys-loop receptors: new twists and turns. Trends Neurosci. 27, 329-336 https://doi.org/10.1016/j.tins.2004.04.002
  25. Lyford, L.K., Sproul, A.D., Eddins, D., McLaughlin, J.T., and Rosenberg, R.L. (2003). Agonist-induced conformational changes in the extracellular domain of alpha 7 nicotinic acetylcholine receptors. Mol. Pharmacol. 64, 650-658 https://doi.org/10.1124/mol.64.3.650
  26. Miyazawa, A., Fujiyoshi, Y., and Unwin, N. (2003). Structure and gating mechanism of the acetylcholine receptor pore. Nature 42, 949-955 https://doi.org/10.1038/nature01748
  27. Nah, S.Y., Kim, D.H., and Rhim, H. (2007). Ginsenosides: are any of them candidates for drugs acting on the central nervous system? CNS Drug Rev. 13, 381-404 https://doi.org/10.1111/j.1527-3458.2007.00023.x
  28. Nashmi, R., and Lester, H.A. (2006). CNS localization of neuronal nicotinic receptors. J. Mol. Neurosci. 30, 181-184 https://doi.org/10.1385/JMN:30:1:181
  29. Orr-Urtreger, A., Broide, R.S., Kasten, M.R., Dang, H., Dani, J.A., Beaudet, A.L., and Patrick, J.W. (2000). Mice homozygous for the L250T mutation in the alpha7 nicotinic acetylcholine receptor show increased neuronal apoptosis and die within 1 day of birth. J. Neurochem. 74, 2154-2166 https://doi.org/10.1046/j.1471-4159.2000.0742154.x
  30. Palma, E., Mileo, A.M., Eusebi, F., and Miledi, R. (1996). Threoninefor-leucine mutation within domain M2 of the neuronal $\alpha$7 nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist. Proc. Natl. Acad. Sci. USA 93, 11231-11235 https://doi.org/10.1073/pnas.93.20.11231
  31. Revah, F., Bertrand, D., Galzi, J.L., Devillers-Thiery, A., Mulle, C., Hussy, N., Bertrand, S., Ballivet, M., and Changeux, J.P. (1991). Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature 353, 846-849 https://doi.org/10.1038/353846a0
  32. Sala, F., Mulet, J., Choi, S., Jung, S.Y., Nah, S.Y., Rhim, H., Valor, L.M., Criado, M., and Sala, S. (2002). Effects of ginsenoside $Rg_{2}$ on human neuronal nicotinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 301, 1052-1059 https://doi.org/10.1124/jpet.301.3.1052
  33. Sali, A., and Blundell, T.L. (1993). Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815 https://doi.org/10.1006/jmbi.1993.1626
  34. Seguela, P., Wadiche, J., Dineley-Miller, K., Dani, J.A., and Patrick, J.W. (1993). Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. J. Neurosci. 13, 596-604
  35. Sine, S.M., and Taylor, P. (1982). Local anesthetics and histrionicotoxin are allosteric inhibitors of the acetylcholine receptor. Studies of clonal muscle cells. J. Biol. Chem. 257, 8106-8114
  36. Stierand, K., Maass, P.C., and Rarey, M. (2006). Molecular complexes at a glance: automated generation of two-dimensional complex diagrams. Bioinformatics 22, 1710-1716 https://doi.org/10.1093/bioinformatics/btl150
  37. Unwin, N. (2005). Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J. Mol. Biol. 346, 967-989 https://doi.org/10.1016/j.jmb.2004.12.031
  38. Verdonk, M.L., Cole, J.C., Hartshorn, M.J., Murray, C.W., and Taylor, R.D. (2003). Improved protein-ligand docking using GOLD. Proteins 52, 609-623 https://doi.org/10.1002/prot.10465
  39. Weiland, S., Bertrand, D., and Leonard, S. (2000). Neuronal nicotinic acetylcholine receptors: from the gene to the disease. Behav. Brain Res. 113, 43-56 https://doi.org/10.1016/S0166-4328(00)00199-6

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