Functional Analysis of Olfactory Receptors Expressed in a HEK-293 Cell System by Using Cameleons

  • Ko, Hwi-Jin (School of Chemical and Biological Engineering, Seoul National University) ;
  • Park, Tai-Hyun (School of Chemical and Biological Engineering, Seoul National University)
  • Published : 2007.06.30

Abstract

Cameleon is a genetically engineered $Ca^{2+}$ sensing molecule consisting of two variants of the green fluorescent protein (GFP), calmodulin and calmodulin-binding protein, M13. HEK-293 cells stably expressing three types of cameleons, yellow cameleon-2, cameleon-3er, and cameleon-2nu, were constructed, and the expression and localization of these cameleons were confirmed by fluorescent imaging. Among the cameleons, the yellow cameleon-2 was selected for analyzing the change in $Ca^{2+}$ induced by the olfactory receptor-mediated signal transduction, because it is localized in the cytosol and binds to cytosolic $Ca^{2+}$ ions. Cells stably expressing yellow cameleon-2 were transfected with each of the test olfactory receptor genes, odr-10 and 17, and the expression of the olfactory receptor genes were examined using immunocytochenmical methods and RT-PCR. Stimulating each olfactory receptor with its specific odorant caused an increase in the intracellular $Ca^{2+}$ level, which was measured using yellow cameleon-2. These results demonstrate that yellow cameleon-2 can be conveniently used to examine the function of the olfactory receptors expressed in heterologous cells.

Keywords

References

  1. Bae, Y. M., K.-W. Park, B.-K. Oh, and J.-W. Choi. 2006. Immunosensor for detection of Escherichia coli O157:H7 using imaging ellipsometry. J. Microbiol. Biotechnol. 16: 1169-1173
  2. Choi, S. H., J. W. Lee, and S. J. Sim. 2004. Enhancement of the sensitivity of surface plasmon (SPR) immunosensor for the detection of anti-GAD antibody by changing the pH for streptavidin immobilization. Enzyme Microbial Technol. 35: 683-687 https://doi.org/10.1016/j.enzmictec.2004.08.022
  3. Choi, J.-W., Y.-K. Kim, H.-J. Kim, W. C. Lee, and G. H. Seong. 2006. Lab-on-a-chip for mornitoring the quality of raw milk. J. Microbiol. Biotechnol. 16: 1229-1235
  4. Golovina, V. A. and M. P. Blaustein. 1997. Spatially and functionally distinct $Ca^{2+}$ stores in sarcoplasmic and endoplasmic reticulum. Science 275: 1643-1648 https://doi.org/10.1126/science.275.5306.1643
  5. Grynkiewicz, G., M. Poeniem, and R. Y. Tsien. 1985. A new generation of $Ca^{2+}$ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450
  6. Hofer, A. M. and I. Schulz. 1996. Quantification of intraluminal free [$Ca^{2+}$] in the agonist-sensitive internal calcium store using compartmentalized fluorescent indicators: Some considerations. Cell Calcium 20: 235-242 https://doi.org/10.1016/S0143-4160(96)90029-9
  7. Hwang, S. Y., C. H. Yoo, J. Y. Jeon, S. C. Choi, and E. K. Lee. 2005. Quantitative assay of hepatitis B surface antigen by using surface plasmon resonance biosensor. Biotechnol. Bioprocess Eng. 10: 309-314 https://doi.org/10.1007/BF02931847
  8. Ikura, M., G. M. Clore, A. M. Gronenborn, G. Zhu, C. B. Klee, and A. Bax. 1992. Solution structure of a calmodulintarget peptide complex by multidimensional NMR. Science 256: 632-638 https://doi.org/10.1126/science.1585175
  9. Kang, C. D., S. W. Lee, T. H. Park, and S. J. Sim. 2006. Performance enhancement of real-time detection of protozoan parasite, Cryptosporidium oocyst by a modified surface plasmon resonance (SPR) biosensor. Enzyme Microbial Technol. 39: 387-390 https://doi.org/10.1016/j.enzmictec.2005.11.039
  10. Kendall, J. M., M. N. Badminton, G. B. Sala-Newby, A. K. Campbell, and C. M. Rembold. 1996. Recombinant apoaqueorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free $Ca^{2+}$ of intact cells. Biochem. J. 318: 383-387 https://doi.org/10.1042/bj3180383
  11. Kim, E. J. and T. H. Park. 2003. Antiapoptosis engineering. Biotechnol. Bioprocess Eng. 8: 76-82 https://doi.org/10.1007/BF02940260
  12. Kim, N. S., I.-S. Park, and D.-K. Kim. 2006. Optimization of quartz crystal microbalance-precipitation sensor measuring acetylcholinesterase activity. J. Microbiol. Biotechnol. 16: 1523-1528
  13. Ko, H. J. and T. H. Park. 2005. Piezoelectric olfactory biosensor: Ligand specificity and dose-dependence of an olfactory receptor expressed in a heterologous cell system. Biosens. Bioelectron. 20: 1327-1332 https://doi.org/10.1016/j.bios.2004.05.002
  14. Ko, H. J. and T. H. Park. 2006. Dual signal transduction mediated by a single type of olfactory receptor expressed in a heterologous system. Biol. Chem. 387: 59-68 https://doi.org/10.1515/BC.2006.009
  15. Krautwurst, D., K. W. Yau, and R. R. Reed. 1998. Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 95: 917-926 https://doi.org/10.1016/S0092-8674(00)81716-X
  16. Lee, J. Y., H. J. Ko, S. H. Lee, and T. H. Park. 2006. Cellbased measurement of odorant molecules using surface plasmon resonance. Enzyme Microbial Technol. 39: 375- 380 https://doi.org/10.1016/j.enzmictec.2005.11.036
  17. Levasseur, G., M.-A. Persuy, D. Grebert, J.-J. Remy, R. Salesse, and E. Pajot-Augy. 2003. Ligand-specific doseresponse of heterologously expressed olfactory receptors. Eur. J. Biochem. 270: 2905-2912 https://doi.org/10.1046/j.1432-1033.2003.03672.x
  18. Miyawaki, A., J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and R. Y. Tsien. 1997. Fluorescent indicators for $Ca^{2+}$ based on green fluorescent proteins and calmodulin. Nature 388: 882-887 https://doi.org/10.1038/42264
  19. Miyawaki, A., O. Griesbeck, R. Helm, and R. Y. Tsien. 1999. Dynamic and quantitative $Ca^{2+}$ measurements using improved cameleons. Proc. Natl. Acad. Sci. USA 96: 2135-2140
  20. Montero, M., M. Brini, R. Marsault, J. Alvarez, R. Sitia, T. Pozzan, and R. Rizzuto. 1995. Monitoring dynamic changes in free $Ca^{2+}$ ion concentration in the endoplasmic reticulum of intact cells. EMBO J. 14: 5467-5475
  21. Petrou, S., D. N. Bowser, R. A. Nicholls, R. G. Panchal, M. L. Smart, A. M. Reilly, and D. A. Williams. 2000. Genetically targeted calcium sensors enhance the study of organelle function in living cells. Clin. Exp. Pharmacol. Physiol. 27: 738-744 https://doi.org/10.1046/j.1440-1681.2000.03327.x
  22. Sengupta, P., J. H. Chou, and C. I. Bargmann. 1996. odr-10 encodes a seven transmembrane domain olfactory receptor required for responses to the odorant diacetyl. Cell 84: 899- 909 https://doi.org/10.1016/S0092-8674(00)81068-5
  23. Sung, J. H., H. J. Ko, and T. H. Park. 2006. Piezoelectric olfactory biosensor using olfactory receptor protein expressed in Escherichia coli. Biosens. Bioelectron. 21: 1981-1986 https://doi.org/10.1016/j.bios.2005.10.002
  24. Tse, F. W., A. Tse, and B. Hille. 1994. Cyclic $Ca^{2+}$ changes in intracellular stores of gonadotropes during gonadotropinreleasing hormone-stimulated $Ca^{2+}$ oscillations. Proc. Natl. Acad. Sci. USA 91: 9750-9754
  25. Zhang, Y., J. H. Chou, J. Bradley, C. I. Bargmann, and K. Zinn. 1997. The Caenorhabditis elegans seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells. Proc. Natl. Acad. Sci. USA 94: 12162-12167
  26. Zhao, H., L. Ivic, J. M. Otaki, M. Hashimoto, K. Mikoshiba, and S. Firestein. 1998. Functional expression of a mammalian odorant receptor. Science 279: 237-242 https://doi.org/10.1126/science.279.5348.237