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KMID : 0545120070170060928
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Journal of Microbiology and Biotechnology
2007 Volume.17 No. 6 p.928 ~ p.933
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Functional Analysis of Olfactory Receptors Expressed in a HEK-293 Cell System by Using Cameleons
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Ko Hwi-Jin
Park Tai-Hyun
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Abstract
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Cameleon is a genetically engineered Ca2+ 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 Ca2+ induced by the olfactory receptor-mediated signal transduction, because it is localized in the cytosol and binds to cytosolic Ca2+ ions. Cells stably expressing yellow cameleon-2 were transfected with each of the test olfactory receptor genes, odr-10 and I7, and the expression of the olfactory receptor genes were examined using immunocytochemical methods and RT-PCR. Stimulating each olfactory receptor with its specific odorant caused an increase in the intracellular Ca2+ 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.
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KEYWORD
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Cameleon, olfactory receptor, HEK-293 cells, Ca2+
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