Here, we provide the first evidence for functional expression of a human olfactory receptor protein (OR17-40) and show that recombinant olfactory receptors can be functionally expressed in heterologous systems. A mixture of 100 different odorants (Henkel 100) elicited a transient increase in intracellular [Ca(2+)] in human embryonic kidney 293 (HEK293) cells stably or transiently transfected with the plasmid pOR17-40. By subdividing the odorant mixture into progressively smaller groups, we identified a single component that represented the only effective substance: helional. Only the structurally closely related molecule heliotroplyacetone also activated the receptor. Other compounds, including piperonal, safrole, and vanillin, were completely ineffective. Mock-transfected cells and cells transfected with other receptors showed no change in intracellular [Ca(2+)] in response to odor stimulation. We were also able to functionally express OR17-40 in Xenopus laevis oocytes. Coexpression of a "reporter" channel allowed measurement of the response of oocytes injected with the cRNA of the human receptor to the odor mixture Henkel 100. The effective substances were the same (helional, heliotropylacetone) as those identified by functionally expressing the receptor in HEK293 cells and were active at the same, lower micromolar concentration. These findings open the possibility of now characterizing the sensitivity and specificity of many, if not all, of the hundreds of different human olfactory receptors.

Odorant receptors of zebrafish and C elegans were functionally expressed in vertebrate kidney cells (HEK293) using the eucaryotic expression vector pSMyc. Receptor-encoding cDNA cloned into this vector was expressed as a fusion protein with the N-terminal membrane import sequence of the guinea-pig serotonin receptor followed by a myc tag. Immunocytochemical evidence indicates that this strategy directs a protein with the predicted immunoreactivity and approximate molecular weight to the plasma membrane. Fish food extract (TetraMin) evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for three fish odorant receptors and converted to stable cell lines. The effect of the extract was concentration dependent and limited to the fraction of the extract < 5 kDa. Pretreating the transfected cells with the PLC inhibitor U73122 reduced the odor-evoked signal. Fish food extract also evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for single fish odorant receptors. Diacetyl evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids encoding the cDNA of ODR10, an odorant receptor of C. elegans suggested in other work to be specific for diacetyl. These results strongly imply that odorant receptors can be functionally expressed in HEK293 cells using this novel expression protocol.

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Ca2+ transients evoked by endothelin-1 (ET-1) were measured in single cells of a human tracheal epithelial cell line using the fluorescent Ca2+ indicator fura-2. In line with a previous study, a single exposure to ET-1 (10 nM) for 10-20 s resulted in a long-lasting desensitization to a subsequent challenge by the peptide, without affecting sensitivity to agonists for other Ca2+-mobilizing receptors such as P2y or H1, respectively. In the absence of extracellular Ca2+ ET-1 elicited a Ca2+ signal of comparable amplitude as in the presence of extracellular Ca2+ but of shorter duration. Exposure to ET-1 in the absence of Ca2+ caused significantly less desensitization. Inhibition of the Ca2+ entry component of the Ca2+ transient by means of SK&F 96365, an inhibitor of Ca2+ entry, had effects comparable to Ca2+ removal. The Ca2+ transient was shortened but not significantly reduced in amplitude, and desensitization was reduced in the presence of the compound. These data demonstrate that desensitization of ET(A) receptors (ET(A)R) is promoted by transmembrane Ca2+ entry but not by Ca2+ release.