FUNCTIONAL EXPRESSION OF ODORANT RECEPTORS OF THE ZEBRAFISH DANIO-RERIO AND OF THE NEMATODE C-ELEGANS IN HEK293 CELLS

Odorant receptors of zebrafish and C. elegans were functionally expres sed in vertebrate kidney cells (HEK293) using the eucaryotic expressio n vector pSMyc. Receptor-encoding cDNA cloned into this vector was exp ressed as a fusion protein with the N-terminal membrane import sequenc e of the guinea-pig serotonin receptor followed by a myc tag. Immunocy tochemical evidence indicates that this strategy directs a protein wit h the predicted immunoreactivity and approximate molecular weight to t he plasma membrane. Fish food extract (TetraMin) evoked a transient in crease in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for three fish odorant receptors and co nverted to stable cell lines. The effect of the extract was concentrat ion dependent and limited to the fraction of the extract <5 kDa. Pretr eating the transfected cells with the PLC inhibitor U73122 reduced the odor-evoked signal. Fish food extract also evoked a transient increas e 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 tr ansiently transfected with plasmids encoding the cDNA of ODR10, an odo rant 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 pro tocol.