Specificity and Sensitivity of a Human Olfactory Receptor Functionally Expressed in Human Embryonic Kidney 293 Cells andXenopus LaevisOocytes

Abstract

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.

The human olfactory receptor gene family

“…To identify the closest human homologs of rodent ORs with known odor ligands (9)(10)(11)(12)(13)(14)(16)(17)(18), the sequence of each rodent OR was used as a query to search (TBLASTN) the NCBI human genome sequence database (build 32).…”

“…Ligands have been reported for a total of two human ORs (13,18) and 23 rodent ORs (9-12, 14, 16, 17). This set includes ORs that recognize odorants with diverse structures as well as a variety of perceived odors, including, sweet, floral, herbal, woody, rancid, and sweaty.…”

“…Odor detection and coding by the OR family are combinatorial: each OR recognizes multiple odorants (9)(10)(11)(12)(13)(14)(15)(16)(17)(18), but different odorants are detected, and thereby encoded, by different combinations of ORs (11). The OR family can be divided into subfamilies whose members have related protein sequences (1).…”

“…The OR family can be divided into subfamilies whose members have related protein sequences (1). Odorants detected by the same OR have related structures (9)(10)(11)(12)(13)(14)(15)(16)(17)(18). In addition, ORs that belong to the same subfamily can detect odorants with similar structures (11,14).…”

Complex Transcription and Splicing of Odorant Receptor Genes

“…However, several lines of evidence argue in favor of the functionality of OR in testis: (i) promiscuous gene expression is also a feature of certain thymic medullary cells, where it serves a purpose within negative T-cell selection (50); (ii) certain OR have been detected on spermatozoa (13), proving testicular translation of OR transcripts; (iii) the expression levels of OR genes within the testis appear to be comparable with that in the main olfactory epithelium (7) 4 ; and (vi) the G-protein used by OR for signal transduction is expressed in testis (45), and spermatozoa seem to be endowed also with proteins involved in olfactory desensitization (51), implying that testicular OR expression is a meaningful event. Specific ligands are known only for very few OR (52)(53)(54)(55), and these odorants are without exception volatile, favoring their interaction with OR residing on cells of the main olfactory epithelium. However, because OR also play a major role in axonal targeting of olfactory neurons to specific glomeruli in the olfactory bulb (19, 56 -58), it may well be that OR interact with nonvolatile molecules as well, e.g.…”