Molecular bases of odor discrimination: Reconstitution of olfactory receptors that recognize overlapping sets of odorants

Abstract

The vertebrate olfactory system discriminates a wide variety of odorants by relaying coded information from olfactory sensory neurons in the olfactory epithelium to olfactory cortical areas of the brain. Recent studies have shown that the first step in odor discrimination is mediated by approximately 1000 distinct olfactory receptors, which comprise the largest family of G-protein-coupled receptors. In the present study, we used Ca(2+) imaging and single-cell reverse transcription-PCR techniques to identify mouse olfactory neurons responding to an odorant and subsequently to clone a receptor gene from the responsive cell. The functionally cloned receptors were expressed in heterologous systems, demonstrating that structurally related olfactory receptors recognized overlapping sets of odorants with distinct affinities and specificities. Our results provide direct evidence for the existence of a receptor code in which the identities of different odorants are specified by distinct combinations of odorant receptors that possess unique molecular receptive ranges. We further demonstrate that the receptor code for an odorant changes with odorant concentration. Finally, we show that odorant receptors in human embryonic kidney 293 cells couple to stimulatory G-proteins such as Galphaolf, resulting in odorant-dependent increases in cAMP. Odor discrimination is thus determined by differences in the receptive ranges of the odorant receptors that together encode specific odorant molecules.

Fig. 1.

Functional cloning of an olfactory receptor gene expressed in single olfactory neurons that responded to EG or EV. A, The Ca²⁺ response of a single olfactory neuron to EG (shown by an arrow in the top middle panel) in reflected changes in fura-2 fluorescence intensity ratios (340/380 nm). The odorants were applied to olfactory neurons for 4 sec during the times indicated by the black boxes. The cells were washed for 20 sec continuously between odorant applications. Names of odorants are abbreviated as follows: h4, 1-butanol;lCA, l-carvone; LY, lyral;EG, eugenol; GE, geraniol;EV, ethyl vanillin; LI, lilial;CR, cresol; PY, pyridine;dCA, d-carvone; and BZ, benzene (each 1 mm). HK stands for high KCl buffer. Pseudocolored images of Ca²⁺ measurements were taken at three representative time points (top panel). White cells indicate high intracellular Ca²⁺ levels, and blue cells represent the basal level and outlines the shape of each cell. B, The Ca²⁺ response of a single olfactory neuron to EV. C, The predicted amino acid sequences of the olfactory receptor genes that were isolated from single olfactory neurons depicted in A and B, which correspond to mOR-EG and mOR-EV, respectively. The putative TMs areunderlined. In the alignment of the mOR-EG and mOR-EV, residues that differ are highlighted.

Fig. 2.

Odorant-induced Ca²⁺responses of the functionally cloned receptors in HEK293T cells. The lyral response to the chimeric MOR23 (A), the EG response to chimeric mOR-EG (B), and the EV response to chimeric mOR-EV (C) in fura-2-loaded HEK293T cells cotransfected with the Gα15/16 subunits. Odorants (lyral and eugenol, 100 μm; ethyl vanillin, 1 mm) were applied for 20 sec at the times indicated byarrows. Carbamylcholine (CCh) (10 μm) was applied to verify the viability of cells as a control. Isoproterenol (Iso) (10 μm) serves as a control for Gα15/16 cotransfection. Themiddle and bottom panels show responses of cells that were not transfected with Gα15/16 (indicated as−Gα15/16) and that were transfected only with Gα15/16 (indicated as no receptor +Gα15/16). ΔF, ΔF ratio (340/380 nm).

Fig. 3.

A, Odorant responses of the mOR-EG. Various odorants (1 mm) were applied for 20 sec at the time indicated by arrows. The mOR-EG shows responses to EG, vanillin, and EV in HEK293T cells cotransfected with Gα15/16, as shown by changes in fura-2 fluorescence intensity ratios. Isoproterenol (Iso) (10 μm) serves as a control for Gα15/16 cotransfection. 1, Isoeugenol;2, guaiacol; 3, safrol; 4, allylbenzene; 5, syringic aldehyde; 6, vanillic acid. The structures of these compounds were drawn in Figure5. B, Dose-dependent Ca²⁺ responses of the mOR-EG to various ligand odorants. The responses of mOR-EG to increased concentrations of EG, vanillin, and EV in HEK293T cells cotransfected with Gα15/16, as shown by changes in fura-2 fluorescence intensity ratios.

Fig. 4.

A, Dose–response curves of the mOR-EG to EG, EV, and vanillin, obtained from Ca²⁺increases as a percentage of the responses at the highest concentrations of odorants. The maximum responses by ligand odorants were approximately the same. Each point represents the mean ± SE from at least 20 responding cells. B, Dose–response curves of Ca²⁺ responses of the mOR-EV to EV and vanillin. The data are shown as a percentage of the responses at 3 mm odorant concentrations. Eachpoint represents the mean ± SE from 11–36 cells in three to six separate experiments starting at different concentrations as described in Materials and Methods.

Fig. 5.

The mOR-EG and mOR-EV recognize overlapping sets of odorants with different affinities and specificities. Dose dependencies of mOR-EG and mOR-EV for various odorant molecules that share some structural similarities with EG or EV were obtained by Ca²⁺ response assays in HEK293T cells. Thesizes of the circles corresponds to the magnitude of Ca²⁺ responses normalized as a percentage of the responses at 3 mm ligand: from thesmallest circle for 0–25%, to the biggest circle for 75–100% as indicated. EG and vanillin have the highest affinity for mOR-EG among the odorants tested in this study. The mOR-EG also recognizes odorants 8, 9,10, and 11 at a threshold concentration of 300 μm. The mOR-EV recognizes vanillin and EV with different affinities from those of mOR-EG. Compounds1–7 did not activate mOR-EG or mOR-EV.1, Isoeugenol; 2, guaiacol;3, safrol; 4, allylbenzene;5, syringic aldehyde; 6, vanillic acid;7, heliotropyne; 8, 2-methoxy-4-ethylphenol; 9, 2-methoxy-4-methylphenol;10, eugenol acetate; 11, eugenol ethyl ether.

Fig. 6.

Odorant receptors couple to stimulatory G-proteins Gαs or Gαolf and induce cAMP increases in HEK293T cells.A, EG (300 μm) induced a 2.75-fold cAMP increase in HEK293T cells transfected with mOR-EG. EV (1 mm) induced a 1.3-fold cAMP increase in HEK293T cells transfected with mOR-EV. The data are mean ± SE of five and seven independent transfection experiments for mOR-EG and mOR-EV, respectively (*p < 0.05). B, Dose-dependent cAMP elevation as a percentage of the response to 1 mm EG by mOR-EG-transfected HEK293T cells. Thepoints represent the mean ± SE of three experiments. C, Gαolf coupling to the mOR-EG. The cAMP increase by EG (300 μm) in mOR-EG-transfected HEK293T cells was completely inhibited by cotransfection of Gα15/16. As the ratio of Gαolf to Gα15/16 was increased by cotransfection of Gαolf (2–8 times equivalent of the plasmid amount), the cAMP responses were rescued. Data are representative of three independent transfection experiments.

Fig. 7.

Dose–responses of vanillin and EV for odorant receptors (mOR-EG and mOR-EV). The response curves shown in Figure 4are redrawn as an odorant-based comparison.