FIGURE 2.

Functional expression and characterization of mosquito ORs. A, selective responses to 4-methylphenol (4MP) and indole (IN) can be monitored from cells expressing OR1/Orco and OR2/Orco, respectively, upon challenging with the cognate ligand at 10 μm concentration (shown are time course of luminescence changes over time; RLU, relative light units). No responses are measured from ORx alone- or Orco alone-expressing cells, even at higher agonist concentration, showing that coexpression of ORx and Orco is required. B, the presence of Gα₁₆ protein does not stimulate agonist-dependent activity of OR2 to any appreciable degree in cells expressing OR2/Gα₁₆ challenged with indole at 10 μm. C, responses of cells expressing OR1/δOR/Gα₁₆ to sequential administration of 4-methylphenol at 100 μm (arrowhead 1) and the δ-opioid receptor ligand DPDPE at 1 μm (arrowhead 2) are shown. In the latter case, Gα₁₆-dependent phospholipase C coupling of the co-expressed murine δ-opioid receptor was clearly observed, as expected. The later calcium response is also much slower than direct calcium ion flux through ion channels, as expected for G-protein-coupled receptor downstream signaling, whereas lower concentrations of agonist, in the nm range (data not shown), were sufficient for activation of the G-protein-coupled receptor relative to those required for OR activation. D, responses are ORx-specific and odorant-selective. Cells expressing OR1/Orco or OR2/Orco were challenged with 4-methylphenol, indole, benzaldehyde (BA), and ethyl butyrate (EB) at 100 μm concentration. E, dose-response curves for OR1, OR2, and Orco. The red curves correspond to the most potent ligands, 4-methylphenol for OR1 (EC₅₀ = 2.8 × 10⁻⁶, n = 2) and indole for OR2 (EC₅₀ = 3.4 × 10⁻⁶, n = 2). The maximal response of each receptor to the administration of its cognate agonist was used for normalization of all other responses for the same receptor. 3MP denotes 3-methylphenol; 2MP denotes 2-methylphenol; OA denotes Orco agonist (OrcoRAM2). % max response, percentage of maximum response. Error bars indicate mean ± S.D.