Thus, IR84a and IR8a are together both necessary and sufficient to reconstitute a cilia-localized and physiologically active olfactory receptor in Drosophila neurons. We extended our investigation of the sufficiency of IR84a and IR8a to form a functional receptor by determining their ability to confer phenylacetaldehyde responsiveness in an ex vivo non-neuronal system. We chose Xenopus laevis oocytes, which are commonly used for functional expression of iGluRs ( Walker et al., 2006). In these cells, single or combinations of IR complementary RNAs (cRNAs) can be injected, and odor-evoked current responses across the oocyte

membrane can be measured by two-electrode voltage clamp. When cRNAs for IR84a DAPT supplier or IR8a were injected alone into oocytes, we observed no responses to phenylacetaldehyde (Figures 4D and 4E). By contrast, when IR84a and IR8a cRNAs were coinjected, phenylacetaldehyde induced an inward current of several hundred nA in these cells (Figures 4D and 4E). We further tested the functional properties of a different odor-specific receptor, IR75a, which is expressed in the IR8a-dependent propionic acid-sensitive neuron in ac3 sensilla (Figures 2B and 2C). Oocytes

expressing IR75a and IR8a together, but not either GSK126 supplier receptor alone, exhibited robust propionic acid-evoked current responses (Figures 4D and 4E). Odor-induced current responses were highly specific to each receptor pair and displayed concentration dependency (Figures 4D–4F). The concentration response curves for both phenylacetaldehyde and propionic acid did not saturate at the highest concentrations obtainable without changing the osmolarity of the solution, preventing our determination of 50% effective concentration (EC50) values. Baseline currents measured in the absence of either agonist were similar between IR84a+IR8a-expressing, IR75a+IR8a-expressing, and uninjected oocytes (data many not shown), suggesting that these receptors do not have detectable constitutive activity, at least in these cells. The codependency of IR84a and IR8a for cilia localization and

odor-evoked responses suggested that these proteins might form a complex. We tested this possibility through optical imaging of fluorescent protein-tagged receptors. We first generated an mCherry-tagged IR8a fusion protein and confirmed that this promotes cilia targeting of EGFP:IR84a in OR22a neurons (Figure 5A). In these cells, we observed precise colocalization and consistent relative intensities of EGFP and mCherry fluorescence throughout the cell bodies, inner dendrites, and cilia (Figure 5A). Odor-evoked responses conferred on these neurons by the fluorescent protein-tagged IRs were indistinguishable from those generated by untagged receptors (Figure 5B), indicating that the fluorescent tags do not interfere with their function.