e., had a single clear bleaching step). This analysis revealed that the entire population of spots had

an average of 1.57 EGFP molecules in EGFP:IR8a+mCherry:IR84a complexes and 1.49 EGFP molecules in EGFP:IR84a+mCherry:IR8a Epacadostat research buy complexes (Figures S3D and S3E). Because only 80% of EGFP tags are fluorescent (Ulbrich and Isacoff, 2007), some complexes with two EGFP-tagged subunits would have only one visible EGFP. Therefore, from the normalized integrated EGFP intensities, we calculated that 83% of EGFP:IR8a+mCherry:IR84a complexes and 70% of EGFP:IR84a+mCherry:IR8a complexes had two EGFP-tagged subunits per spot (see Experimental Procedures). Together, these analyses are consistent with IR complexes containing up to two subunits each Ceritinib solubility dmso of IR8a and IR84a, similar to

the dimer-of-dimers structure of many types of iGluR (Gereau and Swanson, 2008). The similarity in the domain organization of IRs and iGluRs led to the hypothesis that these olfactory receptors function as odor-gated ion channels (Benton et al., 2009). Our functional reconstitution of IRs in heterologous cells, in the absence of other Drosophila proteins, supports this model of signaling. We wished, however, to test the ionotropic activity of IRs. We first examined the ion conduction properties of phenylacetaldehyde- or propionic acid-evoked currents induced by IR84a+IR8a and IR75a+IR8a, respectively. This was performed in oocytes by measuring current/voltage (IV) relationships when the extracellular solution contained primarily Na+, K+, or Ca2+ as the cationic charge carrier (Figure 6A). For both receptor combinations, the reversal potential with Na+ or K+ solutions

was slightly negative and not different between the two cations (IR84a+IR8a: −8 ± 7 mV (Na+) and −11 ± 5 mV SB-3CT (K+); IR75a+IR8a: −6 ± 2 mV (Na+) and −4 ± 1 mV (K+); n = 8–13; paired t test, p > 0.05). Current amplitudes were similar for K+ and Na+ for both IR84a+IR8a and IR75a+IR8a (Figure 6A), except when measured at −100 mV, where they were higher for K+ than for Na+ for IR84a+IR8a (paired t test, p = 0.03), but indistinguishable for IR75a+IR8a (paired t test, p = 0.20) (Figure 6A). In the presence of extracellular Ca2+ and absence of extracellular Na+ and K+, activation of IR84a+IR8a, but not IR75a+IR8a, also induced inward currents (Figure 6A). These currents were, however, abolished by pre-injection of IR84a+IR8a-expressing oocytes with the Ca2+ chelator BAPTA, which prevents indirect activation of endogenous Ca2+-dependent chloride channels (Kuruma and Hartzell, 1999). Together, these experiments indicate that odor-evoked IR-dependent currents are carried principally by monovalent cations, but that IR84a+IR8a-dependent activation also leads to low Ca2+ entry, which is amplified by oocyte Ca2+-dependent channels to produce a measurable current.