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Figure 2. Responsiveness of various Or-Orco complexes to cyclic nucleotides.(A) Representative current traces of oocytes expressing various insect Or-Orco complex in response to their cognate ligands and cyclic nucleotide reagents. Pheromone/odorant was applied to the oocytes at the time indicated by the arrowhead (for 5 sec), and cyclic nucleotide reagent was applied during the time indicated by colored square (for 30 sec), respectively. Concentrations of ligands are as follows: E11–14:OH, 10 µM, E11–14:OAc, 10 µM, pentylacetate, 100 µM, 2-methylphenol, 100 µM, cyclic nucleotide reagents, 100 µM. (B) Summary of the average responses to cyclic nucleotide reagents. Each response was normalized to a response to the cognate ligand of each Or. The bar graphs show mean ± S.E.M. from 3–4 independent oocytes.
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Figure 3. Cyclic nucleotides interact with BmOr-1 subunit of the BmOr-1-BmOrco complex.(A) Representative current traces of oocytes expressing various combinations of the insect Or-Orco complex in response to their cognate ligand or cGMP. Odorant was applied to the oocytes at the time indicated by an arrowhead (for 5 sec), and cGMP was applied during the time indicated by colored square (for 30 sec), respectively. Concentrations of ligands were as follows: bombykol, 10 µM, bombykal, 3 µM, cis-Jasmone, 100 µM, cGMP, 100 µM. (B) Summary of the average response amplitudes of the Or-Orco complex to cGMP. The Y-axis is normalized to the response to their cognate ligand. The bar graphs represent mean ± S.E.M. from 3–4 independent oocytes. One-way ANOVA with Tukey’s multiple comparison test, ***p<0.001. (C) Representative current traces of oocytes expressing the Orco family alone in response to cyclic nucleotide reagents or VUAA1 (for 30 sec or 10 sec, respectively). All reagents were applied at the concentration of 100 µM.
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Figure 4. Cyclic nucleotides non-competitively inhibit the response of BmOr-1-BmOrco to bombykol.(A) A representative current trace of an oocyte expressing BmOr-1-BmOrco as it responds to bombykol in the presence of 100 µM cGMP. Bombykol was applied to the oocyte at the time indicated by the arrowhead, and cGMP was applied during the time indicated by light blue square, respectively. The trace for the inhibition of a response to bombykol by cGMP is magnified in an inset figure wherein the response to cGMP is distinguished from that to bombykol in the presence of cGMP. (B) Summary of average responses of BmOr-1-BmOrco to 10 µM bombykol in the absence (+None) or presence (+cGMP, +cAMP) of cyclic nucleotides (100 µM). Each bar represents mean ± S.E.M. from 3–4 independent oocytes. The Y-axis is normalized to the response amplitude of 10 µM bombykol+None condition. Unpaired Student’s t-test, ***p<0.001 vs ‘+None’. (C) Dose-dependent inhibition of BmOr-1-BmOrco by cyclic nucleotides as a percentage of the response to 10 µM bombykol. Each point represents mean ± S.E.M. from 4 independent oocytes. The IC50 value is 217 nM. (D) Dose-response curves of responses of BmOr-1-BmOrco to bombykol in the absence or presence of 100 µM cyclic nucleotides. Each point represents mean ± S.E.M. from 4–5 independent oocytes. EC50 values are as follows: ‘+None’, 8.2 µM, ‘+cGMP’, 2.8 µM, ‘+cAMP’, 2.7 µM.
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Figure 5. Cyclic nucleotides do not inhibit the responses of Or-Orco types other than BmOr-1-BmOrco.(A) Representative current traces of oocytes that each expressed a different Or-Orco complex; these oocytes were responding to their cognate ligands in the absence or presence of 100 µM cGMP. Odorant (100 µM cis-Jasmone, 100 µM pentylacetate, 100 µM 2-methylphenol) or pheromone (1 µM bombykol, 3 µM bombykal, 10 µM E11–14:OH, and 1 µM E11–14:OAc) was applied to the oocytes at the time indicated by the arrowhead, and cGMP was applied during the time indicated by light blue square. (B) Summary of average responses of Or-Orco to their cognate ligands in the absence (+None) or presence (+cGMP, +cAMP) of cyclic nucleotides (100 µM). Each bar represents mean ± S.E.M. from 3–4 independent oocytes. The Y-axis is normalized to the response amplitude of 10 µM bombykol+None condition. Unpaired Student’s t-test, **p<0.01 vs ‘+None’.
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Figure 6. Structure-activity relationship of BmOr-1-BmOrco to cyclic nucleotides and related reagents.(A) Structural formulae of reagents examined in this assay. (B) Summary of the average responses of BmOr-1-BmOrco-injected oocytes (left) and none-injected oocytes (right) to various nucleotide-related reagents (100 µM). Each response was normalized to the response of BmOr-1-BmOrco-injected oocytes to 10 µM bombykol. Each bar represents mean ± S.E.M. from 3 independent oocytes. (C) Summary of inhibition of the response to 10 µM bombykol by various nucleotide-related reagents (100 µM). The Y-axis is normalized to the response to 10 µM bombykol. Each bar represents mean ± S.E.M. from 3 independent oocytes. One-way ANOVA with Tukey’s multiple comparison test, **p<0.01, ***p<0.001. (D) A model showing the mechanism of action of cyclic nucleotides to the bombykol receptor (BmOr-1-BmOrco) complex. Cyclic nucleotides modulate the activity of the complex by binding to the allosteric site in the extracellular domain of the BmOr-1 subunit.
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Figure 1. Cyclic nucleotides weakly activate the BmOr-1-BmOrco complex from outside of the cell.(A) Summary of the average responses of BmOr-1-BmOrco-injected oocytes (left) and none-injected oocytes (right) to various cyclic nucleotide reagents (100 µM). Each response was normalized by the response amplitude of BmOr-1-BmOrco-injected oocytes to 10 µM bombykol. The bar graphs show mean ± S.E.M. from 3–4 independent oocytes. (B) Representative current traces of oocytes expressing BmOr-1-BmOrco in response to bombykol and various concentrations of cyclic nucleotides. Bombykol was applied to the oocytes at the time indicated by the arrowhead (for 5 sec), and cyclic nucleotides were applied during the time indicated by colored square (for 30 sec), respectively. Concentrations of ligands were as follows: bombykol, 10 µM, cyclic nucleotides, 100 nM, 1 µM, 10 µM, 100 µM. (C) Dose-dependent responses of BmOr-1-BmOrco to cAMP (yellow) or cGMP (blue). Each point represents the mean current ± S.E.M. from 3 independent oocytes. Each response was normalized to the response to 10 µM bombykol. (D) Representative current traces of oocytes expressing rat olfactory cyclic nucleotide-gated channel complex (CNGA2+A4+B1b) in response to 8-Br-cGMP or cGMP (100 µM). n = 4. (E) Current-voltage relationships of oocytes expressing BmOr-1-BmOrco in response to bombykol (black), cGMP (blue), and cAMP (yellow).
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