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Figure 3. A screen of OX1a analogs for Orco antagonist activity.A. An example of the antagonist screening protocol. An oocyte expressing Cqui\Orco+Cqui\Or21 was exposed to 60 sec applications of 3 µM OLC12 with 4 min washed between applications. 100 µM OX1l was applied for 90 sec preceding the third application of OLC12 and then co-applied during the OLC12 application. B. Responses of Cqui\Orco+Cqui\Or21 to 3 µM OLC12 (~EC25) in the presence of 100 µM of each analog are presented as a percentage of the average of the two preceding responses to OLC12 alone (mean ± SEM, n = 3-7). Inhibition by OX1d and OX2 did not differ from inhibition by OX1a. Inhibition by all other compounds was significantly different from inhibition by OX1a: OX1e (p<0.05); OX1b-c,f-n, OX3a (p<0.001). C. A screen of additional OX1a analogs for Orco antagonist activity. Responses of Cqui\Orco+Cqui\Or21 to 3 µM OLC12 in the presence of 30 µM of each candidate antagonist are presented as a percentage of the average of two preceding responses to OLC12 alone (mean ± SEM, n=3-5). Inhibition by all compounds was significantly different from inhibition by OX1a (p<0.001). Inhibition by OX1i was significantly different (p<0.001) from values for OX1r and OX1s. Inhibition by OX1l was significantly different from values for OX1o (p<0.001) and OX1p (p<0.05). Inhibition by OX1m was significantly different (p<0.001) from values for OX1o and OX1p. Inhibition values that did not differ are: OX1g and OX1q, OX1j and OX4, OX1m and OX1q.
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Figure 4. Concentration-inhibition curves for OX1a, OX1g, OX1i, OX1j, OX1k, OX1l, OX1o, OX1p, OX1q, OX3a, OX3b and OX4 inhibition of Cqui\Orco+Cqui\Or21 activated by 3 µM OLC12.IC50 and nH values may be found in Table 1.
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Figure 5. The potent Orco antagonists OX1l, OX3a and OX4 non-competitively inhibit odorant activation of a mosquito OR.A. Altering the concentration of Orco agonist (OLC12) shifts the OX1l inhibition curve. The IC50 for OX1l inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (8.3 ± 0.5 µM, n = 4) is significantly different from the IC50 for OX1l inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (3.8 ± 0.3 µM, n = 9) (p<0.0001, F-test). The IC50 for OX1l inhibition of Cqui\Orco+Cqui\Or21 activation by 30 µM OLC12 (24 ± 3 µM, n = 6) is significantly different from the IC50 for OX1l inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (p<0.0001, F-test) and from the IC50 for OX1l inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (p<0.0001, F-test). B. Altering odorant (3MI) concentration fails to alter the inhibition curve for OX1l antagonism of Cqui\Orco+Cqui\Or21 activated by 3MI. The IC50 values for OX1l inhibition of responses to 10 nM 3MI (17 ± 1 µM, n = 3), 100 nM 3MI (16 ± 1 µM, n = 3), and 1µM 3MI (19 ± 2 µM, n = 3) did not differ (p=0.3605, F-test). C. Altering the concentration of Orco agonist (OLC12) shifts the OX3a inhibition curve. The IC50 for OX3a inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (12 ± 1 µM, n = 3) is significantly different from the IC50 for OX3a inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (1.7 ± 0.2 µM, n = 4) (p<0.0001, F-test). The IC50 for OX3a inhibition of Cqui\Orco+Cqui\Or21 activation by 30 µM OLC12 (28 ± 2 µM, n = 3) is significantly different from the IC50 for OX3a inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (p<0.0001, F-test) and from the IC50 for OX3a inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (p<0.0001, F-test). D. Altering odorant (3MI) concentration fails to alter the inhibition curve for OX3a antagonism of Cqui\Orco+Cqui\Or21 activated by 3MI. The IC50 values for OX3a inhibition of responses to 10 nM 3MI (2.6 ± 0.3 µM, n = 3), 100 nM 3MI (2.3 ± 0.2 µM, n = 4), and 1µM 3MI (1.9 ± 0.1 µM, n = 3) did not differ (p=0.07, F-test). E. Altering the concentration of Orco agonist (OLC12) shifts the OX4 inhibition curve. The IC50 for OX4 inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (8.8 ± 1.2 µM, n = 3) is significantly different from the IC50 for OX4 inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (2.1 ± 0.3 µM, n = 5) (p<0.0001, F-test). The IC50 for OX4 inhibition of Cqui\Orco+Cqui\Or21 activation by 30 µM OLC12 (73 ± 5 µM, n = 3) is significantly different from the IC50 for OX4 inhibition of Cqui\Orco+Cqui\Or21 activation by 3 µM OLC12 (p<0.0001, F-test) and from the IC50 for OX4 inhibition of Cqui\Orco+Cqui\Or21 activation by 10 µM OLC12 (p<0.0001, F-test). F. Altering odorant (3MI) concentration fails to alter the inhibition curve for OX4 antagonism of Cqui\Orco+Cqui\Or21 activated by 3MI. The IC50 values for OX4 inhibition of responses to 10 nM 3MI (3.4 ± 0.6 µM, n = 3), 100 nM 3MI (2.7 ± 0.4 µM, n = 3), and 1µM 3MI (2.5 ± 0.3 µM, n = 3) did not differ (p=0.42, F-test).
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Figure 1. Non-competitive inhibition of odorant activation of an insect OR by an Orco antagonist.
A. Structure of N-(4-ethylphenyl)-2-thiophenecarboxamide (OX1a). B. Increasing the concentration of Orco agonist (OLC12) decreases the effectiveness of OX1a. Oocytes expressing Cqui\Orco+Cqui\Or21 were activated with 3 µM or 30 µM OLC12, in the absence or presence of 30 µM or 100 µM OX1a. Responses in the presence of OX1a are presented as a percentage of the average of the two preceding responses to OLC12 alone (mean ± SEM, n=3). Statistical significance: **p<0.01, ***p<0.001 (two-tailed, unpaired t-test). C. OX1a inhibition of Cqui\Orco+Cqui\Or21 activation by 3-methylindole (3MI) is not altered by changes in odorant (3MI) concentration. IC50 values for OX1a inhibition of responses to 10nM 3MI (47 ± 5 µM), 100nM 3MI (42 ± 7 µM) and 1uM 3MI (50 ± 4 µM) did not differ (p=0.2970, F-test). D. Co-application of 50 µM OX1a significantly reduces the maximal response to 3MI, as compared to the response to 3MI in the absence of OX1a (p<0.0001, F-test). The EC50 for 3MI activation of Cqui\Orco+Cqui\Or21 in presence of 50 µM OX1a (37 ± 7 nM) did not differ (p=0.57, F-test) from the EC50 for activation of Cqui\Orco+Cqui\Or21 by 3MI alone (32 ± 7 nM).
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