Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors |
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Authors: | Yoshihisa Ozoe Tomo Kita Fumiyo Ozoe Toshifumi Nakao Kazuyuki Sato Kangetsu Hirase |
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Institution: | 1. Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan;2. Agrochemicals Research Center, Mitsui Chemicals Agro, Inc., Mobara, Chiba 297-0017, Japan |
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Abstract: | γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of 3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit 3H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the 3H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. 3H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited 3H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced 3H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of 3H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs. |
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Keywords: | BPB 3-benzamido-N-phenylbenzamide EBOB 4&prime -ethynyl-4-n-propylbicycloorthobenzoate GABA γ-aminobutyric acid GABAR GABA receptor IGluR inhibitory l-glutamate receptor SOS standard oocyte solution TEVC two-electrode voltage clamp |
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