Xu Y et al. (2015), Meroterpenoid Chrodrimanins Are Selective and P...

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PLoS One 2015 Apr 22;104:e0122629. doi: 10.1371/journal.pone.0122629.

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Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels.

Xu Y , Furutani S , Ihara M , Ling Y , Yang X , Kai K , Hayashi H , Matsuda K .

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Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to 10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs.

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	Fig 2. Effects of chrodrimanin B on ACh (A)-, L-glutamate (B)- and GABA (C)-induced currents in the silkworm larval neurons.ACh (10 μM), L-glutamate (30 μM) and GABA (30 μM) were applied for 2 s using a U-tube, whereas chrodrimanin B was bath-applied for 1 min prior to co-application with corresponding agonists. After 3 min wash, each neurotransmitter was challenged again for 2 s.
	Fig 3. Blocking effects of chrodrimanins A, B and D on silkworm RDL GABAR expressed in Xenopus oocytes and concentration-inhibition curves for each compound.(A) After three successive control applications of GABA for 2 s, each chrodrimanin (30 nM) was bath-applied for 5 min and then co-applied with 30 μM GABA to RDL-expressing oocytes for 2 s to record the inward currents in response to GABA. (B) Concentration-normalized response curves for chrodrimanins A, B and D concentration ranges. The peak current amplitude of the response to GABA recorded in the presence of chrodrimanins was normalized to the maximum response to 30 μM GABA recorded before application of chrodrimanins. Each plot represents the mean ± standard error of the mean (SEM) of four experiments. The pIC50 (= -log IC50) values (mean ± SEM, n = 4) of chrodrimanins A, B and D were 6.83 ± 0.03 (IC50 = 148 nM), 8.95 ± 0.04 (IC50 = 1.13 nM) and 8.22 ± 0.04 (IC50 = 6.01 nM), respectively. The rank order of blocking actions of chrodrimanins was similar to that of their insecticidal actions on the silkworm larvae determined by oral application [2,3].
	Fig 4. Effects of repeated application of GABA on the blocking action of chrodrimanin B.(A) Traces of the GABA-induced current responses in the presence of 3 nM chrodrimanin B. After three control responses to GABA at 30 μM, chrodrimanin B was continuously bath-applied at 3 nM, during which GABA was also applied at 30 μM every 5 min. (B) Normalized peak current amplitude of GABA responses recorded during continuous application of chrodrimanin B. Each plot represents the mean ± SEM of four separate experiments.
	Fig 5. Effects of chrodrimanin B on the concentration-response curve for GABA on silkworm RDL GABAR expressed in Xenopus oocytes.The GABA-induced responses were measured at various concentrations in the presence and absence of chrodrimanin B. The EC50 value for GABA shifted from 41.1 μM (n = 4, CI = 37.3–45.2 μM) without chrodrimanin B to 143 μM (n = 4, 121–170 μM) with 3 nM chrodrimanin B and to 565 μM (n = 4, 478–668 μM) with 10 nM chrodrimanin B, respectively.
	Fig 6. Concentration-inhibition curves for chrodrimanin B and fipronil on A282S;T286V mutant silkworm RDL GABAR expressed in Xenopus oocytes.After three control responses to GABA (30 μM for wild-type and 300 μM for A282S;T286V mutant), fipronil was bath-applied for 10 min, during which GABA was also applied every 2 min. The last peak current amplitude recorded in the presence of fipronil was normalized to the maximum response to GABA recorded before application of fipronil. Chrodrimanin B on A282S;T286V mutant was tested using the same method as described for Fig 3. Each plot represents the mean ± SEM of three experiments. The pIC50 values (mean ± SEM) of chrodrimanin B on A282S;T286V mutant and fipronil on wild-type and A282S;T286V mutant were 8.83 ± 0.04 (IC50 = 1.48 nM), 7.41 ± 0.03 (IC50 = 39.4 nM) and 6.24 ± 0.07 (IC50 = 583 nM), respectively.
	Fig 7. Effects of chrodrimanin B on the GABA-induced responses of human α1β2γ2 GABAR expressed in Xenopus oocytes.(A) 1 μM chrodrimanin B was continuously bath-applied for 5 min and then co-applied with 30 μM GABA for 2 s after three successive control applications of GABA. Chrodrimanin B tested at 1 μM blocked the peak current amplitude of the response to GABA of the human GABAR by 38.9 ± 3.9% (n = 3). (B) Chrodrimanin B was bath-applied for 5 min and then co-applied with 30 μM GABA at different concentrations (30 nM—30 μM). Each plot represents the mean ± standard error of the mean of three experiments. The IC50 value of chrodrimanin B was 1.48 μM.
	Fig 1. Structures of chrodrimanins A, B and D.

References [+] :

Akaike, Bicuculline and picrotoxin block gamma-aminobutyric acid-gated Cl- conductance by different mechanisms. 1985, Pubmed