GluCl a target of indole alkaloid okaramines: a 25 year enigma solved

Abstract

In 1989, indole alkaloid okaramines isolated from the fermentation products of Penicillium simplicissimum were shown to be insecticidal, yet the mechanism of their toxicity to insects remains unknown. We therefore examined the action of okaramine B on silkworm larval neurons using patch-clamp electrophysiology. Okaramine B induced inward currents which reversed close to the chloride equilibrium potential and were blocked by fipronil. Thus it was tested on the silkworm RDL (resistant-to-dieldrin) γ-aminobutyric-acid-gated chloride channel (GABACl) and a silkworm L-glutamate-gated chloride channel (GluCl) expressed in Xenopus laevis oocytes. Okaramine B activated GluCl, but not RDL. GluCl activation by okaramines correlated with their insecticidal activity, offering a solution to a long-standing enigma concerning their insecticidal actions. Also, unlike ivermectin, okaramine B was inactive at 10 μM on human α1β2γ2 GABACl and α1β glycine-gated chloride channels and provides a new lead for the development of safe insect control chemicals.

Figure 1. Structures of okaramines A, B, 4′,5′-dihydrookaramine B (okaramine B-H₂), I and Q.

Figure 2. Action of okaramine B on the membrane currents of silkworm larval neurons as measured with whole-cell patch-clamp electrophysiology (a–d) and on silkworm larval RDL γ-aminobutyric-acid-gated chloride channel (GABACl) (e, f) and L-glutamate-gated chloride channel (GluCl) (g) expressed in Xenopus laevis oocytes as measured with two-electrode voltage-clamp electrophysiology.

Okaramine B was applied via U-tube, whereas antagonists were bath-applied to the neurons. (a) Inward current induced by okaramine B at 1 μM and the effect of nicotinic receptor antagonist mecamylamine (1 μM) applied for 1 min prior to co-application with 1 μM okaramine B. (b) Inward current induced by okaramine B at 1 μM and the effect of ligand-gated chloride channel blocker fipronil (10 μM) applied for 1 min prior to co-application with 1 μM okaramine B. (c) Inward current induced by 1 μM okaramine B at various holding potentials. (d) Current-voltage relationship at two different extracellular chloride concentrations. Each plot indicates the mean ± standard error of repeated experiments (n = 3). (e) Five min after recording the response to 30 μM GABA of RDL, 10 μM okaramine was bath-applied. (f) Okaramine B was bath-applied at 10 μM for 1 min prior to co-application with 30 μM GABA. Okaramine B had a minimal impact on the GABA response RDL. (g) Five min after recording the response to 100 μM L-glutamate of GluCl, 1 and 3 μM okaramine B was bath-applied with at 5-min interval.

Figure 3. Concentration-response relationships of okaramines A, B, 4′,5′-dihydrookaramine B (okaramine B-H₂), I and Q for the Cl⁻ current inducing activity on the silkworm neurons and the GluCl activating activity, and correlations of these two activities with the insecticidal activity on the silkworm larvae.

(a) Concentration-response relationship for the chloride-current inducing activity on the silkworm larval neuron. (b) Concentration-response relationship for the GluCl activating activity. Each plot indicates the mean ± standard error of the mean of repeated experiments (n = 4). (c) 3D plot for the relationship of the GluCl activating activity (X axis), and the Cl⁻ current inducing activity on the larval neuron (Y axis) with the insecticidal activity (n = 3) (Z axis). Each sphere plot in (c) is projected to X-Y, X-Z and Y-Z planes to show its position in each plane.

Figure 4. Actions of okaramine B on human α1β2γ2 GABACl and α1β glycine-gated chloride channel (GlyCl) expressed in Xenopus laevis oocytes.

(a) Three minutes after recording a response to 30 μM GABA, 10 μM okaramine B was bath-applied to oocytes expressing the human α1β2γ2 GABACl. After 3 min wash, 10 μM ivermectin was bath-applied. (b) Okaramine B (10 μM) was pre-applied for 1 min prior to co-application with 30 μM GABA to the α1β2γ2 GABACl. (c) Three minutes after recording a response to 100 μM glycine, 10 μM okaramine B was bath-applied to oocytes expressing the human α1β GlyCl. After 3 min wash, 10 μM ivermectin was bath-applied. (d) Okaramine B (10 μM) was pre-applied for 1 min prior to co-application with 100 μM glycine to oocytes expressing the α1β GlyCl. All the results were reproducible (n = 4).