Nodulisporic acid produces direct activation and positive allosteric modulation of AVR-14B, a glutamate-gated chloride channel from adult Brugia malayi

Abstract

Glutamate-gated chloride channels (GluCls) are unique to invertebrates and are targeted by macrocyclic lactones. In this study, we cloned an AVR-14B GluCl subunit from adult Brugia malayi, a causative agent of lymphatic filariasis in humans. To elucidate this channel's pharmacological properties, we used Xenopus laevis oocytes for expression and performed two-electrode voltage-clamp electrophysiology. The receptor was gated by the natural ligand L-glutamate (effective concentration, 50% [EC₅₀] = 0.4 mM) and ivermectin (IVM; EC₅₀ = 1.8 nM). We also characterized the effects of nodulisporic acid (NA) on Bma-AVR-14B and NA-produced dual effects on the receptor as an agonist and a type II positive allosteric modulator. Here we report characterization of the complex activity of NA on a nematode GluCl. Bma-AVR-14B demonstrated some unique pharmacological characteristics. IVM did not produce potentiation of L-glutamate-mediated responses but instead, reduced the channel's sensitivity for the ligand. Further electrophysiological exploration showed that IVM (at a moderate concentration of 0.1 nM) functioned as an inhibitor of both agonist and positive allosteric modulatory effects of NA. This suggests that IVM and NA share a complex interaction. The pharmacological properties of Bma-AVR-14B indicate that the channel is an important target of IVM and NA. In addition, the unique electrophysiological characteristics of Bma-AVR-14B could explain the observed variation in drug sensitivities of various nematode parasites. We have also shown the inhibitory effects of IVM and NA on adult worm motility using Worminator. RNA interference (RNAi) knockdown suggests that AVR-14 plays a role in influencing locomotion in B. malayi.

Keywords: AVR-14B; Brugia malayi; filarial nematode; glutamate-gated chloride channels; nodulisporic acid.

Fig. 1.

Effect of IVM and NA on adult worm motility. (A) Time- and concentration-dependent effects of IVM on adult female (n ≥ 10) and male (n ≥ 10) B. malayi worms in 24-well plates. Motility was recorded with the Worminator system. (B) Time- and concentration-dependent effects of NA on adult female (n ≥ 10) and male (n ≥ 10) B. malayi worms in 24-well plates. Motility was recorded with the Worminator system. (C) Effect of RNAi knockdown of Bma-avr-14 on adult worm motility. Time series of motility for dsRNA-soaked adult female (n = 15) and male (n = 15) worms. Black data points represent untreated control. Error bars represent the SEM.

Fig. 2.

Protein sequence alignment of selected nematode AVR-14B homologs showing ligand-binding loops, transmembrane domain (TM1-TM4), pre-TM1 region, and TM2-TM3 linker region. Residues of the amino acid agonist (AAA) motif in the loops that contact bound glutamate are illustrated in maroon. Residues that reduce agonist sensitivity are highlighted in purple in the pre-TM1 and TM2-TM3 linker region. PAR motif, which is characteristic of LGICs, is coded in orange in the TM2 region. The sites of mutation that reduced IVM sensitivity are illustrated in a black box in the TM regions. Cel, C. elegans; Bma, B. malayi; Hco, H. contortus; Dim, D. immitis; Con, C. oncophora.

Fig. 3.

Pharmacology of B. malayi AVR-14B. (A) Representative traces (Top) for agonist responses and bar chart (Bottom) showing mean ± SEM, %, for normalized current responses to the different agonists tested at 1 mM. Rank order series: L-glutamate (100 ± 0.0, n = 13) > ibotenate (73.4 ± 2.5, n = 6) ⋙ kainate (n = 6) = L-quisqualate (n = 6) = L-aspartate (n = 7) = glycine (n = 7) = GABA (n = 7) = AMPA (n = 7) = NMDA (n = 7). (B) Representative inward current traces and concentration-response plot for L-glutamate (n ≥ 5). L-glutamate produced an EC₅₀ = 0.4 mM (pEC₅₀ of 3.4 ± 0.2) and n_H = 0.8 ± 0.3. (C) Representative trace (Top) and concentration-inhibition curve (Bottom) for L-glutamate (L-glu)–mediated response in the presence of PTX (n = 5). The IC₅₀ and n_H values were 33.1 µM (pIC₅₀ = 4.5 ± 0.2) and −1.2 ± 0.6. (D) Representative trace (Top) and concentration-inhibition curve (Bottom) for L-glutamate–mediated response in the presence of FIP (n = 5). The IC₅₀ and n_H values were 1.8 µM (pIC₅₀ = 5.7 ± 0.1) and −1.3 ± 0.4. L-glu, L-glutamate. Error bars in A–D represent SEM.

Fig. 4.

Activation of B. malayi AVR-14B by IVM. (A) Representative trace showing the cumulative concentration-response relationship for IVM. (B) Cumulative concentration-response relationship curve for IVM. The EC₅₀ and n_H values for IVM were 1.9 nM (pEC₅₀ = 8.7 ± 1.2) and 0.3 ± 0.2. (C and D) Representative trace (C) and scatter plot (D) with bar showing the effect of 0.01 nM IVM (n = 6) on 100-µM, 1-mM, and 10-mM L-glutamate–mediated current responses (expressed as mean ± SEM, %; normalized to 10 mM L-glutamate response); 0.01 nM IVM produced significant inhibition of 1-mM (54.3 ± 9.2 alone; 34.2 ± 7.5 in the presence of IVM) and 10-mM (100.0 ± 0.0 alone; 70.3 ± 7.9) L-glutamate–mediated current responses. (E and F) Representative trace (E) and scatter plot (F) with bar showing the effect of 0.1 nM IVM (n = 6) on 100-µM, 1-mM, and 10-mM L-glutamate–mediated current responses (mean ± SEM %; normalized to 10-mM L-glutamate response); 0.1 nM IVM produced significant inhibition of 100-µM (12.9 ± 3.7 alone; 1.3 ± 0.3 in the presence of IVM), 1-mM (52.8 ± 3.3 alone; 4.0 ± 0.6 in the presence of IVM), and 10-mM (100.0 ± 0.0 alone; 7.7 ± 0.7 in the presence of IVM) L-glutamate–mediated current responses. Two-way ANOVA; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; significantly different as indicated; Bonferroni’s multiple comparisons tests. L-glu, L-glutamate. Error bars in B, D, and F represent SEM.

Fig. 5.

Effect of NA on the Xenopus oocytes expressing B. malayi AVR-14B. (A) Representative inward current responses from oocyte challenged with increasing concentrations of NA (n ≥ 5). (B) Representative inward current response from oocyte used for studying the positive modulatory effect of NA on L-glutamate (30 µM) gated current response (n ≥ 5). (C) Example of peak current and AUC measurements of the traces to quantify the PAM effect of NA on L-glutamate gated current response. (D) Concentration-response relationship curves for L-glutamate, NA, and NA in the presence of 30 µM L-glutamate when normalized to 30-mM L-glutamate current response. EC₅₀ and n_H were 0.4 mM (pEC₅₀ = 3.4 ± 0.2) and 0.8 ± 0.3 for L-glu, respectively; 2.7 µM (pEC₅₀ 5.6 ± 0.2) and 1.6 ± 0.8 for NA, respectively. The estimated minimum EC₅₀ and n_H for NA in the presence of 30 µM L-glu was 0.4 µM (pEC₅₀ = 6.4 ± 0.4) and 0.8 ± 0.4, respectively. Bottom was constrained to zero. (E) Concentration-response relationship curves for L- glutamate, NA, and NA in the presence of 30 µM L- glutamate when normalized to 30-mM L- glutamate AUC response. AUC (max) ± SEM, %, was 154.7 ± 10.4 for L- glutamate; 999.4 ± 227.1 for NA; 1430.0 ± 168.3 for NA in the presence of 30 µM L-glutamate. L-glu, L-glutamate. Error bars in D and E represent SEM.

Fig. 6.

IVM acts as an inhibitor of NA-mediated potentiation of L-glutamate–gated currents in the Xenopus oocytes expressing B. malayi AVR-14B. (A) Representative inward current responses showing the effect of IVM (0.1 nM IVM in top trace, n = 6; 1 pM in bottom trace; n = 6) on combination of NA with L- glutamate (L-glu; 30 µM). (B) Concentration-response curves showing the potent inhibitory effect of 0.1 nM IVM on combination of NA (0.1 to 0.3 µM) with L-glutamate (30 µM) when normalized to 30-mM L-glutamate current response. (C) Concentration-response curves showing effect of 1 pM IVM on combination of NA (0.1 to 3 µM) with L-glutamate (30 µM) when normalized to 30-mM L-glutamate current response. EC₅₀ values were 0.4 µM (pEC₅₀ = 6.4 ± 0.4) for NA and L-glutamate combination in the absence of IVM; 0.3 µM (pEC₅₀ = 6.5 ± 1.0) NA and L-glutamate combination in the presence of IVM. There was no significant difference between the Hill slopes. (D) Concentration-response curves showing effect of 1 pM IVM on combination of NA (0.1 to 3 µM) with L-glutamate (30 µM) when normalized to 30-mM L-glutamate AUC response. AUC (max) ± SEM, %, was 1,430.0 ± 168.3 for NA and L-glutamate combination in the absence of IVM; 295.5 ± 101.0 for NA and L-glutamate combination in the presence of IVM. L-glu, L-glutamate. Error bars in B and C represent SEM.

Fig. 7.

(A) Schematic showing the multiple states of Bma-AVR-14B receptor after ligand binding. (B) Diagrammatic representation of the ligand-binding model for Bma-AVR-14B receptor.