GluClR-mediated inhibitory postsynaptic currents reveal targets for ivermectin and potential mechanisms of ivermectin resistance

Abstract

Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor.

Fig 1. Activity of glutamate and IVM at homo- and heteromeric GluClRs.

A. Structure of the GluClR (3RIF, [5]) showing the large extracellular domain with glutamate bound at interfaces between adjacent subunits. Also shown is the transmembrane domain, which consists of four helices (M1-M1) per subunit and IVM bound between the M1 and M3 of adjacent subunits. B. Sequence alignments of selected invertebrate GluClRs subunits showing the IVM binding site, which consists of the first (M1) to third (M3) transmembrane domains. Boxed in red are sites of missense mutations that reduce IVM sensitivity. Abbreviations are H. cont., H. contortus, C. eleg., C elegans, P. xylos., P. xylostella, T. urtic., T. urticae (isoforms 1 and 3) and D. melan., D. melanogaster. C. Example currents obtained in oocytes injected with the β subunit (above) or the α and β subunits (ratio 1:50, below) in response to the indicated glutamate concentrations. D. Group concentration-response data for the indicated GluClRs. Holding potential was –40 mV.

Fig 2. Activity of IVM at homo- and heteromeric GluClRs.

(A-C) Representative concentration-response experiments at the indicated IVM concentrations in oocytes. IVM-induced currents were normalised to the response elicited by a saturating concentration of glutamate (5 mM) for oocytes injected with cDNA encoding the α subunit (A) or α and β subunits at a ratio of 1:50 (B) or 1:1 (C). D. Group concentration-response data for the indicated GluClRs. Holding potential was −40 mV.

Fig 3. IVM analogue potency.

A. Structures of IVM and the three analogues. Arrows indicate the synthesis reaction steps. MOMO represents a methoxymethyl ether group. A red broken border indicates the change in 5-OH configuration between IVM-2 and IVM-3. B. Example experiments in oocytes expressing α GluClRs that test IVM and analogues, IVM-1 and IVM-3. The IVM or analogue-induced current was normalised to the response elicited by a saturating concentration of glutamate (5 mM). C. Group bar plots comparing the potency between α and αβ (1:50) GluClRs for IVM and the three IVM analogues. *** p < 0.001, * p < 0.05. Numbers within the bars indicate the number of experiments (oocytes). Holding potential was −40 mV.

Fig 4. IPSCs mediated by α and αβ GluClRs.

(A-B). A whole-cell recording from HEK293 cell expressing α GluClRs (A) or αβ GluClRs (B) in co-culture with primary neurons. C. Expanded view of a segment from (A) showing isolated IPSCs that rise rapidly to a peak before decaying back to baseline. D. Expanded view of a segment from (B) showing isolated IPSCs that decay faster than those in (C). E. Inhibition of IPSCs mediated by α GluClRs by picrotoxin. (F-H). Group bar plot showing the mean IPSC decay time constant (F), 10–90% rise times (G) and peak amplitude (H) of α and αβ GluClRs. ** p < 0.01. Holding potential was −70 mV.

Fig 5. Effects of IVM at IPSCs mediated by α and αβ GluClRs.

(A-B). A whole-cell recording from a HEK293 cell expressing α GluClRs (A) or αβ GluClRs (B) in co-culture with primary neurons in the continuous presence of 5 nM IVM. Note the steady increase in inward tonic current, which reaches a steady-state plateau at about 17 min in both (A) and (C). C. Expanded view of a segment from (A) showing isolated IPSCs, which have larger peak amplitudes and rise and decay more slowly than the corresponding IPSCs in the absence of IVM. D. Expanded view of a segment from (B) showing isolated IPSCs, which have larger peak amplitudes and rise and decay more slowly than the corresponding IPSCs in the absence of IVM. Note that the peak amplitude is smaller and the rise and decay times are faster than those in (C). E-G. Group bar plot showing the mean IPSC decay time constant (E), 10–90% rise times (F) and peak amplitude (G) of α and αβ GluClRs in the presence and absence of IVM. ** p < 0.01, * p < 0.05, ## p < 0.01. Holding potential was −70 mV.

Fig 6. Single channel properties of α and αβ GluClRs.

A. Single receptor currents from patches expressing αβ (1:1) GluClRs at 2 μM and 3 mM glutamate and 2 μM glutamate plus 5 nM IVM. Note the increase in the duration of the active periods in the presence of IVM. B. Transfections of α and β subunits produced two distinct types of single channel activity based on current amplitude (1.2 and 0.7 pA). The 1.2 pA activity was the most prominent, accounting for 90% of the openings. The active durations of both types increase at higher glutamate concentrations and in the presence of IVM. C. Single receptors currents mediated by β GluClRs in the presence of the indicated concentrations of glutamate and in 2 μM glutamate plus 5 nM IVM. Note only one amplitude level (0.4 pA). D. Amplitude histograms for single receptor activity from patches expressing αβ GluClRs showing the two main amplitude levels (left) and β GluClRs (left). E. Single receptor mean active period durations (left) and intra-activation open probability (right) for αβ GluClRs in response to the indicated ligands. F. Single receptor mean active period durations (left) and intra-activation open probability (right) for β GluClRs in response to the indicated ligands. * p < 0.05. Numbers within the bars indicate the number of experiments (patches). Holding potential was −70 mV.

Fig 7. IVM-bdpy fluorescent probe.

A. Structures of free bdpy fluorophore and IVM-bdpy. B. The spectral emission properties of IVM-bdpy and the free bdpy fluorophore. C. IVM-bdpy retained activity as shown in the sample oocyte current mediated by α GluClRs. D. Group concentration-response plot (n = 6) for IVM-bdpy at α GluClRs, showing reduced potency relative to IVM.

Fig 8. Membrane partitioning and lateral diffusion of IVM-bdpy.

a. Confocal image of a field of HEK293 cells before (left) and after 30 min exposure of 500 nM IVM-bdpy (right). B. Group plot (n = 5) of the change in fluorescence over time for IVM-bdpy (green) the free bdpy fluorophore (black) and the subtraction (IVM-1, yellow). These plots were fitted to exponential functions to yield time constants of 6.5 min for the IVM-bdpy, 8.2 min for the bdpy fluorophore and 6.0 min for IVM-1. C. An example of a FRAP experiment showing a cell after equilibrating in 500 nM IVM-bdpy for 30 min (left), after a circular region (demarcated) was pholobleached with laser energy (middle) and after recovery of the bleached region (right). D. Group plot (n = 5) of the rate of recovery after pholobleaching. The data were fitted to an exponential with a time constant of 1.3 min.