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Open Access

Peer-reviewed

Research Article

Reconstitution of an N-AChR from Brugia malayi, an evolved change in acetylcholine receptor accessory protein requirements in filarial parasites

  • Jennifer D. Noonan,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing

    Affiliation Institute of Parasitology, McGill University, Ste-Anne-de-Bellevue, Québec, Canada

    ⨯
  • Robin N. Beech

    Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing

    robin.beech@mcgill.ca

    Affiliation Institute of Parasitology, McGill University, Ste-Anne-de-Bellevue, Québec, Canada

    ⨯

Abstract

Neurotransmission is an important target for anthelmintic drugs, where receptor characteristics and response can be examined through reconstitution ex vivo in Xenopus laevis oocytes. The homomeric ACR-16 nicotine sensitive acetylcholine receptors (N-AChRs) of several helminth species have been characterized in this way. Our efforts to reconstitute the N-AChR from the clade III filarial parasite, Brugia malayi using similar conditions, initially produced no detectable response. A robust response to acetylcholine is obtained from the closely related clade III parasite Ascaris suum, suggesting that specific changes have occurred between Ascaris and Brugia. N-AChRs from three species intermediate between A. suum and B. malayi were characterized to provide information on the cause. Maximal response to acetylcholine did not change abruptly, consistent with a discrete event, but rather decreased progressively from A. suum through Dracunculus medinensis, Gonglylonema pulchrum and Thelazia callipaeda. Receptor responses to the characteristic nicotine, and other agonists were generally similar. The decrease in maximal current did correlate with a delayed time to reach larger response. Together, this suggested that the failure to reconstitute the B. malayi N-AChR was one extreme of a progressive decrease and that an issue with synthesis of the receptor in oocytes was responsible. Addition of accessory proteins EMC-6, NRA-2 and NRA-4, in addition to RIC-3, produced a small, but measurable B. malayi N-AChR response. Pharmacological properties of a chimeric B. malayi N-AChR were equivalent to the other species, confirming the receptor response remains unchanged while its production is increasingly dependent on accessory proteins. One possibility is that loss of many subunits for acetylcholine receptors from the filarial nematode genome is linked to new subunit combinations that lead to such a dependence. This novel phylogenetic approach allowed the first characterization of a B. malayi AChR ex vivo and in doing so, provides a framework for the successful characterization of other receptors that have yet to be reconstituted.

Author summary

Many anti-parasitic drugs target neurotransmitter receptor ion-channels in the nervous system. These are commonly examined through expression outside the parasite, in frog oocytes. Many channel types have been characterized this way, including the nicotine-sensitive acetylcholine receptor (N-AChR) in nematodes. Initial attempts to reconstitute an N-AChR from the human parasite, Brugia malayi, were unsuccessful. This was surprising, since the N-AChR from the closely related Ascaris suum produces a robust response and both species respond to nicotine in vivo. We compared N-AChRs from three species intermediate between A. suum and B. malayi and found that all responded similarly to a panel of channel activators, ruling out a change in functional characteristics. The response of each species receptor decreased progressively from A. suum to B. malayi and the time required to produce a response increased. This suggested a change in production of the receptor to maturity. Addition of three known chaperone proteins increased the response of intermediate species N-AChRs and produced a first response of the B. malayi N-AChR. This novel approach to use evolutionary change to characterize functional properties of parasite channels allowed the first characterization of a B. malayi nicotinic receptor and provides a method to systematically address difficulties in receptor reconstitution.

Citation: Noonan JD, Beech RN (2022) Reconstitution of an N-AChR from Brugia malayi, an evolved change in acetylcholine receptor accessory protein requirements in filarial parasites. PLoS Pathog 18(11): e1010962. https://doi.org/10.1371/journal.ppat.1010962

Editor: Richard J. Martin, Iowa State University, UNITED STATES

Received: June 6, 2022; Accepted: October 29, 2022; Published: November 14, 2022

Copyright: © 2022 Noonan, Beech. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: The funding for this research was a Discovery Research Grant (RGPIN/05320-2020) from the Canadian National Sciences and Engineering Research Council to RB. JN was the recipient of an NSERC PGSD Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Random drugs screens for antiparasitic compounds have produced several that target neurotransmission and suggest post-synaptic, neurotransmitter receptors are useful targets for anthelmintic drugs [1–4]. These pentameric ligand-gated ion channels (pLGICs) are composed of five subunits that form a radially symmetrical complex embedded in the post-synaptic membrane [5]. Subunits of these channels are encoded by a gene family, derived from an ancestral gene shared with the prokaryotes [6]. This family has expanded in the nematodes and is characterized by many gene duplication and loss events [7,8]. Their 3D structure is highly conserved where each subunit contains a large extracellular domain (ECD) [9], four transmembrane domains (TM 1–4) [10] and a large, variable intracellular loop (ICL) [11]. Activating ligands bind at the ECD interface between adjacent subunits. Anthelmintics that activate these channels lead to paralysis, death and/or expulsion of the worm [12–14] and so represent a significant biological target for the control of infection [15].

Identification of different target receptors has typically been achieved using genetic techniques in the model nematode Caenorhabditis elegans [3,14,16–19]. Confirmation of the composition and characteristics of specific receptors has then been facilitated by expression of subunits, ex vivo, in the oocytes of Xenopus laevis [1,2,20–22]. More recently, identification of predicted subunit genes in genome data, followed by direct expression in oocytes has proved an effective tool to identify previously unknown receptor classes directly [2]. There are limitations to this technique since expression of receptors ex vivo occurs in the absence of the many accessory proteins they normally interact with during assembly and stabilization of precursors to the mature receptor complex in vivo [23]. More than a dozen accessory proteins have been characterized in C. elegans that interact with the acetylcholine receptors (AChRs) although only a few are required ex vivo. These include RIC-3, that is required for the nicotine sensitive acetylcholine receptor (N-AChR) encoded by acr-16 [1,24], and UNC-50 and UNC-74, together with RIC-3 that are required for the levamisole sensitive receptor (L-AChR) [1]. These proteins have homologs in vertebrates but their expression in the oocyte appears insufficient for reconstitution of the nematode receptors since over-expression of the Xenopus RIC-3 can successfully reconstitute the C. elegans N-AChR [25,26].

The filarial nematodes, such as Brugia malayi, are an important group of parasites where the range of antiparasitic drugs is limited and where the mechanism of action for drugs that are available is much less clear than for species more closely related to C. elegans [27]. Characterization of neurotransmitter receptors in filarial worms ex vivo will therefore be an important resource. Two acetylcholine-sensitive neurotransmitter receptors in the muscle regulate body movement in C. elegans, the N-AChR and L-AChR [28]. In parasitic nematodes, a third is present, encoded by acr-26 and acr-27, that is sensitive to morantel (M-AChR) [2]. While four different acetylcholine receptors have been demonstrated in living B. malayi, representing the L-AChR, N-AChR, M-AChR and one sensitive to pyrantel (P-AChR) [29–31], none have been characterized in recombinant systems.

The N-AChR, a homomeric receptor composed of ACR-16, has been characterized in many different nematode species including Ancylostoma caninum [32], A. ceylanicum [33], Necator americanus [33], Trichuris suis [34], T. muris [35] and two species basal to the filarial nematode clade: Ascaris suum (Asu-ACR-16) [36] and Parascaris equoram [37]. Unfortunately, initial attempts in our hands to reconstitute the B. malayi N-AChR (Bma-ACR-16) in the presence of RIC-3 were unsuccessful. To understand the reason for this, we characterized the N-AChR from three clade III nematode species intermediate between A. suum and B. malayi, namely Dracunculus medinensis (Dme-ACR-16), Gonglyonema pulchrum (Gpu-ACR-16) and Thelazia callipaeda (Tzc-ACR-16) [37]. Each one of these intermediates are independent representatives of N-AChR change along the clade III phylogeny. Using this approach, we were able to show that the response of the N-AChR from these species, to a variety of agonists was not substantially different from each other. The maximal response however, decreased and took longer to achieve progressively from A. suum to T. callipaeda, with no response from B. malayi. This suggested a progressive change along the phylogeny in receptor assembly and/or surface transport was responsible. Addition of accessory proteins EMC-6 [38], NRA-2 and NRA-4 [23,39] allowed reconstitution and characterization of the first B. malayi AChR ex vivo. The use of an evolutionary approach to follow the change in N-AChR function was a novel and effective way to investigate the mechanism responsible for the failure to reconstitute an N-AChR from B. malayi. This method may prove useful for the study of other helminth AChRs as drug targets.

Results

ACR-16 is conserved across nematodes

An N-AChR has been reconstituted and characterized as a homopentamer of ACR-16 from several different nematode species [24,32–34,36,40]. In each case, co-expression of the RIC-3 accessory protein is required for a robust response. These include the clade IIIb nematode, A. suum that encodes an N-AChR substantially similar to that found in the clade V nematode C. elegans [1,24,36]. The genome of the clade IIIc filarial parasite, B. malayi [41], also contains an acr-16 that is expected to encode a functional N-AChR since the worm responds to nicotine (NIC) in vivo with an influx of cations as expected from functional AChRs [29,42]. Perhaps surprisingly, the B. malayi N-AChR failed to reconstitute a functional homomeric receptor ex vivo in the presence of RIC-3. Two likely explanations for this are either reconstitution of a receptor whose functional characteristics have changed sufficiently for the receptor to no longer respond to a panel of conventional ligands or that no functional receptor is produced in the oocyte expression system. The fact that the A. suum N-AChR can be reconstituted [36], but not B. malayi indicates a specific change has occurred in the more derived clade III ACR-16. To identify this change and when it occurred along filarial phylogeny, we characterized the N-AChR from three intermediate species (Fig 1).

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Fig 1. ACR-16 phylogeny in the nematodes.

ACR-16 is conserved across worms with short branches observed within the clade III worms. Clade III and clade V ACR-16 sequence group as expected following the worm phylogeny [37]. B. malayi ACR-16 groups within the later clade III nematodes, showing no unexpected branch topology that would explain the inability to measure a B. malayi N-AChR receptor ex vivo. Clade V sequences shaded in blue, clade IIIa in light yellow, and clade IIIb in dark yellow. Clade III ACR-16 sequences used in this study indicated in blue. Bootstrap resampling branch support values are shown. Scale bar represents substitution rate.

https://doi.org/10.1371/journal.ppat.1010962.g001

The phylogeny and aligned primary sequence of ACR-16 for C. elegans, A. suum, D. medinensis, G. pulchrum, T. callipaeda and B. malayi in order of divergence from A. suum are shown in Figs 1 and 2. A majority of changes occurred within the intracellular loop, as expected since this region is commonly found to be the most variable region within pLGIC receptors [11]. The transmembrane domains had few changes, confined to TM2 and TM4. The ion charge filter motif, located at the start of TM2, (GEK) was conserved throughout, as such receptors were expected to be cation permeable [10]. Substitution within TM2 is similar in side chain chemistry (I287V) and is therefore not expected to alter ion-selectivity. Substitutions within the six agonist binding loops were not residues that interact with cholinergic ligands, so it seems unlikely that a significant change in activating ligand has occurred [43,44]. Overall, no substitutions within functionally important regions were found within the B. malayi ACR-16 sequence that could explain the inability to detect a receptor in oocytes.

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Fig 2. ACR-16 is conserved in clade III worms.

Five clade III ACR-16 were used to identify why no B. malayi N-AChR can be measured in oocytes. Predicted coding sequences were synthesized from their respective genomes obtained from WormBase ParaSite WBPS13 [81,87] with primary protein sequence alignment shown. C. elegans ACR-16 was included for reference as it has been previously characterized [1,24]. All functionally important regions and motifs are conserved within the B. malayi sequence and implies that no specific substitutions explain the inability of measuring an N-AChR. Transmembrane domains are shown in blue, signal peptide in pink, acetylcholine binding loops in brown, cys-loop in green and residues that interact with agonist in red [43,44].

https://doi.org/10.1371/journal.ppat.1010962.g002

Clade III N-AChRs show progressive declining maximal response

Each ACR-16 subunit was co-injected with H. contortus RIC-3 and, except for B. malayi, produced measurable agonist-induced currents. A progressive decline in maximal response to both acetylcholine (ACh) and nicotine (NIC) was observed from A. suum to B. malayi (Fig 3A–3B). Receptor currents were compared to Asu-ACR-16 as it lies at the base of the clade and has been previously characterized [36,37]. For acetylcholine-induced currents, Asu-ACR-16 (2052 ± 66 nA) and Dme-ACR-16 (2150 ± 59 nA, p = 0.2787) produced the largest and did not differ statistically (Fig 3A). In phylogenetic order, Gpu-ACR-16 produced smaller currents (1401 ± 91 nA, p<0.0001) and Tzc-ACR-16 produced even smaller measurable currents (563 ± 39 nA, p<0.0001). Bma-ACR-16 produced no detectable response (0 ± 0 nA, p< 0.0001). For nicotine-induced currents, Asu-ACR-16 (2067 ± 74 nA) and Dme-ACR-16 (2025 ± 81 nA, p = 0.7134) again produced the highest currents that were not statistically different. Gpu-ACR-16 produced significantly smaller currents (1231 ± 98 nA, p<0.001), and Tzc-ACR-16 produced the smallest measurable currents (481 ± 45 nA, p<0.0001). Bma-ACR-16 again produced no measurable current (0 ± 0 nA, p< 0.0001). The same decline in response was observed when B. malayi RIC-3 replace H. contortus RIC-3 and was therefore not caused by incompatibility with the clade V accessory protein (Figs 3C and S1A). Endogenous calcium-gated chloride channels in oocytes have been found to alter current response [1]. Preliminary assessment with the intracellular calcium chelator, BAPTA-AM, showed no difference in response (S1B Fig), however a noticeable decline in oocyte quality was observed, therefore BAPTA-AM was omitted in further analysis. The gradual decline in ACR-16 current along its phylogeny ruled out that a discrete evolutionary event was responsible.

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Fig 3. N-AChR has phylogenetic declining response and delayed expression in oocytes.

To identify why no B. malayi N-AChR was measured in a heterologous expression system, N-AChRs from related species, along the clade III phylogeny, were reconstituted in oocytes. (A) A declining response to 100 μM acetylcholine was measured when co-injected with H. contortus RIC-3. A. suum served as the positive reference receptor. In order of phylogeny, responses to acetylcholine were 2052 ± 66 nA (n = 13, black) for A. suum; 2150 ± 59 nA (n = 15, p = 0.2787, blue) for D. medinensis; 1403 ± 91 nA (n = 14, p<0.0001, orange) for G. pulchrum; 563 ± 39 nA (n = 13, p<0.0001, green) for T. callipaeda, 0 ± 0 nA (n = 12, p<0.0001, grey) for B. malayi. ****p<0.0001. (B) Responses to 100 μM nicotine were 2067 ± 74 nA (n = 12, black) for A. suum; 2025 ± 81 nA (n = 15, p = 0.7134, blue) for D. medinensis; 1231 ± 98 nA (n = 14, p<0.0001, orange) for G. pulchrum; 481 ± 45 nA (n = 13, p<0.0001, green) for T. callipaeda, 0 ± 0 nA (n = 12, p<0.0001, grey) for B. malayi. The declining response indicates a progressive adaptation has occurred within the clade that decreases response to ligand. Error bars represent standard error, circles represent recordings from individual oocytes. ****p<0.0001. (C) A declining response to 100 μM acetylcholine was obtained with the addition of other accessory protein conditions: B. malayi RIC-3; B. malayi RIC-3 + UNC-50 + UNC-74; B. malayi RIC-3 + C. elegans EMC-6; and B. malayi RIC-3 + C. elegans NRA-2 + NRA-4. Small B. malayi N-AChR responses were measured when including EMC-6 (17 ± 4 nA, n = 13, p<0.01) or NRA-2 + NRA-4 (4 ± 2 nA, n = 12, p<0.01). Error bars represent standard error. See S1 Fig for additional accessory protein conditions and S1 Table for values. **p<0.01; ***p<0.0005; ****p<0.0001. (D) An expression analysis was carried out to measure the time taken to reach larger response for each N-AChR. Responses to 100 μM acetylcholine was measured for three consecutive days post-injection and normalized to responses obtained on Day 3. In order of phylogeny, 44 ± 5% (n = 12, p<0.0001) and 88 ± 5% (n = 10, p = 0.0841) for A. suum in black, 49 ± 7% (n = 10, p<0.0001) and 102 ± 7% (n = 10, p = 0.7011) for D. medinensis in blue, 19 ± 6% (n = 17, p<0.0001) and 76 ± 6% (n = 14, p<0.05) for G. pulchrum in orange, 7 ± 1% (n = 11, p<0.0005) and 47 ± 12% (n = 10, p<0.05) for T. callipaeda in green, for Days 1 and 2, respectively. A. suum and D. medinensis reached full response by Day 2 that is not significantly different from Day 3, in contrast to G. pulchrum and T. callipaeda responses on Day 2 that were significantly smaller. The delayed time to reach larger responses confirms changes to receptor functional synthesis contribute to the declining response observed. Error bars represent standard error. *p<0.05; ***p<0.0005; ****p<0.0001.

https://doi.org/10.1371/journal.ppat.1010962.g003

Clade III N-AChRs have similar pharmacological response in oocytes

Agonist affinity was measured with half maximal effective concentration (EC50) for both ACh (Fig 4A) and NIC (Fig 4B). Concentration-response curves were comparable between species. Consistent with previous characterization of Asu-ACR-16 [36], EC50 for Asu-ACR-16 was 8.9 μM, 95% CI [8.2, 9.5] for ACh and 5.1 μM, 95% CI [4.6, 5.6] for NIC. EC50 for Dme-ACR-16 was 12.8 μM, 95% CI [12.0, 13.6] and 3.8 μM, 95% CI [3.5, 4.1], for Gpu-ACR-16 was 10.5 μM, 95% CI [9.6, 11.4] and 2.1 μM, 95% CI [1.9, 2.3] and for Tzc-ACR-16 was 17.4 μM, 95% CI [15.9, 19.0] and 4.9 μM, 95% CI [4.2, 5.6], for ACh and NIC respectively. A higher affinity was observed for NIC compared to ACh for all N-AChRs, agreeing with the published characterization of Asu-ACR-16 [36] and all receptors reached saturation (example recordings are shown in Fig 4C–4F).

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Fig 4. Clade III N-AChR show similar agonist affinities.

Concentration- response curves were measured to determine if declining maximal response is due to decreasing ligand affinity. Concentration-response curves for clade III N-AChRs for (A) acetylcholine and (B) nicotine. In order of phylogeny; A. suum in black, D. medinensis in blue, G. pulchrum in orange, T. callipaeda in green. EC50 were A. suum 8.9 μM, 95% CI [8.2, 9.5] (hill coefficient = 1.8, 95% CI [1.6, 2.1]; n = 11) and 5.1 μM, 95% CI [4.6, 5.6] (hill coefficient = 2.0, 95% CI [1.7, 2.3]; n = 13), D. medinensis 12.8 μM, 95% CI [12.0, 13.6] (hill coefficient = 1.7, 95% CI [1.6, 1.9]; n = 11) and 3.8 μM, 95% CI [3.5, 4.1] (hill coefficient = 1.6, 95% CI [1.4, 1.8]; n = 14), G. pulchrum 10.5 μM, 95% CI [9.6, 11.4] (hill coefficient = 2.1, 95% CI [1.7, 2.5]; n = 15) and 2.1 μM, 95% CI [1.9, 2.3] (hill coefficient = 2.3, 95% CI [1.9, 2.7]; n = 13), T. callipaeda 17.4 μM, 95% CI [15.9, 19.0] (hill coefficient = 2.1, 95% CI [1.7, 2.4]; n = 10) and 4.9 μM, 95% CI [4.2, 5.6] (hill coefficient = 1.9, 95% CI [1.5, 2.3]; n = 14), for acetylcholine and nicotine, respectively. Representative recordings in response to acetylcholine are shown for (C) A. suum. (D) D. medinensis, (E) G. pulchrum and (F) T. callipaeda. Similar concentration-response curves confirm the declining response is not due to changes in agonist affinity. Error bars represent standard deviation.

https://doi.org/10.1371/journal.ppat.1010962.g004

Responses to each of the characteristic ligands for the four classes of AChRs identified in B. malayi were determined, namely levamisole (LEV), NIC, morantel (MOR), and pyrantel (PYR), [2,29,30,45,46]. Responses relative to ACh showed similar profiles across the species (Fig 5). Each receptor had a large response to NIC, smaller response to dimethylphenylpiperazinium (DMPP) and oxantel (OXA), and minimal response to MOR and pyrantel (PYR). No response was measured for LEV, epinephrine (EPI), norepinephrine (NOR), dopamine (DA), tyramine (TYR), betaine (BET) or bephenium (BEPH) for any receptor. Ligand-response profiles coincided with previous clade III N-AChR characterization [36,40] and confirm no meaningful change in ligand interaction has occurred. Taken together, the agonist-response panel and concentration-response curves ruled out a pharmacological explanation for the trend observed in Fig 3A.

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Fig 5. No change in ligand response profile is observed in clade III N-AChRs.

N-AChR responses to different pLGIC agonists were measured to determine if declining maximal response is due to changing ligand preference. Current responses to 100 μM for all ligands are shown normalized to 100 μM acetylcholine for (A) A. suum, (B) D. medinensis, (C) G. pulchrum, (D) T. callipaeda. All four receptors had similar response profiles: ACh ≥ NIC >> DMPP> OXA = MOR = PYR > LEV = EPI = NOR = DA = TYR = BET = BEPH. Circles represent responses from individual oocytes. Similar ligand response profile indicates no change in ligand occurs down clade III receptor phylogeny. **p<0.01; ***p<0.0005; ****p<0.0001. Error bars represent standard error. n>6 for all.

https://doi.org/10.1371/journal.ppat.1010962.g005

Later clade III N-AChRs show delayed response

Different classes of AChR require different expression times, typically two days for the N-AChR and five days for the L-AChR [1,33,47,48]. A standard of two days was chosen for expression in this study based on this previous work. Once characteristics of the receptor had been ruled out as an explanation for decreased response in later Clade III ACR-16, this suggested an issue with expression, and if so would require a longer expression time. To evaluate this, ACh-induced responses were measured each day after injection of receptor cRNA, for three consecutive days and normalized to responses obtained on Day 3. Receptors for each species took progressively longer to reach the largest response along the phylogeny (Fig 3D). After Day 1, both Asu-ACR-16 and Dme-ACR-16 had nearly half of their Day 3 maximal response; 44 ± 5%, p<0.0001 and 49 ± 7% p<0.0001, respectively. By Day 2 both receptors had reached responses that were no longer significantly smaller than Day 3, 88 ± 5%, p = 0.0841 for Asu-ACR-16 and 102 ± 7%, p = 0.7011 for Dme-ACR-16. This in contrast to G. pulchrum and T. callipaeda that did not reach largest response by Day 2. On Day 1, Gpu-ACR-16 had only 19 ± 6%, p<0.0001 of its Day 3 response, and on Day 2 had reached 76 ± 6%, p<0.05 response. On Day 1, Tzc-ACR-16 had 7 ± 1% p<0.0005 of its Day 3 response, and by Day 2 had only reached 47 ± 12% p<0.05. These results indicate that earlier clade III N-AchRs reach larger responses more efficiently compared to later clade III receptors.

B. malayi ACR-16 requires at least three additional accessory proteins

No meaningful difference in the way the N-AChR from different species responded to various agonists, coupled with delayed maximal responses in more derived receptors suggested that a decrease in overall receptor function was likely responsible for the decreased response. The nicotine response of B. malayi in vivo [29,42], the failure to reconstitute a receptor ex vivo, together with a delayed time to maximal response for the species closest to B. malayi suggests a change that affected expression ex vivo specifically. This could be due to an increased dependence on one or more members of the network of regulatory accessory proteins that were not included in the cRNA injected into oocytes. A review of accessory proteins known to be involved in C. elegans AChR expression identified UNC-50, UNC-74, MOLO-1, EAT-18, EMC-6, NRA-2 and NRA-4 as possible candidates [1,38,39,49,50]. These accessory proteins were then included with each N-AChR individually or in a series of combinations to determine if a specific combination was needed to produce the B. malayi N-AChR. UNC-50 and UNC-74 were co-injected because of their joint requirement for expression of the L-AChR in oocytes [1,48,51]. NRA-2 and NRA-4 were co-injected because they function in tandem for subunit assembly [39].

Receptor responses in the presence of different accessory protein conditions are shown in Figs 3C and S1, and S1 Table. The response of each receptor with B. malayi RIC-3 was used as the reference condition for the other accessory protein conditions. In general, the same progressive decline was observed, regardless of accessory protein combination, however some combinations produced significant deviations from the B. malayi RIC-3 reference condition. Responses for Asu-ACR-16 were higher under all additional accessory protein conditions except for when MOLO-1 was added which decreased the response (1525 ± 5 nA, p<0.0001, S1 Fig). The absence of any accessory protein produced an inconsistent, little to no response (24 ± 10 nA, p<0.0001, S1 Fig). None of the additional accessory proteins affected Dme-ACR-16 responses. Interestingly, when no accessory protein was included, it still produced large consistent current (1290 ± 70 nA, p<0.0001, S1 Fig). Current responses for Gpu-ACR-16 were increased when RIC-3, UNC-50, UNC-74 and EAT-18 were combined (1854 ± 83, p<0.01, S1 Fig) and when RIC-3, NRA-2 and NRA-4 were combined (2553 ± 123 nA, p<0.0001, Fig 3C). As expected, current responses were completely inhibited without any RIC-3 (0 ± 0 nA, p<0.0001, S1 Fig). Current responses for Tzc-ACR-16 were decreased when RIC-3, UNC-50 and UNC-74 were combined (367 ± 43nA, p<0.0005, Fig 3C), increased when RIC-3, NRA-2 and NRA-4 were combined (1263 ± 88 nA, p<0.01, Fig 3C) and abolished when no accessory protein was included (0 ± 0 nA, p<0.0001, S1 Fig). BMA-ACR-16 only produced measurable currents when RIC-3 + EMC-6 (17 ± 4, p<0.01, Fig 3C) or RIC-3, NRA-2 + NRA-4 (4 ± 2, p<0.05, Fig 3C) were combined. Therefore, EMC-6, NRA-2, and NRA-4 accessory proteins play a more critical role for functional expression of more derived clade III receptors. This is seen by decreased receptor response in a heterologous system where the native accessory proteins are not present.

The increased dependence on EMC-6, NRA-2 and NRA-4 suggests an evolved change in the ACR-16 subunit itself. In order to determine approximately where in the subunit sequence these changes occur, a series of chimeras were made between A. suum and T. callipaeda ACR-16 by exchanging each of their three main structural regions (S2A Fig). T. callipaeda was chosen rather than B. malayi so that the small current observed for T. callipaeda could be used to verify that the chimeras were in fact functional. The response of the chimeras was not straightforward, however they suggested all main structural regions contribute to the reduced responses to varying degrees, with the ECD having the largest effect (S2C Fig). Replacing the T. callipaeda ECD into Asu-ACR-16 (TZC-ECD) inhibited nearly all response to ACh (42 ± 9 nA, p<0.0001) whereas presence of the A. suum ECD in Tzc-ACR-16 (ASU-ECD) significantly increased the response (1819 ± 115 nA, p<0.0001). Both ICL chimeras produced large responses. The T. callipaeda ICL in Asu-ACR-16 (TZC-ICL) was not significantly different from native Asu-ACR-16 (1959 ± 78 nA, p = 0.593). The A. suum ICL in Tzc-ACR-16 (ASU-ICL) significantly increased the response (2237 ± 98 nA, p<0.0001). The A. suum TM regions in Tzc-ACR-16 (ASU-TM) severely decreased maximal response (24 ± 8 nA, p<0.0001), while the Tzc-TM region in the A. suum ACR-16 (TZC-TM) increased the response (2917 ± 105 nA, p<0.0001). To summarize, later clade III N-AChR current responses can be increased with the addition of new accessory proteins EMC-6 or NRA-2 + NRA-4, and they are sufficient to produce a measurable B. malayi N-AChR. Chimeras indicate that all regions may contribute to this requirement, with the ECD having a majority of the effect.

Pharmacology of chimeric B. malayi ACR-16

The original goal of this work was to characterize the response of the B. malayi N-AChR. Initially no response could be seen from Bma-ACR-16. Addition of EMC-6, NRA-2 and/or NRA-4 led to a detectable response to ACh but the currents remained small for all batches of oocytes. The highest responses for Bma-ACR-16 were obtained with RIC-3 + NRA- 2 (33 ± 9 nA, p<0.05, Fig 6B), RIC-3 + NRA-4 (31 ±13 nA, p = 0.0698, Fig 6B) or RIC-3 + EMC-6 (17 ± 4 nA, p<0.05, Fig 3C). Other combinations reduced measurable currents; RIC-3 + NRA-2 + NRA-4 + EMC-6 (11 ± 9 nA, n = 11, p = 0.3108, Fig 3C) and RIC-3 + NRA-2 + NRA-4 EMC-6 + EAT-18 (2 ± 1 nA, p = 0.2019, Fig 6B), and one case abolished the response entirely RIC-3 + NRA-2 + NRA-4 + EMC-6 + EAT-18 + UNC-50 + UNC-74 (0 ± 0 nA, Fig 6B). A similar phenomenon has been seen previously for other N-AChRs [1,32,36].

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Fig 6. malayi N-AChR requires additional accessory proteins and has unchanged pharmacology compared to other clade III N-AChR.

The B. (A) Bma-ACR-16-asuICL and Asu-ACR-16-bmaICL chimeras. (B) Bma-ACR-16-asuICL and Bma-ACR-16 were co-injected with a variety of accessory protein combinations to identify those that produces the highest current response. Bma-ACR-16-asuICL chimera in black, Bma-ACR-16 in blue. Circles represent measured currents from individual oocytes to 100 μM acetylcholine. Bma-ACR-16 responses (compared to RIC-3) to different combinations of EMC-6, NRA-2 and NRA-4 were: RIC-3 + NRA- 2 (33 ± 9 nA, p<0.05); RIC-3 + NRA-4 (31 ±13 nA, p = 0.0698); RIC-3 + EMC-6 (17 ± 4 nA, p<0.05); RIC-3 + NRA-2 + NRA-4 EMC-6 (11 ± 9 nA, n = 11, p = 0.3108); RIC-3 + NRA-2 + NRA-4 EMC-6 + EAT-18 (2 ± 1 nA, p = 0.2019); and RIC-3 + NRA-2 + NRA-4 + EMC-6 + EAT-18 + UNC-50 + UNC-74 (0 ± 0 nA). Bma-ACR-16-asuICL chimera responses under the different accessory protein conditions were: RIC-3 (59 ±43 nA); RIC-3 + NRA-2 (256 ± 295 nA, p<0.05); RIC-3 + NRA-2 + NRA-4 (47 ± 66 nA, p = 0.5887); RIC-3 + EMC-6 (37 ± 46 nA, p = 0.1957); RIC-3 + NRA-2 + NRA-4 + EMC-6 (1 ± 2 nA, p<0.005). RIC-3 + NRA-2 was therefore included for acetylcholine and nicotine concentration-response curves and for ligand panel analysis. Error bars represent standard error. *p<0.05; **p<0.01; ***p<0.0005; ****p<0.0001. (C) Sample recordings of Bma-ACR-16 with various accessory protein conditions in response to 100 μM acetylcholine. (D) concentration-response curves were measured for the Bma-ACR-16-asuICL chimera to compare to the other clade III ACR-16. concentration-response curves for acetylcholine in pink (EC50 = 15.7 μM, 95% CI [12.3, 20.2], n = 6) and nicotine in purple (EC50 = 2.3 μM, 95% CI [1.9, 2.6], n = 8). Hill coefficients were 1.1, 95% CI [0.85, 1.4] for ACh and 1.3, 95% CI [1.0, 1.6] for NIC. Sample recording to acetylcholine is shown. Similar EC50 to other clade III ACR-16 indicates the small currents obtained with Bma-ACR-16 is not due to changes in ligand affinity. Error bars represent standard error. (E) Bma-ACR-16-asuICL chimera response to 100 μM ligands relative to acetylcholine. Bma-ACR-16-asuICL chimera had a comparable response profile to other N-AChR. Relative responses to ACh were NIC (16 ± 3% p<0.05), DMPP (30 ± 3%, p<0.05), OXA (5 ± 2%, p<0.05), MOR (2 ± 1%, p<0.05), and PYR (3 ± 1%, p<0.05), LEV (1 ± 1%, p<0.05),) and no responses to NOR (0 ± 0%, p<0.05), DA (0 ± 0%, p<0.05), BET (0 ± 0%, p<0.05), BEPH (0 ± 0%, p<0.05) and TYR (0 ± 0%, p<0.05). Sample recording to each ligand is shown. Similar ligand response profile to other clade III N-AChRs indicates the small currents obtained with Bma-ACR-16 is not due to changes in ligand preference. Error bars represent standard error. *p<0.05.

https://doi.org/10.1371/journal.ppat.1010962.g006

The ability of the A. suum ACR-16 ICL to increase overall response in the reciprocal chimera (S2C Fig) suggested that a chimera of Bma-ACR-16 containing the A. suum ICL (Bma-ACR-16-asuICL) may produce larger responses overall to be used for B. malayi N-AChR pharmacology characterization since ligand-interaction and gating are determined predominantly by the ECD and TM regions [9,52–55]. Chimeras where the ICL has been exchanged have previously shown not to affect receptor pharmacology [56,57]. A comparison between the Asu-ACR-16 and Asu-ACR-16 containing the B. malayi ICL (Asu-ACR-16-bmaICL) was used as a control to verify that the receptor response was in fact determined by the ECD and TM regions (S3 Fig). Asu-ACR-16-bmaICL EC50 for ACh was 4.3 μM, 95% CI [3.3, 5.5] and for NIC was 2.3 μM, 95% CI [1.9, 2.6] (S3C Fig) and had similar ligand response profile as the full-length Asu-ACR-16 receptor (S3D Fig). The Asu-ACR-16-bmaICL chimera’s similarity in pharmacology to the native Asu-ACR-16 receptor confirms that the ICL exchange in the Bma-ACR-16 chimera provides accurate receptor pharmacology.

The Bma-ACR-16-asuICL chimera responses were small, but RIC-3 + NRA-2 produced the largest and most reproducible currents (256 ± 295 nA, p<0.05, Fig 6B). Other combinations were smaller; RIC-3 + NRA-2 + NRA-4 (47 ± 66 nA, p = 0.5887, Fig 6B); and RIC-3 + EMC-6 (37 ± 46 nA, p = 0.1957, Fig 6B), and RIC-3 + NRA-2 + NRA-4 + EMC-6 (1 ± 2 nA, p<0.005, Fig 6B) (see Fig 6C for sample recordings). Receptor characterization was therefore carried out with the Bma-ACR-16-asuICL chimera co-injected with RIC-3 + NRA-2 and its pharmacology was similar to other N-AChR studied here. The EC50 for ACh and NIC of the Bma-ACR-16-asuICL chimera was 15.7 μM, 95% CI [12.3, 20.2] and 2.3 μM, 95% CI [1.9, 2.6], respectively (Fig 6C). Relative responses to ACh (Fig 6D) were NIC (16 ± 3% p<0.05), DMPP (30 ± 3%, p<0.05), OXA (5 ± 2%, p<0.05), MOR (2 ± 1%, p<0.05), and PYR (3 ± 1%, p<0.05), LEV (1 ± 1%, p<0.05),) and no responses to NOR (0 ± 0%, p<0.05), DA (0 ± 0%, p<0.05), BET (0 ± 0%, p<0.05), BEPH (0 ± 0%, p<0.05) and TYR (0 ± 0%, p<0.05). To summarize, we were successful in measuring the first recombinant B. malayi AChR by including new accessory proteins EMC-6, NRA-2 and NRA-4. Furthermore, our characterization of a chimeric Bma-ACR-16 confirmed this acquired accessory protein requirement occurs independent of pharmacology. Despite the success in measuring the B. malayi N-AChR, its small current responses suggest other accessory proteins are still missing.

Discussion

Reconstitution of anthelmintic target receptors ex vivo has proven critical for understanding the neuromusculature of nematodes. While the response of tissues [30,49], and even single receptors can be determined in vivo [46,58,59], it remains challenging to identify the specific components of these receptors and their detailed characteristics [1]. The N-AChR, a homopentamer of ACR-16 has been characterized through reconstitution for many different nematodes, both parasitic [32–34,36,40] and free-living [24]. An initial attempt to characterize the B. malayi N-AChR failed when co-expressed with RIC-3, an ER localized accessory protein involved in subunit assembly required for other species N-AChR [1,25,32–36,60]. The absence of stop codons or amino acid substitutions at positions known to be critical for receptor function suggested the failure may not have been an experimental artefact.

A. suum produces a robust N-AChR current in Xenopus oocytes [36] and is basal to the clade of filarial nematodes to which B. malayi belongs [37]. We therefore characterized five clade III N-AChRs: A. suum, B. malayi, and three intermediate species in order of phylogeny, D. medinensis, G. pulchrum and T. callipaeda. These species represent independent samples of the ancestral state between A. suum and B. malayi ACR-16 and so may shed light on the changes leading to an inability to reconstitute the receptor. Two possibilities were expected, either a discrete change that would suggest a single mutational event may be responsible, or a gradual decline that would suggest an adaptive change. The maximal response to ACh and NIC declined progressively through the clade of filarial nematodes, with Asu- and Dme-ACR-16 responding the most (~2000–2200 nA), followed by Gpu-ACR-16 (~1400 nA), then Tzc-ACR-16 (~400–600 nA) and Bma-ACR-16 with no measurable current. Therefore, the change appears to be an evolved characteristic.

The progressive decrease in measurable response could be due to a decreased affinity for the activating ligands, possibly with changes in the profile of response to other agonists. This could be ruled out for all since the EC50 for ACh and NIC did not change meaningfully between species and were similar to the other N-AChRs characterized so far (Table 1) [36]. In addition, the relative order of response to a panel of agonists was the same for each species. This is consistent with the fact that a response to nicotine in living B. malayi is found and its characteristics are unremarkable [29,42].

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Table 1. Comparison of pharmacology and accessory proteins required for nematode N-AChR.

https://doi.org/10.1371/journal.ppat.1010962.t001

An alternative to pharmacology changes could be that fewer receptors were expressed at the surface of oocytes ex vivo. If so, the ACR-16 subunit may have become more dependent on components of the processing and transport machinery in B. malayi to the point where receptor assembly and surface expression cannot be measured without additional components ex vivo. This is true more generally for the L-AChR that requires three accessory proteins co-injected within the oocyte system to produce measurable currents [1,14,48,61,62]. An increased dependence on missing factors would make expression less efficient and so take longer for receptors be expressed at the surface. Our expression analysis further supported this idea with progressively longer times to reach large responses. Asu- and Dme-ACR-16 reached largest currents by Day 2 whereas Gpu- and Tzc-ACR-16 did not.

Characterization of accessory proteins associated with expression of cationic pLGICs in nematodes has focused primarily on the L-AChR of C. elegans. However, since the N-AChR and the L-AChR are expressed in the same tissue in C. elegans [3] and other nematodes including B. malayi [29,31], we hypothesized that ACR-16 would potentially interact with those that are co-expressed in vivo. A literature search for characterized C. elegans L-AChR accessory proteins identified seven: UNC-50, UNC-74, MOLO-1, EAT-18, EMC-6, NRA-2 and NRA-4 [1,38,39,49,50,63]. We included EAT-18 that is expressed in the C. elegans pharynx and is required for expression of the EAT-2 receptor that is within the ACR-16 clade [50,64]. B. malayi has lost the gene encoding eat-2 [41] and retention of eat-18 may suggest it has acquired other functions. The choice of species of origin of accessory proteins was based on their availability in the lab as follows: H. contortus RIC-3 B. malayi RIC-3 were both used, B. malayi MOLO-1, EAT-18, UNC-50 and UNC-74 were used, and C. elegans EMC-6, NRA-2 and NRA-4 were used. The location and expected function of these accessory proteins on receptor synthesis is shown in S4 Fig. RIC-3 is an ER-resident protein that directly binds to nascent subunits in a 1-to-1 fashion promoting their assembly [60]. It is a highly versatile accessory protein that regulates the expression of not only invertebrate AChRs, but vertebrate and 5-HT3 receptors as well [65,66]. Although its structure is not known, sequence predictions confirm that the main functional regions are conserved across phyla with 1–2 transmembrane domains followed by an intracellular coiled-coil region [67]. X. laevis RIC-3 is expressed in oocytes [26] however the fact that the co-injection of X. laevis RIC-3 is required to produce nematode ACR-16 [25] indicates it is not expressed in sufficient quantities to enable receptor expression at the cell surface. In our case, the use of B. malayi RIC-3 instead of H. contortus RIC-3 marginally increased the response size of TZC-ACR-16 (S1A Fig), suggesting the species of origin of RIC-3 can be exchanged without affecting receptor function in a meaningful way. Currents of the other ACR-16 were not affected, therefore the observed decline in current sizes across clade III phylogeny is not due to RIC-3 compatibility differences. UNC-74 is a luminal ER-resident protein that acts as a it is required for the L-AChR in oocytes, but not the ACR-16 [1]. EMC-6, NRA-2 and NRA-4 regulate receptor expression in the ER and are discussed in detail below. UNC-50 is a transmembrane protein involved in Golgi-mediated transport and/or modification of the L-AChR [1], while MOLO-1 and EAT-18 are present in surface membrane and modulate the response of receptors. MOLO-1 is a single pass transmembrane protein that associates with and increases the current sizes of the L-AChR by promoting the open probability of the receptor [49]. EAT-18 is required for current responses from the EAT-2 receptor, in the absence of EAT-18, EAT-2 is present at the membrane but in a non-functional form [50]. Only the inclusion of EMC-6 or NRA-2 + NRA-4 together with RIC- 3 resulted in a measurable B. malayi N-AChR.

EMC-6, NRA-2 and NRA-4 all act within various stages of ER receptor synthesis and thus suggests severe changes to ER stages of B. malayi ACR-16 receptor synthesis. EMC-6 is one protein within a larger ER membrane protein complex (termed EMC) that is conserved throughout the eukaryotes and is estimated to contain upwards of 10 different proteins [68]. The EMC functions as an insertase by directing the appropriate insertion of transmembrane domains through the membrane [69,70]. In C. elegans, EMC-6 knock-out led to decreased responses from all three body-wall muscle receptor types (L-AChR, N-AChR and UNC-49) and decreased expression of at least the L-AChR [38]. Including EMC-6 in oocytes may promote the appropriate folding and processing of the B. malayi N-AChR, thus increasing the amount of functional protein at the membrane surface. In the absence of EMC-6, is it possible that the B. malayi N-AChR is not inserted into the membrane properly, which would target it for degradation. Five other EMC-6 paralogs are predicted in the C. elegans genome [38], and it is possible their co-injection may further increase B. malayi N-AChR responses in oocytes. NRA-2 is a nicalin-like homolog and NRA-4 is a NOMO-like homolog [23,39]. In B. malayi, NRA-2 is thought to sense intracellular influx of calcium and regulate the levels of the different L-AChR subunits to produce receptors with different subunit combinations [42]. In C. elegans, NRA-2 and NRA-4 KO worms had reduced L-and N- AChR responses and the L-AChR had altered properties [39]. It was proposed that they function together by determining the subunits that form a channel and exit the ER [39]. In another study, NRA-2 was found to act as a quality control check to retain mutant DEG/ENaC channels in the ER [71]. Their precise function remains unclear but based on current knowledge, including NRA-2 and NRA-4 with more derived clade III N-AChR may promote correct assembly and/or exit of receptors in the ER.

Our strategy to explore the reason for failure to reconstitute a B. malayi N-AChR was to examine intermediate species between A. suum, which reconstitutes well, and B. malayi. The progressive decreased response towards B. malayi suggested that the phenomenon was one extreme of an adaptive response within the filarial nematodes, rather than a discrete event limited to B. malayi. This appears confirmed by the fact that inclusion of NRA-2 and NRA-4 led to a detectable Bma-ACR-16 response and also produced the largest responses of Gpu-ACR-16 and Tzc-ACR-16. Inclusion of EMC-6 also produced a Bma-ACR-16 response. The largest response from the B. malayi N-AChR occurred in combinations of RIC-3 with NRA-2 or NRA-4. Even so, the response was far below what can be expected for an N-AChR expressed in oocytes. We expect other, as yet unidentified accessory proteins are required to obtain a robust receptor ex vivo.

Addition of other accessory proteins that were tried here reduced the observed responses of some ACR-16. A similar phenomenon has been reported previously for other N-AChRs [1,32,36]. We know that the receptor synthesis machinery is complex and involves a network of more than 200 proteins [23]. Interaction between these proteins may influence the overall expression of a specific channel. The presence of only a few of these in the oocytes may dis-regulate their normal function and lead to decreased expression. Two species-specific effects of accessory proteins were also observed. First, MOLO-1 significantly decreased responses for Asu-ACR-16, but not the others. This suggests MOLO-1 interacts with the Asu-ACR-16 receptor in such a way that may decrease the open probability of the channel at the surface membrane [49]. Second, D. medinensis N-AChR no longer required any RIC-3 for expression in oocytes. This was unexpected because all previously characterized N-AChRs require RIC-3 [1,32–36,40] and indicates that specific changes occurred in Dme-ACR-16 altering its interaction with RIC-3.

The increased dependence on accessory proteins for the N-AChR in the filarial nematodes is novel. Since the accessory proteins included were the same for each species, the change in requirement must be due to the specific subunit sequence. Chimeras are a common tool for identifying functional regions within receptor subunits [55,72,73]. Associating the receptor phenotype with the presence of a specific region can infer that the relevant sequence lies within that region. In this case, the phenotype associated with the B. malayi N-AChR is an absence of response that would be indistinguishable from a cloning artefact that led to a non-functional construct. For this reason, the major subunit structural domains were exchanged between A. suum and T. callipaeda ACR-16. These chimeras suggested all regions may contribute to the change in maximal responses. Exchanging the ECD alone reversed the receptor response indicating that the ECD confers the majority of the increased accessory protein requirement. This coincides with the fact that many expression and synthesis signals are found within this region [74–76]. Exchanging the ICL alone was not clear since the constructs with either the Asu-ICL or Tzc-ICL both produced a relatively high response. This may be an indication that a second region conferring increased dependence on additional accessory proteins exists within the ICL, but that it requires a matching region. This may be due to the region being distributed to some extent rather than being discrete. Regardless, the eukaryotic ICLs are highly variable in sequence [11], and their precise structure and function remains unclear. The chimera results presented here agree with previous work showing that the ICL may be involved in regulating receptor synthesis and/or interactions with accessory proteins [77–79].

We could expect that an increased dependence for specific accessory proteins may be due to a physical interaction with them, and our chimeras identified that the majority of the decline is located in the ECD. Interestingly, few amino acid changes were found within the ACR-16 ECD. The Bma-ACR-16 ECD has 9 amino acids substitutions with the Tzc-ACR-16 ECD and 16 with the Asu-ACR-16 ECD. This implies few amino acid changes have a large effect on receptor synthesis and that some of these residues may be critical for receptor synthesis. The ultimate goal of characterizing the B. malayi N-AChR remained challenging due to the small responses observed (<35 nA). To overcome this a chimera of Bma-ACR-16 containing the A. suum ICL (Bma-ACR-16-asuICL) was characterized instead when co-expressed with RIC-3 and NRA-2. We were able to confirm that the complementary chimera of Asu-ACR-16 containing the B. malayi ICL was not substantially different from the Asu-ACR-16, as expected from other similar studies [9,52–54]. These chimeras therefore provide accurate estimates of receptor pharmacology. The Bma-ACR-16-asuICL chimera had similar EC50 and agonist response profiles compared to other clade III receptors suggesting that B. malayi ACR-16 indeed functions as a homomeric nicotinic acetylcholine receptor with an increased dependence on accessory proteins.

Although an increased dependence on additional accessory proteins appears to be a feature of N-AChRs within the filarial nematodes, the reason for this remains unclear. One possibility is that it is a response to the many pLGIC subunit gene loss and duplication events observed within this clade. The filarial parasites have lost subunit genes acr-2, acr-3 and lev-1 and have duplicated unc-38 and unc-63 [7,37,80]. Subunits that comprise pLGICs are subject to a rigorous synthesis pathway with as little as 5–30% of subunits becoming mature receptors at the cell surface [81,82]. A changing receptor subunit landscape means changes to how the subunits interact with each other, potentially leading to increased production of non-functional receptors. Structural change in subunit interaction may be enabled through accessory proteins that stabilize assembly intermediates. This dependence may have led to subunits that enhance the interaction with accessory proteins that would manifest as an increased requirement ex vivo. This could suggest a dynamic and inter-dependent relationship between receptor subunits and their synthesis machinery. This agrees with the fact that all three additionally required accessory proteins act within the subunit folding and/or assembly stages of expression.

Here we present a novel phylogenetic approach to studying receptor change. By characterizing N-AChRs from intermediate species, we were able to show a progressive increase in dependence for EMC-6, NRA-2 and NRA-4 in filarial worms, determined most predominantly by the ECD. Using this approach we have successfully measured the first reconstituted B. malayi AChR while also characterizing three new N-AChRs. The N-AChR characteristics of filarial nematodes has not changed substantially along with this change in accessory protein requirement. Focusing future efforts on proteins related to EMC-6, NRA-2 and NRA-4 may uncover other accessory proteins that increase overall expression of the N-AChR and possibly other AChRs from B. malayi ex vivo. This work highlights the importance of accessory proteins for this receptor family in a heterologous system. Our comparative approach, together with the extensive genome data available for the nematodes, provides an approach that may prove fruitful to investigate the basis for other functional differences in neuromuscular drug target receptors.

Methods

Ethics statement

All work involving animals was carried out under Animal Use Protocol 2022–7758 authorized by the Animal Care Committee of the Office of Research Ethics and Compliance, McGill University.

ACR-16 alignment and phylogeny

Acr-16 sequences of the pLGIC family were identified from WormBase ParaSite WBPS13 [83,84]. The complete sequence was identified for each species except for G. pulchrum. Due to the poorer quality of its genome, eight amino acids (SGEWALPM) in the extracellular domain were missing from a region in the ECD that is identical in the other species and was inserted manually. ACR-16 subunit protein sequences were aligned in Geneious (v 9.0.5, Biomatters Ltd) using the MAFFT plugin (v7.017) [85]. The highly variable signal peptide and intracellular loop sections were removed. A maximum likelihood phylogeny was reconstructed using PhyML [86] with branch support values estimated from 1000 bootstrap resamples using the TBE option [84] and the BEST search option, with five random starting trees. A separate alignment of the ACR-16 studied here is shown in Fig 2.

Accession numbers of sequences used: Tca-acr-16 TCNE_0001320001-mRNA-1; Peq-acr-16 MH806894; Asu-acr-16 GS_23665 + GS_03796; Dme-acr-16 DME_0000017801-mRNA-1; Gpu-acr-16 annotated by hand; Tzc-acr-16 TCLT_0000979901-mRNA-1; Bma-acr-16 Bm13586a.1; Eel-acr-16 EEL_0000848201-mRNA-1; Lsi-acr-16 nLs.2.1.2.t06045-RA; Llo-acr-16 EJD73698.1; Avi-acr-16 nAv.1.0.1.t08089-RA; Ovo-acr-16 OVOC12002.1; Pex-acr-16 PEXSPEC000001098.t1; Ppa-acr-16 PPA34546.1; Hba-acr-16 Hba_18455 + Hba_18456 and annotated by hand; Hco-acr-16 HCON_00147330–00001; Nbr-acr-16 NBR_0001473901-mRNA-1 and annotated by hand; Dvi-acr-16 DICVIV_02269 and annotated by hand; Acn-acr-16 ACAC_0001143301-mRNA-1 and annotated by hand; Hbk-acr-16 HPOL_0000512601-mRNA-1; Ace-acr-16 Acey_s0049.g1864.t1; Aca-acr-16 ANCCAN_01899; Cjp-acr-16 CJA08620a.1; Cel-acr-16 F25G6.3.1; Cbr-acr-16 CBG01491.1

Subunit and accessory protein cloning and RNA synthesis

B. malayi acr-16 and accessory proteins ric-3, unc-50, unc-74 and molo-1 were cloned from adult female B. malayi cDNA received as a gift from Dr. T. Geary using the primers described in S1 Table. B. malayi eat-18 was prepared from two overlapping oligonucleotide primers. Other subunits were synthesized from their predicted genome sequence (Gene Universal, USA) (S2 Table). Asu-acr-16 and Tzc-acr-16 sequences were re-engineered to provide unique restriction sites to generate chimeras that did not change the protein sequence (termed Asu-ACR-16-REM and Tzc-ACR-16-REM, respectively). All sequences were cloned into the pTD2 oocyte expression vector. Asu-ACR-16 and Tzc-ACR-16 chimeras were constructed using appropriate restriction enzymes, reciprocal exchange through ligation and confirmed by sequencing (McLab, USA).

cRNA for oocyte injection was prepared with the mMESSAGE mMACHINE T7 kit following manufacturers protocol (Ambion, USA). Concentration was determined using a Nanodrop spectrophotometer. Desired subunit combinations with associated accessory proteins were mixed and diluted with nuclease-free water to a final concentration of 250 ng/uL for each construct.

Oocyte extraction and injection

Adult female X. laevis oocyte extraction was performed in accordance with the McGill University Animal Use Protocol 2015–7758 following standard protocol [87]. Briefly, frog ovary lobes were placed in a Ca2+-free OR2 solution (82 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES buffer, pH 7.3) and manually divided into clusters of <10 oocytes with thin tweezers followed by a 90-minute incubation in 10 mg/mL collagenase type II (Sigma-Aldrich, USA). Oocytes were washed in Ca2+-free OR2 and placed ND96 solution (NaCl 96 mM, KCl 2 mM, CaCl2 1.8 mM, MgCl2 1 mM and HEPES 5 mM, pH 7.3) supplemented with sodium pyruvate 2.5 mM [86] and incubated at 18°C until cRNA injection. Oocytes were injected with 50 nL (12.5 ng of each injected gene) of the desired cRNA subunit and accessory protein combination mix with the NanojectII (Drummond Scientific, USA). Following injection, oocytes were incubated in ND96 buffer at 18°C until electrophysiology. All electrophysiology was conducted two days post-injection, except for the expression analysis assay in which oocytes were measured every day after injection for three consecutive days.

Pharmacological compounds

Receptor ligands acetylcholine (ACh), (-)-tetramisole hydrochloride (LEV), (-)-nicotine hydrogen tartrate (NIC), bephenium (BEPH), betaine (BET), norepinephrine (NOR), epinephrine (EPI), oxantel (OXA), morantel (MOR), pyrantel (PYR), tyramine (TYR), dimethylphenylpiperazinium (DMPP) and dopamine (DA) used for electrophysiology characterization were obtained from Sigma-Aldrich. All drugs were dissolved in recording solution (NaCl 100 mM, KCl 2.5 mM, CaCl2 1 mM, HEPES 5 mM, pH 7.3) to a stock concentration of 10 mM and diluted to their final recording concentrations. Oxantel and bephenium were first dissolved in DMSO to ensure a final recording concentration contained less than 0.1% DMSO. For agonist profiling, all drugs were diluted to a concentration of 100 μM. For EC50, NIC and ACh were diluted to concentrations between 0.3 and 300 μM. For maximum response measurements, 100 μM NIC and ACh was used.

Electrophysiology

Oocytes were placed individually in an RC3Z chamber (Harvard Apparatus, USA) submerged in recording solution (NaCl 100 mM, KCl 2.5 mM, CaCl2 1 mM, HEPES 5 mM, pH 7.3). The RC3Z chamber was connected to a perfusion system containing recording solution and ligand solutions. Pulled glass capillaries (World Precision Instruments, USA) were filled with 3M KCl and the tips clipped and checked for appropriate resistance (between 0.5 and 5 MΩ). A 3M KCl agar bridge grounded the oocyte bath chamber. Measurements were made using an Axon Axoclamp 900A amplifier with Digidata1550M. Oocytes were voltage-clamped at -60 mV and first exposed to recording solution and allowed to stabilize, those with holding currents less than -400 nA or holding currents that did not stabilize were not considered for analysis due to poor oocyte quality. Ligand solutions were applied to the oocytes with an approximate flow rate of 1.2 m/s and current responses measured. Oocytes were exposed to the ligand solution until a distinct current peak was observed followed by a wash with the recording solution until current returned to baseline for a minimum of 10 seconds until the next ligand exposure. For BAPTA-AM exposed oocytes, oocytes were incubated in 100 μM BAPTA-AM for four hours prior to electrophysiology recording [1]. Data was analyzed using Clampex 9.2 (Axon Instruments, USA) and statistical analysis and graphs produced in Prism (GraphPad version 9.1). T-tests were used to compare responses with error bars representing standard error. For concentration-response curves, individual oocyte current response to the different ligand concentrations were normalized to the response obtained with 300 μM. Data was fit using the Graphpad “log(agonist) vs normalized response–variable slope” equation, with 95% confidence intervals being reported. For ligand rank order analysis, individual oocyte current responses to the different ligands were normalized to the current measured with ACh. Significance was determined by comparing the currents to the different ligands to those of ACh prior to normalization. For examples of oocyte responses, recordings were filtered in Clampfit using the lowband pass at 40 Hz cutoff.

Supporting information

S1 Fig. N-AChR responses with additional accessory protein combinations.

(A) Current responses of the clade III N-AChRs A. suum (black), D. medinensis (purple), G. pulchrum (orange), T. callipaeda (green) and B. malayi (grey) to 100 μM ACh when co-injected with either H. contortus RIC-3 (Hco-ric-3) or B. malayi RIC-3 (Bma-ric-3). A. suum ACR-16 with Hco-ric-3 (2052 ± 66 nA) was not different compared to co-injection with Bma-ric-3 (2023 ± 68 nA, p = 0.7581). D. medinensis ACR-16 with Hco-ric-3 (2150 ± 59 nA) was not different compared to co-injection with Bma-ric-3 (2275 ± 157 nA, p = 0.4285). G. pulchrum ACR-16 with Hco-ric-3 (1403 ± 91 nA) was not different compared to co-injection with Bma-ric-3 (1281 ± 197 nA, p = 0.5300). T. callipaeda ACR-16 with Hco-ric-3 (563 ± 39 nA) was smaller compared to co-injection with Bma-ric-3 (818 ± 112 nA, p<0.05). B. malayi ACR-16 produced no responses under both RIC-3 conditions. (B) Effect of BAPTA-AM incubation on current responses of the clade III N-AChRs. A. suum (black), D. medinensis (purple), G. pulchrum (orange) and T. callipaeda (green) responses to 100 μM ACh co-injected with H. contortus RIC-3. A. suum ACR-16 (2052 ± 70 nA) was not different compared to BAPTA-AM incubated ACR-16 (2125 ± 40 nA, p = 0.6193). D. medinensis ACR-16 (2150 ± 59 nA) was not different compared to BAPTA-AM incubated ACR-16 (1910 ± 164 nA, n = 0.0969). G. pulchrum ACR-16 (1403 ± 91 nA) was not different compared to BAPTA-AM incubated ACR-16 (1529 ± 117 nA, n = 0.5044). T. callipaeda ACR-16 (563 ± 39 nA) was not different compared to BAPTA-AM incubated ACR-16 (623 ± 113 nA, n = 0.4829). Note that the BAPTA-AM treated oocytes represents preliminary data. (C) Current responses of clade III N-AChRs A. suum (black), D. medinensis (purple), G. pulchrum (orange), T. callipaeda (green) and B. malayi (grey) to 100 μM ACh with different accessory proteins. Receptor responses are compared to Bma-RIC-3 condition. Error bars represent standard error. n>8, *p<0.05; **p<0.01; ****p<0.0001. See S1 Table for values.

https://doi.org/10.1371/journal.ppat.1010962.s001

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S2 Fig. A. suum and T. callipaeda ACR-16 chimeras.

The three main structural regions (ECD, ICL & TM) were exchanged between A. suum and T. callipaeda ACR-16 to identify the regions mediating the declining responses in later clade III N-AChRs. (A) A. suum and T. callipaeda ACR-16 coding sequences were modified to introduce restriction enzyme sites to constrict chimeras. A. suum shown in red and T. callipaeda in green. The modified sequences introduced complementary restriction enzyme sites termed ACR-16-REM. The three main structural regions (ECD, ICL, TMs) were exchanged between the two receptors. TZC-ECD contains the A. suum sequence with the T. callipaeda ECD. ASU-ECD contains the T. callipaeda sequence with the A. suum ECD. ASU-TM contains the T. callipaeda sequence with the A. suum TM. TZC-TM contains the A. suum sequence with the T. callipaeda TM. TZC-ICL contains the A. suum sequence with the T. callipaeda ICL. ASU-ICL contains the T. callipaeda sequence with the A. suum ICL. (B) Coding-sequence modified ACR-16 receptors (ACR-16-REM, red) produced responses indistinguishable from native ACR-16 sequence (black). Asu-ACR-16 (2023 ± 68 nA, n = 11) versus Asu-ACR-16-REM (2197 ± 383 nA, n = 20); and Tzc-ACR-16 (818 ± 112 nA, n = 13) versus Tzc-ACR-16-REM (587 ± 415 nA, n = 20). B. malayi ric-3 co-injected with all. Circles represent currents measured from individual oocytes. Error bars represents standard error. (C) Chimeras exchanging each of the three structural regions between the receptors identified all regions contribute to the declining current responses measured in later clade III receptors. Circles represent currents measured from individual oocytes. Error bars represents standard error. Significance determined by comparing the current responses to the relevant reference receptor. ****p<0.0001.

https://doi.org/10.1371/journal.ppat.1010962.s002

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S3 Fig. Asu-ACR-16-bmaICL chimera responses and pharmacology.

(A) Chimeras for the A. suum (red) and B. malayi acr-16 (blue) sequences. Asu-ACR-16-bmaICL chimera has the Asu-acr-16 subunit with the intracellular loop of Bma-acr-16, and Bma-ACR-16-asuICL chimera has the Bma-acr-16 subunit with the intracellular loop of Asu-acr-16. Chimeras were made between A. suum and B. malayi ACR-16 to characterize B. malayi ACR-16 in the event that the unmodified B. malayi ACR-16 did not produce robust enough responses for characterization. The intracellular loop was exchanged since it is one of the regions thought to contribute to the declining responses observed in clade III ACR-16 and it does not contain the ligand-binding regions. (B) Asu-ACR-16-bmaICL chimera current responses under different accessory protein conditions: RIC-3 (3021 ± 444 nA, n = 12); RIC-3 + NRA-2 (2181 ± 369 nA, n = 14, p<0.0001); and no accessory protein (22 ± 15 nA, n = 14, p<0.0001). Circles represent current responses from individual oocytes. Error bars represent standard error. ****p<0.0001. (C) Concentration-response curves were measured for the Asu-ACR-16-bmaICL chimera to compare to the Asu-ACR-16 receptor as a control (see Fig 4A and 4B). Concentration-response curves for acetylcholine in purple (EC50 = 4.3 μM, 95% CI [3.3, 5.5], n = 8) and nicotine in green (EC50 = 2.3 μM, 95% CI [1.9, 2.6], n = 10). Hill coefficients were 0.9, 95% CI [0.7, 1.1] for ACh and 1.2, 95% CI [0.9, 1.6] for NIC. Sample recordings to acetylcholine is shown. Error bars represent standard error. (D) Asu-ACR-16-bmaICL chimera response to ligands relative to acetylcholine. Concentrations of 100 μM was used for all ligands. Apart from higher responses to DMPP, Asu-ACR-16-bmaICL chimera had a comparable response profile compared to Asu-ACR-16 (see Fig 5A): ACh = DMPP> NIC> OXA = MOR = PYR > LEV = EPI = NOR = DA = TYR = BET = BEPH. Sample recording to each ligand is shown. Error bars represent standard error. *p<0.05; ****p<0.0001.

https://doi.org/10.1371/journal.ppat.1010962.s003

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S4 Fig. Expected effect of accessory proteins on nematode receptor expression.

Expected effect on receptor synthesis of accessory proteins included in the present study. Ric-3 (yellow, blue, red) is required for all nematode ACR-16 and the L-AChR in oocytes and is involved subunit assembly in the ER. EMC-6, NRA-2 and NRA-4 (red) are required for the B. malayi ACR-16 receptor in oocytes and are thought to be involved in subunit folding and oligomerization of the pentamer in the ER. UNC-74 (blue) is required for disulfide bond formation in the ER for the L-AChR. UNC-50 (blue) is involved in L-AChR receptor trafficking through the Golgi. EAT-18 (green) is required for the EAT-2 receptor at the membrane and MOLO-1 (green) is required for the L-AChR in vivo and modulates receptor response. See text for detailed discussion.

https://doi.org/10.1371/journal.ppat.1010962.s004

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S1 Table. ACR-16 response (nA) to 100 μM acetylcholine with various accessory protein combinations.

Clade III N-AChR responses (nA) to 100 μM acetylcholine under various accessory protein combinations. The receptor responses are compared to the reference condition of the N-AChR + B. malayi RIC-3. The clade III receptors showed declining currents along their phylogeny with the RIC-3 accessory protein; A. suum and D. medinensis N-AChR produced the largest response currents, G. pulchrum N-AChR produced smaller responses, T. callipaeda N-AChR had even smaller responses, and the B. malayi N-AChR had none. This decline was seen even with the addition of accessory proteins EAT-18, UNC-50, UNC-74 and/or MOLO-1. A B. malayi N-AChR response was only measured upon addition of EMC-6, NRA-2 or NRA-4, however despite multiple combinations of these three accessory proteins, small responses were still obtained indicating that other critical accessory proteins are still missing from the injection mixture. Chimera Bma-ACR-16-asuICL was used to approximate B. malayi N-AChR receptor pharmacology. Error represents standard error. *p<0.05; **p<0.01; ***p<0.0005; ****p<0.0001.

https://doi.org/10.1371/journal.ppat.1010962.s005

(DOCX)

S2 Table. Primers used for cloning.

Receptor subunit and accessory protein coding sequences used in this study were either cloned from worm cDNA or synthesized based on their genome-predicted sequences. Bma-acr-16 and B. malayi accessory proteins were cloned from adult female cDNA using the following primers. All other accessory proteins and subunit sequences were synthesized. Experimental procedure is described in Methods. a received from Dr. C. Neveu and Dr. C. Charvet.

https://doi.org/10.1371/journal.ppat.1010962.s006

(DOCX)

Acknowledgments

We thank Dr. Thomas Duguet for cloning B. malayi acr-16 and Dr. Tim Geary for generously providing us with B. malayi cDNA.

References

  1. 1. Boulin T, Gielen M, Richmond JE, Williams DC, Paoletti P, Bessereau JL. Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor. Proc Natl Acad Sci U S A. 2008;105(47):18590–5. pmid:19020092
  2. 2. Courtot E, Charvet CL, Beech RN, Harmache A, Wolstenholme AJ, Holden-Dye L, et al. Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes. PLoS Pathog. 2015;11(12):1–29. pmid:26625142
  3. 3. Touroutine D, Fox RM, Von Stetina SE, Burdina A, Miller DM, Richmond JE. acr-16 encodes an essential subunit of the levamisole-resistant nicotinic receptor at the Caenorhabditis elegans neuromuscular junction. J Biol Chem. 2005;280(29):27013–21. pmid:15917232
  4. 4. Cully DF, Paress PS. Solubilization and characterization of a high affinity ivermectin binding site from Caenorhabditis elegans. Mol Pharmacol. 1991;40(2):326–32. pmid:1875915
  5. 5. Unwin N. Acetylcholine receptor channel imaged in the open state. Vol. 373, Nature. 1995. p. 37–43. pmid:7800037
  6. 6. Tasneem A, Iyer LM, Jakobsson E, Aravind L. Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels. Genome Biol. 2005;6(1):1–12. pmid:15642096
  7. 7. Beech RN, Neveu C. The evolution of pentameric ligand-gated ion-channels and the changing family of anthelmintic drug targets. Parasitology. 2015;142(2):303–17. pmid:25354656
  8. 8. Dent JA. The evolution of pentameric ligand-gated ion channels. Adv Exp Med Biol. 2010;683:11–23. pmid:20737785
  9. 9. Corringer PJ, Poitevin F, Prevost MS, Sauguet L, Delarue M, Changeux JP. Structure and pharmacology of pentameric receptor channels: From bacteria to brain. Structure. 2012;20(6):941–56. pmid:22681900
  10. 10. Keramidas A, Moorhouse AJ, Schofield PR, Barry PH. Ligand-gated ion channels: Mechanisms underlying ion selectivity. Vol. 86, Progress in Biophysics and Molecular Biology. 2004. 161–204 p. pmid:15288758
  11. 11. Langlhofer G, Villmann C. The intracellular loop of the glycine receptor: It’s not all about the size. Front Mol Neurosci. 2016;9(JUNE):1–14.
  12. 12. Aubrey ML, Cowell P, Davey MJ, Shevde S. Aspects of the pharmacology of a new anthelmintic: pyrantel. Br J Pharmacol. 1970;38(2):332–44. pmid:5417856
  13. 13. Aceves J, Erlij D, Martinez-Maranon R. The mechanism of the paralysing action of tetramisole on Ascaris somatic muscle. Br J Pharmacol. 1970;38(3):602–7. pmid:5445688
  14. 14. Lewis JA, Wu CH, Berg H, Levine JH. The genetics of levamisole resistance in the nematode Caenorhabditis elegans. Genetics. 1980;95(4):905–28. pmid:7203008
  15. 15. Wolstenholme AJ. Ion channels and receptor as targets for the control of parasitic nematodes. Int J Parasitol Drugs Drug Resist. 2011;1(1):2–13. pmid:24533259
  16. 16. Culetto E, Baylis HA, Richmond JE, Jones AK, Fleming JT, Squire MD, et al. The Caenorhabditis elegans unc-63 gene encodes a levamisole-sensitive nicotinic acetylcholine receptor α subunit. J Biol Chem. 2004;279(41):42476–83.
  17. 17. Kaminsky R, Ducray P, Jung M, Clover R, Rufener L, Bouvier J, et al. A new class of anthelmintics effective against drug-resistant nematodes. Nature. 2008;452(7184):176–80. pmid:18337814
  18. 18. Dent JA, Smith MM, Vassilatis DK, Avery L. The genetics of ivermectin resistance in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2000;97(6):2674–9. pmid:10716995
  19. 19. Baylis Howard A. matsuda K. Squire MD. Fleming JT. Harvey RJ. Darlison MG. Bernard EA. Sattelle D. ACR-3, a Caenorhabditis elegans nicotinic acetylcholine receptor subunit. Molecular cloning and functional expression. Recept Channels. 1997;5:149–58. pmid:9606719
  20. 20. Cully DF, Wilkinson H, Vassilatis DK, Etter A, Arena JP. Molecular biology and electrophysiology of glutamate-gated chloride channels of invertebrates. Parasitology. 1996;113(S1):S191–200. pmid:9051935
  21. 21. Rufener L, Baur R, Kaminsky R, Mäser P, Sigel E. Monepantel allosterically activates DEG-3/DES-2 channels of the gastrointestinal nematode Haemonchus contortus. Mol Pharmacol. 2010;78(5):895–902. pmid:20679419
  22. 22. Rufener L, Bedoni N, Baur R, Rey S, Glauser DA, Bouvier J, et al. acr-23 Encodes a Monepantel-Sensitive Channel in Caenorhabditis elegans. PLoS Pathog. 2013;9(8). pmid:23950710
  23. 23. Gottschalk A, Almedom RB, Schedletzky T, Anderson SD, Yates JR, Schafer WR. Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans. EMBO J. 2005;24(14):2566–78. pmid:15990870
  24. 24. Ballivet M, Alliod C, Bertrand S, Bertrand D. Nicotinic acetylcholine receptors in the nematode Caenorhabditis elegans. J Mol Biol. 1996;258(2):261–9. pmid:8627624
  25. 25. Bennett HM, Lees K, Harper KM, Jones AK, Sattelle DB, Wonnacott S, et al. Xenopus laevis RIC-3 enhances the functional expression of the C. elegans homomeric nicotinic receptor, ACR-16, in Xenopus oocytes. J Neurochem. 2012;123(6):911–8. pmid:22970690
  26. 26. Nishtala SN, Mnatsakanyan N, Pandhare A, Leung C, Jansen M. Direct interaction of the resistance to inhibitors of cholinesterase type 3 protein with the serotonin receptor type 3A intracellular domain. J Neurochem. 2016;137(4):528–38. pmid:26875553
  27. 27. Moreno Y, Nabhan JF, Solomon J, MacKenzie CD, Geary TG. Ivermectin disrupts the function of the excretory- secretory apparatus in microfilariae of Brugia malayi. Proc Natl Acad Sci U S A. 2010;107(46):20120–5. pmid:21041637
  28. 28. Richmond JE, Jorgensen EM. One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat Neurosci. 1999;2(9):791–8. pmid:10461217
  29. 29. Verma S, Kashyap SS, Robertson AP, Martin RJ. Functional genomics in Brugia malayi reveal diverse muscle nAChRs and differences between cholinergic anthelmintics. Proc Natl Acad Sci U S A. 2017;114(21):5539–44. pmid:28487481
  30. 30. Robertson AP, Buxton SK, Martin RJ. Whole-cell patch-clamp recording of nicotinic acetylcholine receptors in adult Brugia malayi muscle. Parasitol Int. 2013;62(6):616–8. pmid:23562945
  31. 31. Li BW, Rush AC, Weil GJ. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms. Int J Parasitol Drugs Drug Resist. 2015;5(3):100–9. pmid:26199859
  32. 32. Choudhary S, Tipton JG, Abongwa M, Brewer MT, Chelladurai JJ, Musselman N, et al. Pharmacological characterization of a homomeric nicotinic acetylcholine receptor formed by Ancylostoma caninum ACR-16. Invertebr Neurosci. 2019;19(4):1–10. pmid:31486912
  33. 33. Kaji MD, Geary TG, Beech RN. A Functional Comparison of Homopentameric Nicotinic Acetylcholine Receptors (ACR-16) Receptors From Necator americanus and Ancylostoma ceylanicum. Front Mol Neurosci. 2020;13(November):1–19. pmid:33324163
  34. 34. Hansen TVA, Cirera S, Neveu C, Courtot E, Charvet CL, Calloe K, et al. The narrow-spectrum anthelmintic oxantel is a potent agonist of a novel acetylcholine receptor subtype in whipworms. PLoS Pathog. 2021;17(2):1–26. pmid:33544769
  35. 35. Hansen TVA, Grencis RK, Issouf M, Neveu C, Charvet CL. Functional characterization of the oxantel-sensitive acetylcholine receptor from trichuris muris. Pharmaceuticals. 2021;14(7):1–15. pmid:34358124
  36. 36. Abongwa M, Buxton SK, Courtot E, Charvet CL, Neveu C, McCoy CJ, et al. Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues. Br J Pharmacol. 2016;2463–77. pmid:27238203
  37. 37. Coghlan A, Tyagi R, Cotton JA, Holroyd N, Rosa BA, Tsai IJ, et al. Comparative genomics of the major parasitic worms. Nat Genet. 2019;51(1):163–74. pmid:30397333
  38. 38. Richard M, Boulin T, Robert VJP, Richmond JE, Bessereau JL. Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex. Proc Natl Acad Sci U S A. 2013;110(11). pmid:23431131
  39. 39. Almedom RB, Liewald JF, Hernando G, Schultheis C, Rayes D, Pan J, et al. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse. EMBO J. 2009;28(17):2636–49. pmid:19609303
  40. 40. Charvet CL, Guégnard F, Courtot E, Cortet J, Neveu C. Nicotine-sensitive acetylcholine receptors are relevant pharmacological targets for the control of multidrug resistant parasitic nematodes. Int J Parasitol Drugs Drug Resist. 2018;8(3):540–9. pmid:30502120
  41. 41. Ghedin E, Wang S, Spiro D, Caler E, Zhao Q, Crabtree J, et al. Draft genome of the filarial nematode parasite Brugia malayi. Science (80-). 2007;317(5845):1756–60. pmid:17885136
  42. 42. Kashyap SS, Verma S, McHugh M, Wolday M, Williams PD, Robertson AP, et al. Anthelmintic resistance and homeostatic plasticity (Brugia malayi). Sci Rep. 2021;11(1):1–17.
  43. 43. Bisson WH, Westera G, Schubiger PA, Scapozza L. Homology modeling and dynamics of the extracellular domain of rat and human neuronal nicotinic acetylcholine receptor subtypes α4β2 and α7. J Mol Model. 2008;14(10):891–9.
  44. 44. Zheng F, Robertson AP, Abongwa M, Yu EW, Martin RJ. The Ascaris suum nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators from virtual screening. Int J Parasitol Drugs Drug Resist. 2016;6(1):60–73. pmid:27054065
  45. 45. Martin RJ, Robertson AP, Buxton SK, Beech RN, Charvet CL, Neveu C. Levamisole receptors: A second awakening. Trends Parasitol. 2012;28(7):289–96. pmid:22607692
  46. 46. Qian H, Martin RJ, Robertson AP, Qian H, Martin RJ, Robertson AP. Pharmacology of N -, L -, and B -subtypes of nematode nAChR resolved at the single-channel level in Ascaris suum. FASEB J. 2006;20(14):2606–8. pmid:17056760
  47. 47. Duguet TB, Charvet CL, Forrester SG, Wever CM, Dent JA, Neveu C, et al. Recent Duplication and Functional Divergence in Parasitic Nematode Levamisole-Sensitive Acetylcholine Receptors. PLoS Negl Trop Dis. 2016;10Alan L.(7):1–26. pmid:27415016
  48. 48. Boulin T, Fauvin A, Charvet CL, Cortet J, Cabaret J, Bessereau JL, et al. Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance. Br J Pharmacol. 2011;164(5):1421–32. pmid:21486278
  49. 49. Boulin T, Rapti G, Briseño-Roa L, Stigloher C, Richmond JE, Paoletti P, et al. Positive modulation of a Cys-loop acetylcholine receptor by an auxiliary transmembrane subunit. Nat Neurosci. 2012;15(10):1374–81. pmid:22922783
  50. 50. Choudhary S, Buxton SK, Puttachary S, Verma S, Mair GR, McCoy CJ, et al. EAT-18 is an essential auxiliary protein interacting with the non-alpha nAChR subunit EAT-2 to form a functional receptor. PLoS Pathog. 2020;16(4):1–23.
  51. 51. Williamson SM, Robertson AP, Brown L, Williams T, Woods DJ, Martin RJ, et al. The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: Formation of two distinct drug targets by varying the relative expression levels of two subunits. PLoS Pathog. 2009;5(7):1–11. pmid:19609360
  52. 52. Smart TG, Paoletti P. Synaptic neurotransmitter-gated receptors. Cold Spring Harb Perspect Biol. 2012;4(3). pmid:22233560
  53. 53. Thompson AJ, Lester HA, Lummis SCR. The structural basis of function in Cys-loop receptors. Vol. 43, Quarterly Reviews of Biophysics. 2010. 449–499 p. pmid:20849671
  54. 54. Dacosta CJB, Baenziger JE. Gating of pentameric ligand-gated ion channels: Structural insights and ambiguities. Structure. 2013;21(8):1271–83. pmid:23931140
  55. 55. Duret G, Van Renterghem C, Weng Y, Prevost M, Moraga-Cid G, Huon C, et al. Functional prokaryotic-eukaryotic chimera from the pentameric ligand-gated ion channel family. Proc Natl Acad Sci U S A. 2011;108(29):12143–8. pmid:21730130
  56. 56. Eiselé JL, Bertrand S, Galzi JL, Devillers-Thiéry A, Changeux JP, Bertrand D. Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities. Nature. 1993;366(6454):479–83. pmid:8247158
  57. 57. Mnatsakanyan N, Nishtala SN, Pandhare A, Fiori MC, Goyal R, Pauwels JE, et al. Functional chimeras of GLIC obtained by adding the intracellular domain of anion- and cation-conducting Cys-loop receptors. Biochemistry. 2015;54(16):2670–82. pmid:25861708
  58. 58. Robertson AP, Puttachary S, Martin RJ. Single-channel recording from adult brugia malayi. Invertebr Neurosci. 2011;11(1):53–7. pmid:21590329
  59. 59. Qian H, Robertson AP, Powell-Coffman JA, Martin RJ. Levamisole resistance resolved at the single-channel level in Caenorhabditis elegans. FASEB J. 2008;22(9):3247–54. pmid:18519804
  60. 60. Wang Y, Yao Y, Tang XQ, Wang ZZ. Mouse RIC-3, an endoplasmic reticulum chaperone, promotes assembly of the α7 acetylcholine receptor through a cytoplasmic coiled-coil domain. J Neurosci. 2009;29(40):12625–35.
  61. 61. Fleming JT, Squire MD, Barnes TM, Tornoe C, Matsuda K, Ahnn J, et al. Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits. J Neurosci. 1997;17(15):5843–57. pmid:9221782
  62. 62. Lewis JA, Wu CH, Levine JH, Berg H. Levamisole-resitant mutants of the nematode Caenorhabditis elegans appear to lack pharmacological acetylcholine receptors. Neuroscience. 1980;5(6):967–89.
  63. 63. D’alessandro M, Richard M, Stigloher C, Gache V, Boulin T, Richmond JE, et al. CRELD1 is an evolutionarily-conserved maturational enhancer of ionotropic acetylcholine receptors. Elife. 2018;7:1–29. pmid:30407909
  64. 64. McKay JP, Raizen DM, Gottschalk A, Schafer WR, Avery L. eat-2 and eat-18 are Required for Nicotinic Neurotransmission in the Caenorhabditis elegans Pharynx. Genetics. 2004;166(1):161–9. pmid:15020415
  65. 65. Millar NS. RIC-3: A nicotinic acetylcholine receptor chaperone. Br J Pharmacol. 2008;153(SUPPL. 1):177–83.
  66. 66. Treinin M. RIC-3 and nicotinic acetylcholine receptors: Biogenesis, properties, and diversity. Biotechnol J. 2008;3(12):1539–47. pmid:18956371
  67. 67. Halevi S, Yassin L, Eshel M, Sala F, Sala S, Criado M, et al. Conservation within the RIC-3 gene family: Effectors of mammalian nicotinic acetylcholine receptor expression. J Biol Chem. 2003;278(36):34411–7.
  68. 68. Wideman JG. The ubiquitous and ancient ER membrane protein complex (EMC): Tether or not? F1000Research. 2015;4(May):1–12. pmid:26512320
  69. 69. Bai L, You Q, Feng X, Kovach A, Li H. Structure of the ER membrane complex, a transmembrane-domain insertase. Nature. 2020;584(7821):475–8. pmid:32494008
  70. 70. O’donnell JP, Phillips BP, Yagita Y, Juszkiewicz S, Wagner A, Malinverni D, et al. The architecture of EMC reveals a path for membrane protein insertion. Elife. 2020;9:1–56. pmid:32459176
  71. 71. Kamat S, Yeola S, Zhang W, Bianchi L, Driscoll M. NRA-2, a nicalin homolog, regulates neuronal death by controlling surface localization of toxic caenorhabditis elegans DEG/ENaC channels. J Biol Chem. 2014;289(17):11916–26. pmid:24567339
  72. 72. Martinez-Torres A. GABArho 1/GABAAalpha 1 receptor chimeras to study receptor desensitization. Proc Natl Acad Sci. 2000;97(7):3562–6. pmid:10725369
  73. 73. Tillman TS, Seyoum E, Mowrey DD, Xu Y, Tang P. ELIC-α7 nicotinic acetylcholine receptor (α7nAChR) chimeras reveal a prominent role of the extracellular-transmembrane domain interface in allosteric modulation. J Biol Chem. 2014;289(20):13851–7. pmid:24695730
  74. 74. Verrall S, Hall ZW. The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly. Cell. 1992;68(1):23–31. pmid:1370654
  75. 75. Eertmoed AL, Green WN. Nicotinic receptor assembly requires multiple regions throughout the γ subunit. J Neurosci. 1999;19(15):6298–308.
  76. 76. Sumikawa K, Nishizaki T. The amino acid residues 1–128 in the α subunit of the nicotinic acetylcholine receptor contain assembly signals. Mol Brain Res. 1994;25(3–4):257–64.
  77. 77. Boulter J, O’Shea-Greenfield A, Duvoisin RM, Connolly JG, Wada E, Jensen A, et al. α3, α5, and β4: Three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster. J Biol Chem. 1990;265(8):4472–82.
  78. 78. Kneussel M, Betz H. Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: The membrane activation model. Trends Neurosci. 2000;23(9):429–35. pmid:10941193
  79. 79. Baptista-Hon DT, Deeb TZ, Lambert JJ, Peters JA, Hales TG. The minimum M3-M4 loop length of neurotransmitteractivated pentameric receptors is critical for the structural integrity of cytoplasmic portals. J Biol Chem. 2013;288(30):21558–68. pmid:23740249
  80. 80. Williamson SM, Walsh TK, Wolstenholme AJ. The cys-loop ligand-gated ion channel gene family of Brugia malayi and Trichinella spiralis: A comparison with Caenorhabditis elegans. Invertebr Neurosci. 2007;7(4):219–26. pmid:17952476
  81. 81. Carlin BE, Lawrence JC, Lindstrom JM, Merlie JP. An acetylcholine receptor precursor α subunit that binds α-bungarotoxin but not d-tubocurare. Proc Natl Acad Sci U S A. 1986;83(2):498–502.
  82. 82. Gu Y, Forsayeth JR, Verrall S, Yu XM, Hall ZW. Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells. J Cell Biol. 1991;114(4):799–807. pmid:1869588
  83. 83. Bolt BJ, Rodgers FH, Shafie M, Kersey PJ, Berriman M, Howe KL. Using WormBase ParaSite: An integrated platform for exploring helminth genomic data. Methods Mol Biol. 2018;1757:471–91. pmid:29761467
  84. 84. Howe KL, Bolt BJ, Shafie M, Kersey P, Berriman M. WormBase ParaSite − a comprehensive resource for helminth genomics. Mol Biochem Parasitol. 2017;215:2–10. pmid:27899279
  85. 85. Katoh K, Misawa K, Kuma KI, Miyata T. MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30(14):3059–66. pmid:12136088
  86. 86. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307–21. pmid:20525638
  87. 87. Goldin A. Xenopus laevis: Practical Uses in Cell and Molecular Biology. Chapter 25 Expression of Ion Channels by Injection of mRNA into Xenopus Oocytes. 1991.