Pharmacological profile of Ascaris suum ACR‐16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues

Abstract

SummaryBackground and PurposeControl of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR‐16, from a nematode parasite.Experimental ApproachUsing RT‐PCR, molecular cloning and two‐electrode voltage clamp electrophysiology, we localized acr‐16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr‐16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested.Key ResultsAmino acid sequence comparison with vertebrate nAChR subunits revealed ACR‐16 to be most closely related to α7 receptors, but with some striking distinctions. acr‐16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, bephenium, tribendimidine and pyrantel) did not activate Asu‐ACR‐16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3‐bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu‐ACR‐16 was insensitive to α‐bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu‐ACR‐16 had lower calcium permeability than α7 receptors.Conclusions and ImplicationsWe suggest that ACR‐16 has diverse tissue‐dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.

EAT-18 is an essential auxiliary protein interacting with the non-alpha nAChR subunit EAT-2 to form a functional receptor

“…Cel-EAT-18 functions as an obligate auxiliary protein and modifies the pharmacological properties of this cys-loop ion channel. Several previously characterized nAChRs require ancillary proteins, either RIC-3 alone or in combination with UNC-50 and UNC-74, for successful in vitro expression [6,12,16,17,39]. In vitro expression of Cel-EAT-2 receptors instead only required co-expression with EAT-18, which has little similarity to ancillary proteins.…”

Functional genomics in Brugia malayi reveal diverse muscle nAChRs and differences between cholinergic anthelmintics

“…In Ascaris suum, there are three separable muscle subtypes [N-, L-, and B- (27)] and four types in Oesophagostomum dentatum (11). Xenopus expression experiments suggest in Haemonchus contortus and Oesophagostomum dentatum that: the L-type (L-AChR-1) is composed of UNC-63, UNC-38, UNC-29, and ACR-8 subunits; the P-type (L-AChR-2) is composed of UNC-63, UNC-38, and UNC-29 subunits (10,25); and the N-type in Ascaris suum is composed of ACR-16 subunits (14,35,50). A fourth type of muscle nAChR selectively activated by morantel and composed of ACR-26 and ACR-27 subunits has been described in Haemonchus contortus (51).…”

“…To study the functional genomics of nAChRs of B. malayi, we used the following: (i) video capture for quantitative phenotyping (23,24); (ii) single-cell PCR to identify nAChR subunit genes expressed in muscle (25); (iii) dsRNA for RNAi knockdown of unc-38+unc-29 of nematode levamisole and pyrantel receptors (26,27), and (iv) whole-cell patch clamp to record functional properties of muscle nAChRs (10,28). Knockdown of acr-16+acr-26 had little effect on motility, but knockdown of unc-38+unc-29 abolished motility, suggesting in B. malayi that different physiological functions may be ascribed to different receptors.…”

(S)-5-ethynyl-anabasine, a novel compound, is a more potent agonist than other nicotine alkaloids on the nematode Asu -ACR-16 receptor

“…Asu- ACR-16 is widely distributed in A. suum tissues but its physiological function remains to be determined (Abongwa et al., 2016, Zheng et al., 2016). As one of the recently characterized nematode parasitic nAChRs, Asu- ACR-16 is pharmacologically different to its host α7 nAChR and may be exploited as an anthelmintic drug target to counter resistance to cholinergic anthelmintics directed at other pharmacological types of nAChR (Holden-Dye et al., 2013, Zheng et al., 2016).…”

“…We know that the Asu- ACR-16 receptor is sensitive to six nicotinic agonists: nicotine, ACh, cytisine, 3-bromocytisine, epibatidine, dimethyl-4-phenyllpiperazinium iodide (DMPP), but insensitive to other cholinergic anthelmintic agonists (Abongwa et al., 2016). All six of the Asu- ACR-16 agonists share the nicotinic pharmacophore: a cationic nitrogen separated by ∼5 Å from a hydrogen bond acceptor.…”