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

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.

Two residues determine nicotinic acetylcholine receptor requirement for RIC-3

“…This is in contrast to the closely related pig parasite A. suum ACR-16 which requires its co-injection for function 56,61 . A. suum and D. medinensis ACR-16 receptors have similar pharmacology and identical maximal response, yet they have dramatically different requirements for the accessory protein 61 . Since the co-injected RIC-3 was constant, the cause must be encoded in their sequences, which differ by only 16 %.…”

“…We previously reported that D. medinensis ACR-16 no longer required RIC-3 co-injection for function in the oocyte expression system, whereas the closely related A. suum ACR-16 did 61 . Their high sequence identity (84%) provided an opportunity to identify the sequence responsible (Figure S1A).…”

“…We previously reported that D. medinensis ACR-16 no longer required RIC-3 co-injection for function in the oocyte expression system, whereas the closely related A. suum ACR-16 did 61 .…”

“…An alignment of nematode ACR-16 that have been previously characterized in the oocyte system identified two positions in the cys-loop motif with substitutions, positions 157 and 159 in Figure 4A. Gonglyonema pulchrum is a parasitic nematode phylogenetically related to both A. suum and D. medinensis, and although its ACR-16 sequence contains I504, it requires RIC-3 for function 61 . The G. pulchrum cys-loop substitutions were therefore used to investigate a potential interaction between the cys-loop and tail for RIC-3 requirement.…”

“…Receptor current in response to acetylcholine (ACh) and RIC-3 requirement from these engineered sequences (ACR-16-REM) were indistinguishable from their unmodified counterparts (Figure 1A) 61 . Therefore, any changes observed in responses without RIC-3 in the chimeras would be due to the exchanged region of interest.…”