Noonan, J. D.; Duguet, T. B.; Handy-Hart, C.-J.; Beech, R. N.

The helminth levamisole-sensitive acetylcholine receptor (L-AChR) is a historically significant drug target. This heteromeric receptor belongs to the larger class of pentameric ligand-gated ion channels that has recently expanded within the nematodes and therefore provides the opportunity to study specific examples of how worm receptors evolve. The Caenorhabditis elegans L-AChR can exist in two forms. The first contains three alpha subunits: ACR-13, UNC-38, and UNC-63, and two non-alpha subunits UNC-29 and LEV-1. The second can be formed by replacing ACR-13 with its paralog ACR-8, from either C. elegans or the closely related Haemonchus contortus. This form no longer requires LEV-1, demonstrating an evolved functional difference between ACR-8 and ACR-13. Since the properties of a channel depend on its composition and organization, knowing the underlying mechanisms regulating this would provide valuable insight into one of the least understood aspects of this important drug target. The goal of this study was to identify the subunit stoichiometry of the L-AChR and further elucidate the functional divergence of alpha subunits ACR-8 and ACR-13. Using a series of subunit concatemers, we determined the arrangement of three C. elegans L-AChR subunits in the following N- to C- terminal order; UNC-38 - LEV-1 - UNC-63. We were unable to order the ACR-13 and UNC-29 subunits unambiguously. Additionally, our concatemers provide support for the sequential model of subunit assembly whereby a trimer is formed first, consisting of UNC-38 - LEV-1 - UNC-63, followed by individual addition of the remaining two subunits ACR-13 and UNC-29. Using C. elegans and H. contortus subunit admixtures, we show that replacing ACR-13 with ACR-8 alleviates the requirement for five distinct subunits, as a functional receptor can be measured in the absence of non-alpha LEV-1. We confirm that UNC-29 can replace LEV-1 and that the intracellular loop determines this positional plasticity. This work provides the first evidence of the quaternary structure of the L-AChR which is necessary for future studies on this drug target. It also confirms that subunit arrangement is determined first during receptor heteromerization, followed by functional fine-tuning once subunit types have defined positions.