Two populations of heteromeric acetylcholine receptors are present at Caenorhabditis elegans neuromuscular junctions.

Ligand-gated acetylcholine receptors (AChRs) are pentameric transmembrane proteins that display extensive molecular diversity through combinatorial assembly of subunits. In the nematode Caenorhabditis elegans, this diversity is exceptionally expanded, with nearly 60 AChR subunit genes, yet the composition and function of many receptor subtypes remain poorly defined. At neuromuscular junctions (NMJs), L-AChRs are heteromeric AChRs sensitive to levamisole, a nematode-specific L-AChR agonist, that play a crucial role in locomotion. Forward genetic screens for mutants resistant to levamisole have identified the L-AChR canonical subunits (LEV-1, UNC-29, LEV-8, UNC-63, and UNC-38) along with key biosynthetic regulators. ACR-8 is an alternative L-AChR subunit that has been poorly characterized, largely because acr-8 mutants remain sensitive to levamisole. Using genetic approaches and in vivo imaging, we show that ACR-8-containing L-AChRs (ACR-8*) are present at the NMJ and, surprisingly, they are more abundant than the well-characterized LEV-8-containing L-AChRs (LEV-8*). Although both LEV-8* and ACR-8* are enriched at synapses and rely on the same clustering machinery, they segregate into distinct postsynaptic nanodomains. The 2 populations play a redundant function in worm locomotion, and in mutant conditions, 1 receptor population can compensate for the loss of the other. Interestingly, ACR-8* are already present at high levels at the NMJ from early developmental stages, whereas LEV-8* levels gradually increase throughout development. Our work sheds new light on the molecular landscape of AChRs of the C. elegans NMJ.