The number of receptors present at the postsynaptic terminals is an important parameter that controls the strength of synaptic transmission. At neuromuscular junctions in C. elegans, acetylcholine is released by excitatory motoneurons and activates two types of ionotropic receptors on body-wall muscles; one that is sensitive to levamisole, and one that is insensitive to levamisole but sensitive to nicotine. Levamisole is a nematode-specific cholinergic agonist. Exposure to levamisole causes hypercontraction of body-wall muscles and death within a few hours. Genes that confer strong resistance to levamisole after being mutated encode either acetylcholine receptor subunits or proteins potentially involved in the synthesis and assembly of that receptor - both mutant classes display a strong uncoordinated phenotype. By contrast,
lev-10when mutated, confers weak resistance to levamisole but mutants are pseudo wild-type.
lev-10was initially defined by Jim Lewis (Lewis et al., 1980) as a single allele
x17. To analyze the expression of the levamisole-sensitive receptor in the
lev-10background, we generated an antibody against the UNC-29/LEV-1 subunits. By immunofluorescence, we observed that receptors were no longer detected at neuromuscular junctions. Since
lev-10mutants still hypercontract on levamisole, we hypothesized that dispersed receptors were still present on body-wall muscles. Preliminary electrophysiological recordings of muscle cells support this hypothesis: pressure-applied levamisole-induced current is normal in the mutant compare to the response in the wild-type but miniature postsynaptic excitatory currents generated by activation of levamisole receptors at the synapse are no longer detected. However, both global and miniature currents dependant on the second nicotinic receptor are normal. To further understand the function of
lev-10, we used Mos1mediated mutagenesis to identify a new allele of
lev-10. The mutagenic Mos1insertion was localized by inverse PCR.
lev-10encodes a putative single-pass type I transmembrane protein carrying CUB and LDLa domains. We were able to rescue the levamisole resistance phenotype of
lev-10animals by injecting a PCR fragment corresponding to the predicted
lev-10locus. The Plev-10::GFPtranscriptional fusion drives the expression of the GFP in motoneurons and muscles. Mosaic experiments are in progress to determine if LEV-10 is required in motorneurons or in muscle for receptor clustering. In summary,
lev-10encodes a putative transmembrane protein required specifically for clustering the levamisole receptors at neuromuscular junctions.