Motor neurons release the neurotransmitter acetylcholine (ACh) at the neuromuscular junction (NMJ) resulting in skeletal muscle contraction. Defects in cholinergic signaling at the NMJ can lead to the development of neuromuscular diseases. The body wall muscles of C.elegans are functionally comparable to vertebrate skeletal muscle and are an excellent model for the study of post-synaptic cholinergic signaling. Coordinated body movements results from excitatory signaling through ACh receptors (AChR) and inhibitory signaling through GABA receptors, which causes muscle contraction and relaxation, respectively. Exposure to the AChR agonist levamisole constitutively activates postsynaptic AChRs, leading to hypercontracted paralysis. A forward genetic screen for strong resistance to levamisole identified mutations in 13 different genes [1]. However, mutants with weak resistance or hypersensitivity to levamisole as well as those with sterile and/or lethal phenotypes were not isolated. To identify additional factors that regulate cholinergic signaling at the NMJ, we conducted a genome wide RNAi screen for altered levamisole response and identified 18 gene knockdowns that resulted in resistance and 138 that caused hypersensitivity to levamisole. Knockdown of
mca-3, which encodes a plasma membrane Ca2+ ATPase required for extrusion of intracellular Ca2+ led to levamisole resistance. This was unexpected, as we would predict that an increase in intracellular Ca2+ resulting from loss of
mca-3 would likely lead to hypersensitivity. A second protein, the Na+/Ca2+ exchanger NCX-2 is also important for efflux of intracellular Ca2+. To determine the effect of loss of
mca-3 and
ncx-2 on response to levamisole, we knocked down the genes independently and together and performed time-course assays, counting the number of worms moving in 0.4 M liquid levamisole every five minutes for 60 minutes. Knockdown of
mca-3 led to significant levamisole resistance, however, loss of
ncx-2 resulted in levamisole response indistinguishable from the empty vector control. Loss of both
mca-3 and
ncx-2 resulted in levamisole resistance not significantly different from the
mca-3 knockdown. These results suggest that the mechanism by which loss of
mca-3 causes altered levamisole response may be independent of its role in Ca2+ efflux. References: [1] Lewis, J. A., Wu, C.-H., Berg, H., & Levine, J. H. (1980). The Genetics of Levamisole Resistance in the Nematode CAENORHABDITIS ELEGANS. Genetics, 95(4), 905-928.