| The nematode defecation motor program (DMP) involves three precisely timed sets of muscle contractions concluding with an enteric muscle contraction (EMC) that expels intestinal contents. The neurotransmitter GABA activates contraction of the enteric muscles; nevertheless, null mutants in the GABA biosynthetic enzyme encoded by unc-25 still exhibit an enteric muscle contraction (EMC) in 12% of defecation cycles, indicating the involvement of another excitatory signal. Null aex-2 mutants have no visible EMCs and are extremely constipated. This study demonstrates that aex-2 encodes a putative orphan G protein-coupled receptor of the family A rhodopsin-like receptors, and shows that aex-2 reporter gene fusions are expressed in the enteric muscles.;I investigated genetic interactions between aex-2 and various G protein a subunit alleles by analyzing the defecation phenotypes of double mutants. Gain-of-function alleles of gpa-2 and gsa-1 partially suppress the aex-2 EMC defect. These alleles do not suppress the unc-25 MC defect to the same extent, suggesting the suppression is specific to the aex-2 pathway. Additionally, suppression of the constipation defect allowed me to observe more EMCs than otherwise possible in an aex-2 mutant. I discovered that aex-2 lengthens the EMC latency relative to DMP initiation, an effect that also occurred in gpa-2, but not gsa-1 single mutants.;I also performed experiments that attempted to identify an AEX-2 ligand. Because no clear mammalian orthologue of the receptor exists, the ligand is unlikely to be a well-conserved neurotransmitter; therefore, I looked to the evolutionarily diverged family of neuropeptide genes for candidates. Deletion alleles exist for nine of 54 identified worm neuropeptide genes. I assayed the defecation phenotypes of these strains and found one in which only 58% of defecation cycles exhibited an EMC, identifying it as a candidate AEX-2 ligand.;In summary, I have discovered the identity of aex-2, identified a candidate neuropeptide ligand for AEX-2, identified parallel G protein signaling pathways that regulate the EMC, and illuminated an AEX-2-related function controlling EMC latency. Together these findings strongly suggest a model in which AEX-2 modulates enteric muscle membrane potential to a state that is primed for response to excitatory neurotransmission. |
