Acetylcholine can act as a potent neuromodulator in the brain by activating intracellular G-protein signaling pathways that drive neurotransmission. In C. elegans, agonists for muscarinic AChRs (mAChRs) promote synaptic transmission at the NMJ by activating the heterotrimeric G-protein EGL-30/Gaq. However, the identity of the GPCR activated by acetylcholine and the signaling pathways downstream of muscarinic signaling are not clear. Here, we show that arecoline acts through
gar-3, a mAChR of the M1/M3 subclass, and two EGL-30 effector proteins, EGL-8/PLC and UNC-73/Trio. Furthermore, we identify two novel downstream components activated by this pathway: 1)
sphk-1/sphingosine kinase, an enzyme that converts sphingosine to sphingosine-1-phosphate, and 2)
unc-2, a VGCC required for neurotransmission. We provide evidence that
sphk-1 and
unc-2 are required for arecoline-induced hypersensitivity to aldicarb. Previously, we have shown that SPHK-1 facilitates neurotransmitter secretion by regulating the synaptic vesicle cycle, indicating that muscarinic signaling may utilize SPHK-1 to facilitate ACh release. Indeed, acute treatment of arecoline recruits a GFP-tagged SPHK-1 fusion protein to presynaptic release sites. In contrast, synaptic SPHK-1 levels are decreased in
egl-30 and
unc-73 loss of function mutants, suggesting that SPHK-1 may be activated by this pathway. Arecoline and UNC-73 mediated recruitment of SPHK-1 depends on
unc-2; both
unc-73 and
unc-2 mutants decrease SPHK-1 abundance but
unc-73;
unc-2 double mutants do not have additive effects. Furthermore, the ability of arecoline to recruit SPHK-1 to synapses is completely dependent on
gar-3, which acts cell autonomously in motor neurons. Experiments to identify the source of endogenous ACh are under way, but preliminary data suggests that ACh released from motor neurons themselves is not involved. For example, aldicarb treatment (to promote spillover) or mutations that alter ACh secretion from the NMJ do not have detectable effects on SPHK-1 abundance. Together, our results indicate that acetylcholine facilitates neurotransmitter release at the NMJ by activating GAR-3 on motor neurons. This, in turn, activates an EGL-30 - UNC-73 pathway that leads to Ca++ dependent recruitment of SPHK-1 to sites of neurotransmitter release.