Gaq (EGL-30) is central to neuronal communication, regulating the production and destruction of the 2nd messenger diacylglycerol (DAG). A major target of Gaq is the small GTPase RHO-1. RHO-1 inhibits diacylglycerol kinase (DGK-1) leading to increased levels of DAG at the synaptic membrane, in turn increasing the amount of the neurotransmitter, acetylcholine (ACh) released. Animals over-expressing constitutively active RHO-1 (RHO-1*) in cholinergic motor-neurons have a loopy locomotion and an increased rate of ACh release shown by faster paralysis on 1mM aldicarb effects. RHO-1 still modulates neurotransmission in
dgk-1 mutants identifying that there are other DGK-1 independent pathways. To identify other effectors of RHO-1, we conducted an EMS-mutagenesis screen. 2000 haploid genomes were screened and worms that suppressed the neuronal phenotype of loopy locomotion associated with RHO-1* were identified. A secondary screen was then performed to look for suppression of RHO-1* expressed from the heat shock promoter of a separate transgene, which causes additional non-neuronal phenotypes. From these two screens 12 suppressors were identified that suppressed the neuronal effect of RHO-1* (loopy locomotion) but not the non-neuronal effects. Intriguingly, locomotion and absolute levels of ACh release don't appear to be as strongly correlated as previously thought. Many of our suppressors of neuronal RHO-1* loopy locomotion do not suppress the hypersensitivity to aldicarb, suggesting that levels of ACh release still remain high. Whole genome sequencing of one of these suppressors,
nz99, has identified a premature stop codon in T23G5.5, the gene encoding a dopamine transporter DAT-1. Expressing RHO-1* in the cholinergic motor neurons of a
dat-1 deletion mutant also gives rise to worms with more wildtype locomotion suggesting that DAT-1 is the suppressor identified by the screen. Experiments addressing the role of DAT-1 in RHO-1 signaling will be presented. As dopamine is known to be important in co-ordinating locomotion this raises interesting questions about how DAT-1 and RHO-1 interact.