A genetic suppressor screen identifies a novel, conserved ion channel complex as a new downstream target of RHO-1 signalling.
Doctoral thesis, UCL (University College London).
The small GTPase RHO-1 is an important regulator of neurotransmission. Caenorhabditis elegans nematodes expressing activated RHO-1 (G14V) in their cholinergic motor neurons (nRHO-1*) become hypersensitive to the acetylcholinesterase inhibitor aldicarb, demonstrating increased acetylcholine release, and acquire a highly loopy, uncoordinated locomotion. RHO-1 inhibits diacylglycerol kinase (DGK-1), and so increases the availability of diacylglycerol (DAG), a key second messenger for release at the presynaptic membrane. Inhibiting RHO-1 in a dgk-1 mutant causes a decrease in neurotransmitter release, demonstrating the presence of additional targets downstream of RHO-1. During a forward genetic screen for suppressors of the loopy locomotion of nRHO-1* animals we obtained a mutant, nz94, which carried an additional ‘fainter’ phenotype, helping us identify it as an allele of unc-80, a large, conserved protein, important in the localization of NCA-1 and NCA-2, C. elegans homologues of the novel mammalian ion channel NALCN. RHO-1*;unc-80 double mutants are non-loopy, but still hypersensitive to aldicarb, indicating that the loopy locomotion and high levels of neurotransmitter release can be uncoupled. unc-80 mutants do not suppress non-neuronal phenotypes associated with heat-shock expression of RHO-1*, such as tail swelling and sterility. Expressing an unc-80 transgene under a cholinergic promoter is sufficient to rescue the suppression of loopy locomotion seen in the nRHO-1*;unc-80 double mutants, indicating that unc-80 acts presynaptically in the same cells as nRHO-1* for the generation of this loopy locomotion. Work from other labs shows that unc-80 mutants suppress gain-of-function PPK-1 (PI4P5K) phenotypes, most likely through changes in the localisation of the ion channels. Our current model involves RHO-1 binding to and activating PPK-1, increasing levels of PIP2 and hyperactivating the NCA-1/NCA-2 channels. We suspect this ion channel complex may regulate the release of neuropeptides involved in locomotive behaviour.
|Title:||A genetic suppressor screen identifies a novel, conserved ion channel complex as a new downstream target of RHO-1 signalling|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > MRC/UCL Lab for Molecular Cell Biology|
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