To understand the mechanisms by which phorbol esters and diacylglycerol (DAG) regulate synaptic transmission at the C. elegans neuromuscular junction (NMJ), we have studied a protein kinase C-epsilon (PKC), encoded by the
kin-13ttx-4 gene. We have characterized a putative null allele,
nj3, isolated in the Mori lab, that encodes an early stop. Homozygous
kin-13 mutants are resistant to the acetylcholine (Ach) esterase inhibitor, aldicarb (the Ric phenotype), but are not resistant to the paralytic effects of the ACh agonist levamisole. A
kin-13::GFP transgene is expressed in all of the cholinergic motor neurons in the ventral cord and expression of
kin-13 cDNA under control of a motor neuron-specific promoter rescues the Ric phenotype of
kin-13 mutants. These results suggest that
kin-13 PKC acts in the ventral cord motor neurons to promote Ach release. Our preliminary voltage clamp recordings from
kin-13 mutants show that both the frequency of spontaneous excitatory post-synaptic currents (EPSCs) and the amplitude of electrically evoked EPSCs are reduced while sucrose evoked EPSCs are normal. These results are consistent with
kin-13 mutants having a reduced probability of synaptic vesicle release in the NMJ.
kin-13 mutants are partially resistant to phorbol ester-induced hypersensitivity to aldicarb and partially suppress the hypersensitivity to aldicarb (Hic) phenotype of
dgk-1 DAG kinase null mutants, suggesting that
kin-13 PKC is an important presynaptic target for phorbol esters. We have previously shown that a mutation in the C1 domain of UNC-13 also confers partial resistance to phorbols. In double mutants that lack
kin-13 PKC and that carry the UNC-13 C1 domain mutation, there is a dramatic reduction in phorbol ester sensitivity, suggesting that KIN-13 PKC is a phorbol target that acts in parallel with UNC-13 to facilitate release. Several results suggest that
kin-13 PKC may specifically play a role in dense core vesicle release. First, mutations in the neuropeptide maturation enzyme
egl-21 CPE as well as
unc-31 CAPS cause a Ric phenotype and partial phorbol ester resistance. Second,
kin-13 mutants fail to enhance the Ric or the phorbol resistance phenotype of
egl-21 mutants. Finally, expression of a constitutively active
kin-13 PKC transgene specifically in motor neurons causes a Hic phenotype, which is completely blocked by mutations in either
egl-21 or
unc-31. These data implicate KIN-13 PKC as an important DAG target in motor neurons that facilitates release of synaptic vesicles, possibly by regulating neuropeptide release.