Release of neurotransmitter into the synaptic cleft requires the coordinated action of numerous proteins at the presynaptic terminal. We have taken a genetic approach to identify proteins required for this process. To this end, we are in the process of characterizing two genes,
ric-6 and
ric-7 , that were previously identified in a behavioral screen and appear defective in neurotransmission. Mutations in
ric-7 disrupt both GABAergic and cholinergic neurotransmission.
ric-7 animals lack enteric muscle contractions during defecation and display a weak shrinker phenotype, both features of a loss of GABAergic function. In addition,
ric-7 animals are resistant to the acetylcholinesterase inhibitor aldicarb, although they are sensitive to the acetylcholine receptor agonist levamisole indicating a presynaptic cholinergic defect. The organization of ventral cord motor neurons and the density of neuromuscular junctions appear to be normal in
ric-7 animals and thus, RIC-7 does not function in neuronal development. Taken together, these phenotypes suggest a role for RIC-7 in the release of neurotransmitter at synapses. The
ric-7 phenotype is rescued by an 18kb fragment that is predicted to contain a single open reading frame encoding a 694 amino acid protein with no known homology. Expression of green fluorescent protein (GFP) under the control of the
ric-7 promoter identifies a predominately neuronal expression pattern. This data further supports the hypothesis that
ric-7 encodes a neuronal protein involved in presynaptic function. We are currently in the process of identifying other loci that genetically interact with
ric-7 .
ric-6 , like
ric-7 , also plays a role in both presynaptic cholinergic and GABAergic function. Mutations in
ric-6 result in aldicarb resistant, levamisole sensitive animals that also display a weak shrinker phenotype. The
ric-6 phenotype is rescued by a 5kb fragment that encodes the C. elegans homolog of the vacuolar ATPase B subunit. Vacuolar ATPase function is thought to be required for synaptic vesicle acidification, a process that is essential for loading neurotransmitter into vesicles. We will test this model using electrophysiological techniques recently developed in C. elegans .