GABAA receptors are important inhibitory neurotransmitter receptors in C. elegans and mammals. The strength of the postsynaptic response to the neurotransmitter, GABA, is proportional to the number of receptors in the postsynaptic membrane. Our lab is interested in determining how GABAA receptors are trafficked to, and localized at synapses, using C. elegans as a model system. The C. elegans GABAA receptor is composed of two subunits, UNC-49B and UNC-49C, and is required for coordinated locomotion. This receptor is expressed in muscles and localized to the neuromuscular junctions along the dorsal and ventral nerve cords; an UNC-49B::GFP full-length translational fusion recapitulates this expression pattern. To identify genes required for receptor trafficking and localization, we screened for mutants with altered patterns of UNC-49B::GFP fluorescence, using a dissecting microscope. We isolated five mutant alleles (gr 1, 2, 5, 6 and 8) representing at least two complementation groups. All mutants have GFP fluorescence in vesicular bodies within muscle cells, a phenotype we have termed GABA receptor mislocalized (Grm). Preliminary data, using a pH sensitive dye (Lysotracker), suggests that these vesicles may be acidified compartments. An interesting possibility is that they are endosomes, containing UNC-49B::GFP subunits that have entered into the degradation pathway. We have determined that
gr5 is an allele of
unc-3. UNC-3 is a transcription factor that is transiently expressed in neurons during development.
unc-3 mutants have severely disrupted nervous system structure, characterized by defasciculated ventral and dorsal nerve cords, and ectopic synapses. Both the presynaptic (UNC-47::GFP) and postsynaptic (UNC-49B::GFP) GABAergic markers appear more severely affected dorsally: Large gaps between synapses appear in the dorsal nerve cord, and the majority of UNC-49B::GFP containing vesicles (80%) are found within the dorsal muscles. Because UNC-3 acts presynaptically and the severity of the Grm phenotype correlates with the severity of the synapse spacing defect, we hypothesize that the stability of the GABAA receptor within the postsynaptic membrane depends upon the proper establishment of the synapse. We tested this hypothesis by examining the localization of UNC-49B::GFP in another mutant with a disrupted nervous system structure,
unc-5.
unc-5 encodes an UNC-6/netrin receptor, expressed in neurons, which is required to guide axonal projections to the dorsal nerve cord.
unc-5 mutants also display the Grm phenotype, and the majority of the GFP-containing vesicles (80%) are found within the dorsal muscles. These data suggest that a signal from the presynaptic cell is required to recruit and stabilize postsynaptic GABAA receptors. Analyzing the other mutants from our screen may identify this signal, or the proteins that convey this information to the GABAA receptor trafficking machinery.