In C. elegans, coordinated locomotion requires both excitatory and inhibitory neurotransmission at neuromuscular junctions. The inhibitory neurotransmitter at the neuromuscular junction is GABA, and the
unc-49 locus encodes the GABA receptor.
unc-49 has an unusual gene structure which allows it to generate multiple full-length GABAA receptor-like subunits by splicing a common amino terminus to alternative carboxy termini. Although
unc-49 contains three alternative carboxy-termini, suggesting that three full-length subunits could be encoded, we were only able to isolate cDNAs for two of these subunits. Using RT-PCR, we have recently demonstrated that a third full-length subunits is indeed produced, as predicted from the
unc-49 gene structure. Surprisingly, a fourth subunit is also produced, which is truncated at its amino terminus. The operon-like structure of
unc-49 led us to hypothesize that some of the subunits may function within the same cell, perhaps within a single heteromultimeric receptor complex. This model makes the following two predictions: 1) The
unc-49 subunits should be expressed in the same cells, at about the same levels; and 2) The
unc-49 subunits should form a heteromultimeric ion channel when expressed in heterologous cells. Northern blotting experiments showed that two of the full-length subunits, UNC-49B and UNC-49C are strongly expressed, at about the same level, and the truncated subunit, UNC-49Cshort, is expressed at about 50% of this level. The remaining full-length subunit, UNC-49A, was barely detectable. GFP fusions to the UNC-49B, UNC-49C and UNC-49Cshort subunits showed that all three are co-expressed, primarily in the ventral body wall and head muscles. Thus, subunit expression patterns are consistent with the heteromultimer model. Electrophysiological data also support a model in which these subunits function together to form a GABA receptor. In Xenopus oocytes, UNC-49B forms a homomeric, picrotoxin-sensitive GABA receptor while UNC-49C and UNC-49Cshort are inactive as homomers. However when co-expressed, UNC-49B and UNC-49C co-assemble stoichiometrically to form a heteromultimer with about 6-fold reduced GABA sensitivity1 and greatly-reduced picrotoxin sensitivity. UNC-49Cshort can also co-assemble with UNC-49B, although somewhat inconsistently. We are presently determining whether this heteromultimer forms at the neuromuscular junction in vivo by 1) determining the subcellular localization of the
unc-49-encoded subunits, and 2) analyzing the functional properties of the neuromuscular junction GABA receptor using in vivo patch clamp electrophysiology. 1. Bamber, B. A., Twyman, R. E., and Jorgensen, E. M., submitted.