TheCaenorhabditis elegans unc-49Locus Encodes Multiple Subunits of a Heteromultimeric GABA Receptor

Abstract

Ionotropic GABA receptors generally require the products of three subunit genes. By contrast, the GABA receptor needed for locomotion in Caenorhabditis elegans requires only the unc-49 gene. We cloned unc-49 and demonstrated that it possesses an unusual overlapping gene structure. unc-49 contains a single copy of a GABA receptor N terminus, followed by three tandem copies of a GABA receptor C terminus. Using a single promoter, unc-49 generates three distinct GABAA receptor-like subunits by splicing the N terminus to each of the three C-terminal repeats. This organization suggests that the three UNC-49 subunits (UNC-49A, UNC-49B, and UNC-49C) are coordinately rescued and therefore might coassemble to form a heteromultimeric GABA receptor. Surprisingly, only UNC-49B and UNC-49C are expressed at high levels, whereas UNC-49A expression is barely detectable. Green fluorescent protein-tagged UNC-49B and UNC-49C subunits are coexpressed in muscle cells and are colocalized to synaptic regions. UNC-49B and UNC-49C also coassemble efficiently in Xenopus oocytes and HEK-293 cells to form a heteromeric GABA receptor. Together these data argue that UNC-49B and UNC-49C coassemble at the C. elegans neuromuscular junction. Thus, C. elegans is able to encode a heteromeric GABA receptor with a single locus.

Optogenetic analysis of GABABreceptor signaling inCaenorhabditis elegansmotor neurons

“…2, A-C). Interestingly, when this was repeated in mutants lacking the sole postsynaptic ionotropic GABA A receptor UNC-49 (Bamber et al 1999), the elongation effects were not completely abolished but worms still elongated by up to 2% (Figs. 1, A and B, and 2, A-C; Supplemen- Fig.…”

GABA Is Dispensable for the Formation of Junctional GABA Receptor Clusters inCaenorhabditis elegans

“…In UNC-49B-GFP (green fluorescent protein), GFP is inserted in frame in the M3-M4 cytoplasmic loop of the UNC-49B subunit in a genomic fragment that contains all three GABA receptor subunit open reading frames plus 400 nt of the unc-49 promoter (Bamber et al, 1999). In this construct, we replaced GFP with YFP (yellow fluorescent protein) to generate pJL41.…”

“…Transformation was performed by microinjection of plasmid DNA into the gonad (Mello et al, 1991). unc-49(e407);lin-15(n765ts) worms were injected with a DNA mixture containing pJL37 (Punc-47::snb-1-CFP) (20 ng/ l), a gel-purified AflIIMluI fragment of pJL41 (unc-49B-YFP missing the 5Ј end of the gene) (20 ng/ l) and a 110/38 unc-49 PCR fragment (10 ng/ l) containing a 4 kb promoter fragment plus 2 kb of the 5Ј coding sequence, as described by Bamber et al (1999). pEKL15, a plasmid that contains the wild-type lin-15 gene (Clark et al, 1994), was coinjected at 40 ng/ l as a cotransformation marker.…”

“…In this study, we analyze the differentiation of the postsynaptic domain of GABAergic neuromuscular junctions by visualizing GABA receptors in muscle. The subunits that comprise the GABA A -type receptor expressed in the body muscles are generated by alternative splicing from a single locus called unc-49 (Bamber et al, 1999). Our results demonstrate that innervation from motor neurons is necessary to induce and maintain UNC-49 clusters.…”

TheCaenorhabditis elegans snf-11Gene Encodes a Sodium-dependent GABA Transporter Required for Clearance of Synaptic GABA

“…The 26 C. elegans GABAergic cells modulate locomotion (DD and VD motor neurons), foraging (RMEs), and defecation (AVL and DVB) behaviors (McIntire et al, 1993b;Schuske et al, 2004). Mutations in the GABA biosynthetic enzyme glutamic acid decarboxylase (GAD) (unc-25;Jin et al, 1999), vesicular transporter (unc-47;McIntire et al, 1997), and excitatory and inhibitory receptors (exp-1; Beg and Jorgensen, 2003;unc-49;Bamber et al, 1999), have been described in detail. In addition, earlier studies had demonstrated uptake of exogenous GABA into a subset of C. elegans neurons (McIntire et al, 1993b).…”