g -amino-butyric acid (GABA) is the major fast synaptic inhibitory neurotransmitter and serves as the ligand for GABA receptors. Ligand binding causes a conformational change in GABA receptors forming an ion-channel that is selectively permeable to chloride. In C. elegans, GABA functions as both an inhibitory and excitatory neurotransmitter. The inhibitory action of GABA is mediated through UNC-49, which is an inhibitory GABA receptor that is localized to body wall muscles. UNC-49 provides contralateral inhibition to antagonistic muscle groups ensuring coordinated locomotion. However, GABA also mediates a novel excitatory function. GABA release from the motorneurons AVL and DVB stimulates the contraction of the enteric muscles, causing the expulsion of gut contents. We are interested in the molecular nature and physiology of the GABA receptor that mediates excitatory neurotransmission of the enteric muscles. We have cloned two genes,
exp-1 and Y46G5A.26*, which could potentially encode excitatory GABA receptors subunits in C. elegans .
exp-1 , (expulsion defective), was originally identified by Jim Thomas in screens for mutants defective in the defecation cycle and subsequently in screens looking for mutants in GABA function. Of the six genes required for GABA function in the worm, only
exp-1 specifically eliminates the excitatory function of GABA. We hypothesize that
exp-1 encodes an excitatory GABA receptor expressed in the enteric muscle. We cloned
exp-1 and determined the expression pattern of the protein. Genetic map data place
exp-1 in a small interval between
lin-4 and
lin-23 on chromosome II. A fosmid in this region contains an open reading frame with sequence related to GABA receptors. Transgenes bearing a subclone of the open reading frame rescue
exp-1 mutants. In addition, we identified point mutations in five independent alleles of
exp-1 . Rescuing constructs in which the EXP-1 protein is tagged with GFP indicate that EXP-1 is expressed in the enteric muscles. Consistent with our hypothesis that EXP-1 will form an excitatory GABA receptor, we isolated cDNAs that demonstrate EXP-1 has a completely divergent transmembrane domain 2 (TM2). TM2 is known to confer ion selectivity for the ligand-gated ion channel superfamily, and the numerous negatively charged residues present in TM2 suggest that EXP-1 is a cation selective channel. BLAST searches revealed a second putative excitatory GABA receptor subunit in C. elegans . Y46G5A.26* is 58% identical to EXP-1. Specifically, TM2 of Y46G5A.26* is 85% identical to EXP-1, suggesting that this putative GABA subunit may also form an excitatory receptor. We isolated full-length cDNAs of Y46G5A.26* and have created GFP reporter constructs. Both translational and transcriptional GFP fusions indicate that Y46G5A.26* is expressed in GABA neurons. This putative subunit is not expressed in the enteric muscles as expected, which suggests that GABA may play roles in excitation beyond the enteric muscles. We hypothesize that it might be an excitatory autoreceptor. We are currently characterizing EXP-1 and Y46G5A.26* in Xenopus oocytes . We will determine GABA gating, ion selectivity and primary pharmacology for each putative subunit. Both EXP-1 and Y46G5A.26* appear to be members of a novel class of GABA receptor subunits which may lead to the identifi­cation of homologous proteins in higher vertebrates.