MOD-1 is a serotonin-gated chloride channel that modulates locomotory behaviour in C . elegans

The neurotransmitter and neuromodulator serotonin (5-HT) functions by binding either to metabotropic G-protein-coupled receptors (for example, 5-HT1, 5-HT2, 5-HT4 to 5-HT7), which mediate `slow' modulatory responses through numerous second messenger pathways, or to the ionotropic 5-HT3 receptor, a non-selective cation channel that mediates `fast' membrane depolarizations. Here we report that the gene mod-1 (for modulation of locomotion defective) from the nematode Caenorhabditis elegans encodes a new type of ionotropic 5-HT receptor, a 5-HT-gated chloride channel. The predicted MOD-1 protein is similar to members of the nicotinic acetylcholine receptor family of ligand-gated ion channels, in particular to GABA (g-aminobutyric acid)and glycine-gated chloride channels. The MOD-1 channel has distinctive ion selectivity and pharmacological properties. The reversal potential of the MOD1 channel is dependent on the concentration of chloride ions but not of cations. The MOD-1 channel is not blocked by calcium ions or 5-HT3a-speci®c antagonists but is inhibited by the metabotropic 5-HT receptor antagonists mianserin and methiothepin. mod-1 mutant animals are defective in a 5-HT-mediated experience-dependent behaviour and are resistant to exogenous 5-HT, con®rming that MOD-1 functions as a 5-HT receptor in vivo. The locomotory rate of a well-fed C. elegans hermaphrodite slows when it encounters bacteria (the basal slowing response); this slowing response is enhanced markedly if the animal has been deprived of food for 30 min before the encounter (the enhanced slowing response (Fig. 1a). In a genetic screen for mutants defective in the enhanced slowing response, we identi®ed the gene mod-1, which is de®ned by the mutation n3034 (ref. 3). On Petri dishes with bacteria, the locomotory rate of food-deprived mod-1 mutants is signi®cantly faster than that of the wild type (strain N2) (Fig. 1a, grey bars). On dishes without bacteria, the locomotory rate of fooddeprived mod-1 mutants is not different from that of food-deprived wild-type animals (Fig. 1a, grey bars). Well-fed mod-1 mutants show no defect in their basal slowing response to bacteria (Fig. 1a, black bars). The swimming behaviour of mod-1(n3034) mutants was not affected by exogenous 5-HT treatment, whereas exogenous 5-HT inhibits wild-type C. elegans locomotion; and this phenotype was semi-dominant in a time-dependent manner (Fig. 1b), which aided our cloning of the mod-1 gene (see below). This 5-HT resistance of mod-1 mutants is consistent with the ®nding that the enhanced slowing response is mediated by 5-HTsignalling and the hypothesis that mod-1 mutants are de®cient in 5-HT signalling. We used standard genetic three-factor mapping and scored the 5-HT resistance of mod-1 mutants to locate mod-1 to a ,400 kilobase (kb) region on chromosome V. Cosmid C38E12 from this region rescued the recessive aspect of the Mod-1 mutant phenotype of 5-HT resistance, as assayed by the response to exogenous 5-HT after 5 min (Fig. 1c). We narrowed the rescuing activity to a 5.5-kb fragment (Fig. 1c) containing a single predicted gene. We isolated several complementary DNAs with SL1 transspliced leaders, located at the 59 ends of many C. elegans transcripts, and determined the complete mod-1 transcription unit (Fig. 2a). Genomic subclones lacking all or part of this transcription unit did not rescue the Mod-1 phenotype of 5-HT resistance (Fig. 1c). MOD-1 is similar to members of the nicotinic acetylcholine receptor (nAChR) family of ligand-gated ion channels, in particular to GABAand glycine-gated chloride channels (Fig. 2b). We found a single-base transition mutation in the mod-1 coding sequence in n3034 mutants (Fig. 2b, c), in which Ala 281 (codon GCT) is changed to valine (GTT) in the predicted M2 transmembrane domain of the MOD-1 protein. M2 is thought to be critical for channel function. We used site-directed mutagenesis to introduce this C-to-T (A281V) mutation into the 5.5-kb minimal rescuing