Muscle type acetylcholine gated ion channels are heteropentamers that mediate fast chemical synaptic transmission at the neuromuscular junction. Mature channels are composed of subunits assembled with an invariant stoichometry and in a specific order. These channels belong to a family of ligand gated ion channels that contain an intramolecular cysteine loop. The functional significance of this conserved structural feature is not completely clear, but it is thought to be involved in folding and assembly of cysteine loop channels.Levamisole causes paralysis by activating the levamisole sensitive acetylcholine gated ion channel present on nematode muscle. This channel is composed of UNC-29, UNC-38, UNC-63, and LEV-1 subunits. Genes not encoding subunits (
unc-50,
unc-74, and
ric-3) can be mutated to confer levamisole resistance. These loci are thought to encode proteins required for ion channel formation. In concert with this hypothesis,
unc-74 muscle cells lack levamisole induced electrophysiological responses, and UNC-38:GFP is retained in intracellular compartments in these mutants.We cloned
unc-74 using Mos1 mediated mutagenesis.
unc-74 encodes the worm homologue of TMX3, a novel membrane thioredoxin localized to the ER in human cell lines. Homologues have been identified in other metazoans that express acetylcholine gated ion channels, but not fungi. UNC-74 contains a thioredoxin domain with a conserved CXYC active site, a central region that does not contain any known functional domains, and a single transmembrane spanning region with an ER localization signal.Our working model is that UNC-74 catalyzes cysteine loop formation and thus has a role in ligand gated ion channel formation. An
unc-74:GFP promoter fusion is expressed in neurons and muscle cells, and muscle cell expression of
unc-74 is necessary and sufficient to rescue the locomotion and levamisole resistance phenotypes. A rescuing UNC-74:GFP fusion protein is localized to the ER, which is the site of disulfide bond formation. Although the cysteine loop is a feature of many different types of ligand gated ion channels,
unc-74 appears to be specific for the levamisole sensitive acetylcholine gated ion channel; GABA receptors are localized properly and levamisole insensitive acetylcholine gated ion channels are present in
unc-74 animals. Current experiments are addressing whether the thioredoxin domain of UNC-74 is required for function, and whether
unc-74 has a similar function in other systems.