The mechanisms that allow cys-loop ligand-gated anion channels (e.g. glycine and GABA receptors in vertebrates, as well as glutamate, histidine, acetylcholine and serotonin receptors in invertebrates) to localize to synapses is poorly understood. To develop a system in which we can address this question genetically, we generated a full-length genomic clone of the gene encoding the glutamate gated chloride channel (GluCl) subunit GLC-2 with YFP inserted into the intracellular loop. GLC-2 has been reported to be co-expressed in the pharyngeal muscle (Laughton et al.,1997) with another GluCl subunit, AVR-15, and can form a heteromeric channel with AVR-15 in Xenopus oocytes. AVR-15 is necessary postsynaptically for neurotransmission by the glutamatergic pharyngeal motor neuron M3 (Dent et al., 1997). When expressed in Xenopus oocytes, our GLC-2::YFP fusion protein forms homomeric and heteromeric (with AVR-15) channels with only modest changes in glutamate sensitivity and kinetics. In transgenic animals, the GLC-2::YFP construct expressed in the
pm3 and
pm4 muscles of the pharynx and in the extrapharyngeal neurons RMED and RMEV, a subset of the cells expressing AVR-15. The YFP is in puncta, some of which are localized near to or at the M3 synapse. In an
avr-15 null background there is no M3 neurotransmission and no localization of GLC-2::YFP in the muscle or neurons. To determine whether GLC-2 localization requires presynaptic release of neurotransmitter, we placed GLC-2::YFP in an
eat-4 mutant background. EAT-4 is the vesicular glutamate transporter in M3 (Lee et al., 1999). In the
eat-4(
ky5) background, GLC-2::YFP localization is significantly degraded, though not completely eliminated. Interestingly, in the available
glc-2 deletion mutants, M3 neurotransmission appears normal. We are currently trying to determine whether GLC-2 forms a heteromeric channel with AVR-15 in vivo. We are also developing screens to identify other localization mutants as well as testing candidate genes. Laughton D.L., Lunt, G.G. and Wolstenholme, A.J. (1997) J. Exp Biol.200:1509. Dent, J.A., Davis, M.W. and Avery, L. (1997) EMBO J. 16:5867. Lee, R.Y.N., Sawin, E.R. Chalfie, M., Horvitz, H.R. and Avery, L. (1999) J. Neurosci. 19:159.